Autonomously Designed Free Form 2d Dna Origami

DNA origami has become a valuable tool for basic science in a diversity of research fields, and applications in material sciences, medicine, and in other areas have begun to emerge. (1) The DNA origami design methodology can be used to create custom 2D (2) and 3D shapes (3,4) with nanometer dimensions. DNA origami uses a set of short "staple" DNA oligonucleotides that are designed to fold a long, usually circular "scaffold" DNA single strand into a user-defined shape. The self-assembly can proceed rapidly and with high yields and quality, depending on design and assembly procedures used. During DNA origami design, the scaffold sequence is typically taken as a fixed input from a library of generic scaffold sequences that are available to the community. The sequence string is then routed through a strand diagram that encodes the connectivity of the target object to be made. The sequences for the staple strands are derived by considering local Watson–Crick base complementarity to the scaffold. (2,3)

Generic scaffold sequences, as others have noted previously, (5) may limit the scope of applications that could be addressed with DNA origami. To understand the resulting restrictions, it is helpful to consider the M13 bacteriophage single-strand genome variants which are the most popular generic scaffolds currently used in the field. Many properties of the DNA origami built with M13-based scaffolds are naturally determined by the length and sequence of the M13 phage genome: First, the overall dimensions of a single DNA origami are by default limited by the length of the scaffold strand. Second, designers cannot control global sequence properties such as GC content or sequence redundancy, which could potentially affect the self-assembly behavior as others have speculated. (6) Third, designers cannot avoid undesired sequence motifs such as potentially immunogenic CpG motifs (7) which occur frequently within M13-based scaffolds. Fourth, phage genes may be undesirable in the context of in vivo applications. Fifth, custom sequence motifs can be introduced in DNA origami objects only as appendices or insertions in staple strands, which may negatively affect the yield of incorporation of such motifs, (8) unless the scaffold strand itself is customized by the user. One example of sequence motifs that could be of use at hundreds of sites in the scaffold are AA motifs at all crossovers positions, as this could enable covalent stabilization of the target object via UV point-welding. (9)

We envision that rather than designing objects around generic scaffold sequences, as the current DNA origami paradigm posits, designers should be able to fully specify the target structure not only in terms of desired 3D shape but also in terms of the sequences used. Realizing design-specific scaffolds requires a design tool that can interpret design diagrams and algorithmically build a scaffold sequence, and it also requires scaffold production methods adapted for making fully sequence-customized scaffolds. In order to address real-world applications, it should also be possible to manufacture the resulting design-specific scaffolds in a scalable and cost-efficient process. Whereas M13 scaffolds can be efficiently produced biotechnologically, a substantial portion of the M13 phage genome cannot be modified because these portions are required for the phage lifecycle. Scaffolds for DNA origami may be produced in test tubes using enzymatic reactions (3,6,10−15) or in bacterial cultures via phage-based biotechnological production. (2,3,16−20) Enzymatic production in test tubes affords the possibility of choosing user-defined sequences but is unfortunately economical only on analytical (μg) scales. In general, biotechnological processes using bacterial cultures allow production on much larger scales such as those required for biomedical or materials applications of DNA origami. (18,20,21)

The objective of this work is to provide the missing tools and methods to realize design-specific scaffolds for DNA origami. To this end, we built a tool called "scaffold smith" that can construct design-specific scaffold sequences for DNA origami, and we developed a biotechnological approach to produce fully sequence-customized scaffolds. In total, we produced a library of 17 customized scaffold strands with attractive properties that should be useful to the community as generic scaffolds (plasmids available via Addgene, alongside a target plasmid for construction of other scaffolds). Our design tools and production methods for making design-specific scaffolds allow designing fully user-specified DNA origami while maintaining the possibility to produce materials at larger scales.

Results and Discussion

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Sequence Design and Strand Production

To construct design-specific custom scaffold sequences, we created a design tool termed "scaffold smith" (Figure 1A and Supporting Information Note S1). The tool integrates with the conventional DNA origami design workflow at the point when the user has produced a caDNAno strand diagram. (22) The scaffold smith generates the scaffold string that users will then use subsequently to generate the staple sequences. The user can define sequence motifs that will be excluded entirely, and the user can specify a list of sequence strings to be placed in the scaffold at desired locations in the target object. The tool also enables constructing a design-specific scaffold string for direct, modification-free UV cross-linking of the target object. (9) To this end, the tool automatically identifies all scaffold base indices located at staple termini and at crossovers and places "A" or "AA", respectively, at those positions in the scaffold string. Also, scaffolds may be produced that have fixed scaffold motifs at staple termini so that residual overhangs of staples that are produced biotechnologically and DNAzyme-digested as previously described (21) can directly pair with the scaffold. The scaffold smith can either generate sequences de novo or operate on existing scaffold strings to create new variants of them that include desired motifs at desired locations. For de novo construction, the sequence is built base-by-base with a stochastic Monte Carlo process beginning at a user-defined site in the strand diagram. The algorithm controls the scaffold sequence composition in terms of the statistical weights of base pair steps (e.g., how often A should be followed by A, C, G, or T, respectively), which gives the user control over the thermodynamic properties of the scaffold to be built. It also enables directly reducing or avoiding entirely the occurrence of known immunogenic or UV-radiation-sensitive motifs such as CG or TT, respectively. The tool considers the degree of sequence redundancy that emerges during sequence construction and can build (pseudo-) De Bruijn sequences of user-defined order. It can generate sequences where all strings of a user-defined length (for example 8 bases) appear only as often as the user accepts it in the entire scaffold sequence (for example, not more than once). Finally, the tool computes the overall statistics of the generated scaffold string with respect to composition and redundancy. The user may then adjust parameters and repeat the sequence construction. To summarize, the scaffold sequence construction with the scaffold smith has a deterministic and a stochastic part. The user can define properties, which will be strictly realized, such as exclusion and site-directed inclusion of user-defined sequence motifs. All remaining sites (i.e., sites where the user makes no specific demands) will be filled up stochastically; however, the user has control over the overall statistics of the sequence built in terms of composition and redundancy. The underlying algorithms are described in more detail in Supporting Information Note S1. We created a stand-alone graphical user interface (GUI) for the scaffold smith, but it should be straightforward to introduce the underlying concepts into future caDNAno versions or into future variants of automated design solutions such as DAEDALUS, (23) PERDIX, (24) TALOS, (25) or vHelix. (26)

Figure 1

Figure 1. Design-specific scaffold sequences in minimum-constraint vectors for making fully user-defined DNA origami. (A) Schematic diagram of input for the scaffold smith used for creating custom scaffold sequences: exemplary caDNAno design diagram with scaffold strand indicated in blue and staple strands in multiple colors (I), user-specific constraints (II), and weighting factors for a stochastic base distribution (III). (B) Illustration of scaffold production with helper-plasmid system using phagemids with a split-ori approach (top) and a modified split-ori approach where the backbone sequence is flanked by self-cleaving DNAzymes (bottom). Zn²⁺ addition leads to excision of the backbone and linearization. Black, constant parts for each type of scaffold; gray, user-definable parts; light green, backbone present only in the double-stranded plasmid and not in the single-stranded product; red, self-cleaving DNAzymes.

We now focus on the question of how to practically make fully sequence-customized DNA single strands. A scalable solution for ssDNA production makes use of bacteriophages with fast growing Escherichia coli (E. coli) cells as host, but phage-based ssDNA scaffolds inevitably contain cassettes with sequences that cannot be altered because they are required for the phage production. User-defined insert sequences can only be added to these fixed parts. In fully customizable scaffolds, the length of the fixed part should be negligible compared to the total length of the scaffold. However, in the conventional M13 phage production method, (18,27) the fixed part is approximately 6000 bases long, which is not negligible at all. Phagemids, in combination with helper phages (28) or helper plasmids, (29) allow producing ssDNA with fixed backbones of ∼2000 bases, which is still not negligible. Our goal was thus minimizing the fixed-sequence cassettes to maximize the freedom to design custom scaffold sequences while maintaining the possibility for efficient production in bacterial cultures. To this end, we developed and tested several methods with minimized constant-sequence cassettes (Supporting Information Note S2).

The production method used for most of our custom scaffolds relies on a split origin of replication (split-ori) that was originally developed to produce microphages containing comparably short 221 bases long ssDNA, in combination with helper phages. (30) Here, we integrated our design-specific scaffold sequences as custom inserts into the split-ori system (Figure 1B) and identified a suitable helper plasmid that allows producing pure target ssDNA without contamination of helper phage DNA or other unwanted DNA species (Supporting Information Figure S5). The thus-produced ssDNA scaffold strands are circular with a minimal constant-sequence backbone of 234 bases (Figure 1B). This residual backbone can then also be removed entirely via Zn²⁺-dependent digestion when flanking self-excising DNAzyme cassettes (21) are added during sequence preparation for gene synthesis. As a result, the user obtains linear scaffold molecules with virtually 100% custom sequence (except for two and seven base residuals at the two termini). In support of the robustness of the split-ori/helper-phage approach, we note that, concurrent to our work, Douglas and co-workers produced scaffolds for DNA origami by inserting coding genes or parts of the lambda phage genome into a split-ori backbone, although Douglas et al. used a different helper plasmid. (31)

Sequence Redundancy and Sequence Composition Rules

The commonly used M13-phage-based scaffolds have a comparably high degree of sequence redundancy, and others have speculated that this redundancy may negatively influence the self-assembly behavior of DNA origami. (6) On the other hand, it has also been speculated that the M13-based sequences were particularly well-behaved and thus especially suited for DNA origami. (32) In addition, the influence of sequence composition (e.g., AT vs GC content) on self-assembly remains in the dark. For designing synthetic scaffolds, it is important to understand the impact of sequence redundancy and sequence composition on self-assembly in order to arrive at relevant sequence construction criteria. To study these parameters, we constructed five synthetic 7560 bases long scaffolds (SC2–6) and compared them to a popular M13-based scaffold variant (SC1) of the same length (Figure 2). The designed portions of the custom scaffolds SC2–6 were low redundancy de Bruijn sequences of order 7, which means that sequence strings with length 7 occur exactly once or not at all. (33) All of these scaffolds could be produced in shake flasks with yield and purity similar to that in conventional M13-based production (Supporting Information Note S2 and Figures S4–S6). Four of the scaffolds (SC2, SC4, SC5, SC6) have insert sequences that are orthogonal to each other and to the conventional M13-based scaffolds. Residual sequence overlaps between these four individual scaffolds are determined by details of the constant-sequence cassettes in the phagemids and have lengths between 180 and 426 bases, which is small compared to the total length (7560) of the scaffold variants. Scaffold variant SC3 had a longer 1387 bases long sequence fragment taken from the M13 genome; SC3 has thus a degree of sequence redundancy which fell between the low-redundant de Bruijn scaffolds and the highly redundant M13.

Figure 2

Figure 2. Influence of base composition and sequence redundancy of custom scaffolds on DNA origami self-assembly. Blue indicates M13-based scaffolds; orange, magenta, red, cyan, and green indicate custom scaffolds. (A) Schematic representations of six different 42-helix bundles folded using the six different scaffolds. SC1, M13-based scaffold; SC2, reduced backbone phagemid scaffold with CpG-free de Bruijn insert sequence; SC3, conventional phagemid with high duplicity fragment and de Bruijn insert sequence; SC4, conventional phagemid with de Bruijn insert sequence; SC5 and SC6, split-ori based scaffold with de Bruijn sequence; L, length; GC, GC content of the corresponding scaffold. (B) Electrophoretic mobility analysis of self-assembly reactions of the 42-helix bundles shown in (A) at different temperatures and salt concentrations. SC, scaffold reference; C50 and C20, assembly reactions containing 50 nM (C50) or 20 nM (C20) scaffold, 200 nM staples, and 20 mM MgCl₂ that were subjected to an annealing ramp from 60 to 44 °C (1 h per °C); temperature screen, assembly mixtures as in C50 but subjected to annealing ramps covering the temperature intervals indicated above each lane (1 h per °C); magnesium screen, assembly reactions containing 50 nM scaffold, 200 nM staples, and MgCl₂ concentrations between 5 mM (M5) and 30 mM (M30). P, pocket; F, folded 42-helix bundle. All samples were loaded onto the gel at an approximate scaffold concentration of 20 nM. All temperature ramps contained an initial denaturation step at 65 °C for 15 min. Laser scanned fluorescent images of the electrophoretic analysis were autoleveled. (C) Statistics of sequence duplicates of different scaffold variants as a function of fragment length. Colors as in (A). (D) Experimentally observed optimal folding temperature intervals of the 42-helix bundles plotted against total NN energy of corresponding scaffold variant. Total NN energy was calculated using nearest-neighbor free energy parameters, (36) ignoring edge effects. Dots in red indicate upper, and dots in blue indicate lower limit of the highest folding temperature interval where the sample appeared fully folded. Solid lines represent linear fits.

To test our custom scaffolds, we used them as templates for variants of a previously described brick-like 42-helix bundle (42hb) (34) and synthesized the corresponding sets of staple oligonucleotides (Figure 2A). We analyzed the assembly behavior of the different 42hb variants at different temperatures and salt concentrations using a standardized folding screen. (35) The assembly reactions yielded well-folded products for all six scaffold sequence variants of the 42hb object, as manifested by sharp leading bands in gel electrophoresis (Figure 2B). Contrary to what has been speculated previously, (6) we did not observe systematic quality differences between the scaffold variants with higher or lower degree of sequence redundancy. In particular, we did not detect a beneficial effect on assembly behavior when using the low-redundancy de Bruijn sequences compared to the conventional, much more redundant M13-based scaffold variant (Figure 2C and Supporting Information Figure S7). Similarly, we could not detect any drawbacks of synthetically designed scaffold sequences that are not M13-based.

Sequence composition, however, did have noticeable effects on self-assembly behavior. For example, well-folded objects self-assembled already at lower salt concentrations for sequence variants with higher GC content (Figure 2B, right). As seen previously for other DNA origami objects, (34) each sequence variant assembled successfully in narrowly defined temperature intervals. For our 42hb variants, we found that the sequence composition of the scaffold variant determined the temperature intervals in which the objects folded successfully (Figure 2A,B). In particular, the temperature intervals that yielded the highest folding quality correlated strongly with the scaffold sequence composition in terms of the total nearest-neighbor energy (Figure 2D). (36) In the SC2 sequence, C is never followed by G. As the CG base pair step has a particularly strong stacking energy, the omission of this base pair step leads to a substantially reduced nearest-neighbor energy. Only looking at GC content as predictor is too coarse: SC2 has the lowest temperature interval but the second-lowest GC percentage (44%), whereas SC1 (=M13) has the lowest GC content but does not fold in the lowest temperature interval. Hence, the sequence composition should be considered during sequence construction at the level of base pair step composition. Our design tool scaffold smith was thus built accordingly.

Smaller DNA Origami

Depending on the target application, scaffolds shorter than the conventional M13 variants (∼8000 bases) may be desirable. With the scaffold smith, scaffold sequence strings of any length may now be designed. However, the scaffold production method must be adapted according to the length of the target strand. We thus tested the split-ori approach for its capacity to produce short scaffolds in the ∼1000 bases length range. To this end, we built a circular, 1317 bases long mini-scaffold (Supporting Information Figures S5 and S6D,E). We found that the ssDNA amount per culture volume for this short scaffold was substantially lower (0.38 mg/L) compared to the yields obtained for target strands with lengths between ∼3000 (3.6 mg/L) and ∼9000 bases (2.6 mg/L). We therefore developed an alternative method for the convenient biotechnological production of short linear scaffolds with completely user-definable sequences. The method builds on our recently reported strategy for the biotechnological production of staple strands. (21) We integrated multiple copies of the same target scaffold sequence in one phagemid and interleaved them with Zn²⁺-dependent, self-excising DNAzyme "cassettes". The resulting multi-insert circular DNA single strands have a total size comparable to that of the conventional M13 genome, which is presumably favorable for DNA packaging and phage particle production. Indeed, the multi-insert phagemids can be produced with satisfying yields. Upon incubation with Zn²⁺, the DNAzyme cassettes become catalytically active and the circular ssDNA is digested into excised DNAzyme snippets, residual backbone, and multiple copies of the linear single-stranded target scaffold (Figure 3A). Thus, the multi-insert excision approach effectively allows mass producing homotypic pools of DNA oligonucleotides (as opposed to heterotypic pools as in our previous work (21)). We used our multi-insert excision approach to produce three scaffold variants with lengths of 1024, 1512, and 2048 bases and used them to assemble 13-helix bundles of different lengths. All 13-helix bundle variants self-assembled with excellent yield into the desired shape, as corroborated by gel electrophoresis, transmission electron microscopy (TEM) imaging, and reference-free class averaging (Figure 3A,B). For making scaffolds with lengths between ∼3000 and ∼9000 bases, we found the conventional phagemid approach to be well-suited. As an example, we produced an additional series of synthetic-sequence scaffolds with lengths of 2873, 4536, 6048, and 9072 bases (Figure S6D,E). These variants expand the currently available set of generic scaffolds (17,32) that is available to the community and that may be used to produce DNA origami with corresponding sizes.

Figure 3

Figure 3. DNA origami objects with sizes ranging between 1024 bp (633 kDa) and 37800 bp (23.4 MDa) can be assembled using mini-scaffolds or in one-pot assembly reactions containing multiple scaffolds. Blue indicates M13-based scaffolds; orange, green, cyan, and red indicate custom scaffolds. (A) Schematic representation of a circular DNA single strand (top left) that, in the presence of Zn²⁺, cleaves itself to yield four copies of a short, linear scaffold (top right) that can subsequently be used to assemble a small DNA origami object (bottom). (B) Schematic representation (top) and average TEM images of 13-helix bundle (13hb) variants assembled from linear mini-scaffolds comprising 1024 (I), 1536 (II), or 2048 bases (III). Scale bar: 20 nm. (C) Electrophoretic mobility analysis of mini-scaffolds and 13-helix bundle variants described in (B). (D) Schematic representations, single TEM images, and average TEM images (from top to bottom) of a 42-helix bundle assembled with five scaffolds in one-pot reactions. Scale bar: 50 nm. (E) Schematic representations, single TEM images, and average TEM images (from top to bottom) of an improved 42-helix bundle design with five interlocked scaffolds. Scale bar: 50 nm. (F) Electrophoretic mobility analysis of the two 42-helix bundle versions shown in (D,E). (G) Schematic representation (top), average TEM images with corresponding model views (left), and gel electrophoretic analysis (right) of a 126-helix bundle (126hb) assembled with two interlocked scaffolds. Scale bar: 50 nm. (H) Overlay of a cryo-EM density map fragment and the corresponding scaffold routing diagram. Blue and orange paths indicate the two orthogonal scaffolds. Laser scanned fluorescent images of the electrophoretic analyses were autoleveled. P, pocket; sta, staples.

Larger DNA Origami

Many applications of DNA origami require objects whose sizes exceed the dimensions of conventional M13 scaffolds. (16,37−41) Researchers have thus invested effort into building larger DNA origami to achieve greater overall dimensions and to integrate more features. (41,42) One possibility to build larger DNA origami with sizes beyond 10000 base pairs is to use increasingly long scaffold chains. Consequently, other researchers have reported up to 50000 bases long scaffold strands that were constructed from biological sequences, including E. coli genomic sequences and lambda phage sequences. (16,43) However, for scaffold lengths beyond 10kb assembly, cloning and plasmid handling become challenging. Moreover, when we compared the yield of production of scaffolds of different lengths, a trend emerged indicating that the yield drops for lengths approaching 10000 (Supporting Information Figure S6F), although the data are not entirely conclusive. A second possibility for making larger objects is to form higher-order assemblies from separately folded DNA origami subunits. (40,41,44,45) Oligomerization of individually assembled DNA origami objects can be achieved using sticky-end interactions (37,40) or via shape-complementary surface features and stacking interactions. (38,39,41) When following these routes, the individual building blocks must be produced separately and usually require some type of purification, which in addition to manual labor can negatively affect the overall yield.

Here, we thus pursued a third, complementary strategy to make larger DNA origami which considers the usage of multiple scaffold chains in one-pot assembly reactions, which has been used already exemplarily in our own previous work (41) and in those of others. (31,46) For one-pot assembly of multiscaffold DNA origami, we anticipate that the scaffold sequences must be sufficiently distinct ("orthogonal") to achieve productive folding of the target object. We tested these requirements experimentally and found that successful one-pot coassembly does indeed require orthogonal scaffold sequences (Supporting Information Figures S8 and S9). To enable one-pot coassembly with multiple scaffolds, we thus designed four 7560 bases long scaffolds (SC2, 4, 5, 6, compare Figure 2) that are orthogonal to each other and to the conventional M13-based scaffold (SC1). As a proof-of-concept, we designed a long pentameric 42-helix bundle object (Figure 3D) that self-assembled in a one-pot folding reaction mixture containing the five scaffold chains with distinct sequences and the several hundred staple oligonucleotides. Direct imaging with negative-staining TEM revealed the expected 42-helix bundle pentamers without visible seams between the subunits containing the individual scaffolds (Figure 3D). Reference-free class averages indicated a global twist deformation along the helical axis, which is consistent with recent findings concerning the occurrence of residual twist in honeycomb DNA origami. (41) TEM imaging further revealed higher-order branched networks in which well-folded 42hb pentamers were connected with other 42hb pentamers (Supporting Information Figure S10). We attributed these connected pentamers to design flaws: For this initial demonstration, we simply designed staple strands that connect the individual single-scaffold 42hb blocks across the helical interface. Some of these connecting staple strands featured long binding segments that presumably cause the undesired branched connections. We thus made a second, distinct 42hb pentamer design in which we changed the routing of the five scaffold chains to better interlock the individual chains. We also corrected right-handed twist using base pair deletions in the design, and we included an asymmetric feature. The thus-revised object self-assembled in the expected shape as seen by TEM (Figure 3E), now with reduced twist, and it appears as a single discrete species as seen in gel electrophoresis (Figure 3F). The extent of aggregates was substantially reduced compared to the variant without interlocked scaffolds. Importantly, the folding reaction mixtures for both 42hb pentamer design variants yielded only the pentameric target object in addition to a design-dependent extent of aggregates of intact pentamers, as seen in gel electrophoresis (Figure 3F). Incomplete pentamers were absent in both design versions. To achieve complete pentamers as a single folding product, the scaffold concentrations must be adjusted such that they appear in exactly equivalent amounts in the folding reaction mixture.

To illustrate the excellent potential of using multiple orthogonal scaffold chains for efficiently constructing larger DNA origami with high yield and high quality, we designed a barrel-like 126-helix bundle (126hb) comprising 15120 base pairs distributed over two orthogonal scaffolds that are interlocked in the helical direction (Figure 3G). When the relative scaffold concentrations were properly adjusted, the object formed successfully with close to 100% yield and virtually no side products, as seen in gel electrophoretic mobility analysis and TEM imaging (Figure 3G,H and Figure S11). Reference-free class averages from single-particle micrographs were in very good agreement with the designed shape. Due to the high quality of the object, we were able to solve a structure of this object using cryo-electron microscopy, in which nearly all of the 126 constituent helices were resolved in such detail that the grooves of double helices and all connecting crossovers could be discerned. We analyzed the map with respect to systematic differences at scaffold–scaffold seams and could not find any differences between seams containing one or both scaffolds (Figure 3G). Therefore, given a suitable scaffold routing and properly calibrated strand concentrations, multiscaffold DNA origami objects can be assembled with the high yield and the high quality known from well-behaved single-scaffold DNA origami designs. One-pot assembly of multiscaffold objects represents thus a powerful route for building larger DNA origami.

Functional Scaffolds: Catalytic Motifs and Covalent Cross-Linking

The design of fully synthetic scaffolds enables exclusion of undesired motifs and the inclusion of specific sequence motifs that serve user-defined purposes. As a demonstration for motif exclusion, we built a synthetic de Bruijn scaffold on the order of 7 that lacks CG base pair steps (SC2 from Figure 2). The absence of these CpG motifs could potentially circumvent Toll-like receptor-9-mediated immunogenic reactions in organisms. (7) This CG-free scaffold could be particularly advantageous when exploring in vivo applications of DNA origami. As a demonstration for the site-directed functionalization of synthetic scaffolds with functional sequences, we built two scaffolds that contain catalytic sequence motifs. We included one or two self-excising DNAzyme cassettes during sequence construction. Upon incubation with Zn²⁺, the DNAzymes become catalytically active, causing excision of the DNAzyme cassettes and thus linearization or bisection of the scaffold. Including these 132 bases long DNAzyme cassettes into the scaffold sequence ensures incorporation into every assembled DNA origami.

To illustrate the functionality, we used the self-bisecting scaffold to assemble a variant of a previously published DNA origami switch object (Figure 4A). (38,47) The switch object consists of two rigid beams that are flexibly linked by a single scaffold crossover at the center. The switch features double-helical shape-complementary protrusions and recessions that can dock into each other, stabilizing a closed state of the switch via base stacking interactions. Due to the electrostatic repulsion of the negatively charged DNA arms, the switch will predominantly occupy its open state at low salt concentrations. At higher salt concentrations, the electrostatic repulsion is shielded, and the stacking interactions are sufficient to stabilize the closed state. In our bisectable switch variant, we placed the self-excising DNAzyme cassettes directly at the pivot point, where the scaffold chain crosses from one switch arm to the other (Figure 4A). The thus-designed objects self-assembled with high yield and predominantly populated an open state at <10 mM MgCl₂ and a closed state at >10 mM MgCl₂, as expected. When incubated with Zn²⁺, the switch objects are cut at the pivot point due to the excision of the DNA enzyme cassettes (Figure 4B). Gel electrophoretic mobility analysis (Figure 4C,D) reveals that the bisection reaction goes to completion, and that the kinetics of bisection strongly depends on the state of the switch: at high salt (closed state), the reaction is substantially slower, which we attribute to activity-reducing conformational constraints on the DNAzyme cassettes. A simple Mg²⁺ dependence of the reaction kinetics can be ruled out because the reaction speed is the same in the presence of 1.4 or 5 mM MgCl₂. The cleavage reaction was also faster when residual staple oligonucleotides were removed by PEG precipitation (48) prior to incubation with Zn²⁺ (Figure 4D).

Figure 4

Figure 4. Self-cleaving DNA origami. (A) Schematic representations of circular scaffolds containing two self-excising DNAzyme cassettes (top left) that can be cleaved into two linear scaffolds (bottom left) or assembled into a switch object (top right). Individual switch arms (bottom right) can be obtained by cleavage of assembled switch objects or assembly using cleaved linear scaffolds. (B) Electrophoretic analysis of reaction kinetics of scaffold cleavage. Controls: cleaved scaffold (lane 1), undigested sample (lane 2), and switch arms assembled separately (lane 7) using cleaved scaffold. (C) Field-of-view TEM images of uncleaved (left) and cleaved (right) switch objects. (D) Electrophoretic analysis of cleavage reactions containing unpurified (lanes 1 and 5) and PEG-purified (lanes 2–4, 6–8) switch objects at 1.4, 4, 10, or 20 mM MgCl₂. Laser scanned fluorescent images of the electrophoretic analysis were autoleveled, and the highlighted region was autoleveled individually. P, pocket; U, undigested species; D, digested species. Scale bar: 100 nm.

Synthetic scaffold design also allows integrating hundreds of user-defined motifs site-specifically into a DNA origami, which can be exploited, for example, for sequence-programmable, chemical-modification-free covalent cross-linking of DNA origami objects, (9) termed UV point-welding. UV point-welded DNA origami objects are substantially more durable compared to nontreated objects and can remain stable at temperatures up to 90 °C and in pure double-distilled water with no additional cations present. In our previous work, covalent cross-linking was achieved by placing additional thymidine bases in the staple strand sequences at all termini and at all double-crossover positions. (9) Irradiation of such objects with 310 nm light induces the formation of covalent cyclobutane pyrimidine dimer (CPD) bonds between colocalized thymidine bases. As a result, double-helical domains become topologically trapped, and the constituent strands of thus-treated DNA origami can no longer dissociate, unless covalent bonds are broken. The possibility of making fully customized scaffolds offers an elegant way to realize the formation of UV-induced CPD bonds at desired sites while suppressing the formation of CPD bonds at undesired sites. Using the scaffold smith tool, a scaffold sequence can be designed that does not exhibit any TT motifs and that features AA only at desired crossover sites and strand termini as specified in the strand diagram.

As a demonstration, we constructed a semigeneric scaffold that can be used to create UV-cross-linkable single- or multilayer DNA origami objects in square lattice packing. In this scaffold, AA sites simply appear in regular intervals of eight bases. Given appropriate scaffold routing, all staple crossover sites feature AA motifs on the scaffold, which therefore leads to thymidines in staple strands that can be cross-linked (Figure 5A). We produced the corresponding 7560 bases long welding scaffold using the backbone excision split-ori method described in Figure 1B and used it to assemble a variant of a previously reported multilayer DNA origami object known as the pointer. (49) The UV-welding-ready pointer object self-assembled with satisfyingly high yield, as judged by electrophoretic mobility analysis (Figure 5C, lanes 2 and 12) and TEM imaging (Figure 5BI). We then irradiated the pointer object at 310 nm in the presence of 30 mM magnesium chloride. TEM images of the pointer acquired directly after exposure to UV light compared very well to those acquired prior to irradiation (Figure 5BII), indicating that the object retained its structure. We then incubated the irradiated sample for 48 h in physiological (low) ionic strength conditions (PBS buffer) at 40 °C (Figure 5BIII). Under such low ionic strength conditions, nonirradiated control pointer objects immediately dissociated into staple strands and scaffold strand as seen in gel electrophoresis (Figure 5B, left). By contrast, the irradiated samples remained fully intact, as indicated by the fact that the electrophoretic mobility did not change and by the absence of dissociated staple strand bands (Figure 5C, right). TEM imaging of the 48 h long PBS-incubated UV-welded pointer reveals well-folded objects consistent with the designed shape (Figure 5B, right). We thus conclude that the UV point-welding via scaffold-templated CPD bonds of the pointer was successful.

Figure 5

Figure 5. UV point-welding of DNA origami with a custom scaffold. (A) Section of a multilayer DNA origami strand diagram with a customized scaffold featuring AA motifs every 8 base pairs, which results in adjacent Thymidines in separate staple strands that may be UV-cross-linked. Blue lines, scaffold strand; gray lines, staple strands. (B) Schematic representation (left) and average TEM images of the pointer object assembled with the welding scaffold. Average images of the pointer as obtained in the presence of 30 mM MgCl₂ before irradiation (I), after irradiation for 2 h at 310 nm (II) in the presence of 30 mM MgCl₂, and after irradiation for 2 h at 310 nm and 48 h long incubation in low ionic strength phosphate-buffered saline (PBS) at 40 °C (III). (C) Electrophoretic analysis of nonirradiated and irradiated pointer objects incubated over time in PBS at 40 °C. L, 1kB Ladder; NI, not irradiated; RT, room temperature; P, pocket; F, folded species; sta, staples. Scale bar: 50 nm.

Conclusion

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With the tools and methods presented herein, researchers can now fully specify a target structure not only in terms of desired 3D shape and dimensions but also in terms of the sequences used. There is no longer a need to design objects around generic scaffold sequences as in the original DNA origami procedures. We demonstrated the potential of these tools and methods with a set of synthetic-sequence scaffolds which we used to explore the effects of sequence redundancy and sequence composition on the self-assembly of DNA origami, which is important input for guiding the construction of design-specific scaffolds. We built mini scaffolds as short as 1024 bases and a set of fully orthogonal scaffolds that enable efficient one-pot multiscaffold assembly of DNA origami comprising up to ∼38000 base pairs. We also made scaffolds containing functional motifs that enable DNAzyme-driven linearization and bisection of scaffolds or folded structures, which can enable constructing for example interlocked machine-like objects. Interlocked parts of these objects could be released by DNAzyme cleavage triggered by Zn²⁺-addition. We demonstrate that functional sequence motifs like DNAzymes, which are too long for staple strand synthesis, can be integrated in the scaffold sequence. We also constructed a CpG-free scaffold with presumably lower immunogenicity for future in vivo applications. Finally, we produced a customized scaffold with AA motifs spaced in intervals of 8 base pairs, which enables constructing square-lattice like single- or multilayer DNA origami that can be covalently cross-linked via UV point-welding right after folding. This scaffold can be considered as a demonstration of a fully design-specific scaffold, but the design was done such that the resulting scaffold can be used modularly in many other DNA origami designs.

With the currently available commercial gene synthesis services, our method allows constructing an entirely custom scaffold for less than 1000 € synthesis cost and requiring about 2 weeks of manual labor. We deposited precursor plasmids for all of our scaffolds at Addgene to make them available for the use by other researchers, along with the helper plasmids needed to produce the actual scaffold ssDNA. We also deposited a designated target plasmid containing the split-ori cassette, allowing other researchers to easily create their own custom scaffolds. Synthetic genes or gene fragments can be introduced into our target plasmid using a convenient and robust one-step Golden Gate cloning protocol. (50)

With custom-sequence scaffolds, DNA origami designers may rationally exploit sequence composition as a design parameter. Here, we produced mostly scaffold variants having a total nearest-neighbor energy higher than the conventional M13 variants, which led to assembly at temperatures higher than those of the M13-scaffolded object. It may be beneficial to explore whether the sequence composition may be tuned to push productive assembly temperature intervals down to physiological temperatures and without requiring a prior denaturation step. Furthermore, with full control over sequence design, sets of orthogonal scaffolds may now be produced that enable the direct and efficient assembly of oligomeric superstructures in one pot. For optimized designs such as the 126hb, we observed virtually perfect assembly yield in a one-pot reaction containing multiple scaffolds, which underlines the great potential of the multiscaffold strategy.

DNA origami applications often rely on the positioning of functionalities that typically consist of or are attached to specific ssDNA sequences. When conventional M13 scaffolds or natural sequences are used, these functional sequences must be introduced as extensions of staple strands. The incorporation yield of these extended staple strands may vary and can be unsatisfyingly low (e.g., 48%). (8) If, on the other hand, the desired functional sequences are included in the scaffold strand, the incorporation yield into a folded DNA origami is 100%. As we demonstrated, custom scaffolds can be designed and produced to include functional sequences at user-defined positions. An extreme example is the welding scaffold that contained hundreds of custom AA sites while excluding undesired TT sites. As an example, we integrated self-excising DNAzyme cassettes as functional motifs into our scaffolds. Assembly of mechanically interlocked DNA origami mechanisms (39,51) should become much easier with such bisectable scaffolds because detachment and component release can be achieved through Zn²⁺-induced excision of the DNAzyme cassettes. Self-linearizing scaffolds should be useful for designing multilayer DNA origami with odd-numbered helices and for making objects with applications such as nanopore translocation (52,53) or for tethered fluorophore motion assays (54) that require a linear scaffold. Future custom scaffolds might be designed to include other functional sequence motifs, such as aptamers, recognition sites for DNA-binding proteins, and indicator sites for complementary DNA strands as needed, for example, for DNA paint super-resolution microscopy. (55)

Another attractive aspect of creating design-specific scaffolds is that they lower the barrier to making DNA origami at larger scales. Previously, we reported how to biotechnologically mass produce pools of staple strands. (21) The synthesis of the necessary plasmids with many interleaved self-cleaving DNAzyme cassettes poses an initial obstacle, which may render this method somewhat unattractive at intermediate scales and in situations where design variants will need to be iterated. However, precursor plasmids for custom-sequence scaffolds are easily synthesized as they do not, by default, contain repetitive sequences. Hence, the DNA origami concept can now be inverted: one fixed pool of staple strands could be mass-produced biotechnologically in a lab-scale (or even industrial scale) bioreactor. Then, different custom-sequence scaffolds can be made in shake flasks that fold the set of fixed-sequence staple strands into different structures, thereby allowing to iterate through design versions at scales inaccessible with DNA reagents produced via chemical synthesis. A variant of this idea has been tested presented previously with the goal to reuse chemically produced DNA oligonucleotides. (19)

Methods

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Design and Construction of Scaffold Plasmids

For our custom scaffolds, we designed insert sequences for the variable part using either a python-based de Bruijn sequence generator or by using the scaffold smith GUI (see Supporting Information Note S1). Resulting insert sequences were split into shorter fragments to facilitate gene synthesis either manually or using a gene splitter GUI. Gene fragments were ordered either as linear gene strands or as genes in plasmids from Eurofins genomics (Ebersberg, Germany) or Twist bioscience (San Francisco, CA, USA). Full-length precursor plasmids were assembled using either Gibson assembly (56) or Golden Gate cloning. (50) Correct assembly was verified using restriction digest and DNA sequencing (Eurofins genomics, Ebersberg Germany). Sequences of all plasmids used in this work can be found in an Excel table in the Supporting Information.

Production of ssDNA in Shaker flasks

A detailed overview of the four different production methods is given in Supporting Information Note S2. M13 phage scaffolds were produced as previously described. (18) For custom scaffolds (methods II, III, and IV), chemically competent cells (E. coli DH5α) were cotransformed with the corresponding precursor plasmid and a helper plasmid. The actual ssDNA production for methods II and IV was carried out as previously described. (21) For convenience, the protocol is reprinted below:

"A single clone was picked and grown to saturation in a 5 mL pre-culture in 2xYT medium containing 5 mM MgCl₂, 30 μg/mL kanamycin, and either 50 μg/mL carbenicillin or 30 μg/mL chloramphenicol, depending on the phagemid backbone used; 750 mL of the same medium was inoculated with the preculture and grown overnight at 37 °C in 2.5 L Ultra Yield flasks (Thomson). Bacteria were removed by centrifugation for 30 min at 4000 rcf. Phagemid particles were precipitated by adding 3% polyethylene glycol 8000 (PEG-8000) and 0.5 M NaCl and centrifugation for 30 min at 4000 rcf. The pellet was resuspended in 5 mL of 1× TE buffer (10 mM Tris, 1 mM EDTA, pH 8) and centrifuged again for 15 min at 16000 rcf to remove residual bacterial cell fragments."

For method III, ssDNA production was carried out analogously but using a growth medium containing 16 g/L tryptone, 10 g/L yeast extract, 60 g/L sucrose, and 30 μg/mL kanamycin. (57) For all methods, ssDNA was subsequently isolated from the phagemid particles using alkaline lysis according to standard protocols for M13 ssDNA purification. (58) Purity of ssDNA was analyzed using gel electrophoresis, and sequences of custom scaffolds were verified using DNA sequencing. ssDNA concentrations were determined via the absorbance at 260 nm using extinction coefficients of 9828 M^–1 cm^–1 per base. Extinction coefficients for all scaffolds used in this work can be found in an Excel table in the Supporting Information.

Design, Assembly, and Purification of 3D DNA Origami

DNA origami objects were designed using caDNAno (22) and the designs were evaluated using CanDo. (59,60) Design diagrams can be found in Supporting Information Figures S11–S17 or in the corresponding references. (34,41,49,61) Reaction mixtures contained concentrations of scaffold strands and staples that were optimized for each object (42hb monomers: 50 nM scaffold + 200 nM staples; one-pot reaction mixture of 42hb and 10hb: 10 nM scaffold + 100 nM staples; 94hb: 20 nM scaffold + 100 nM staples; 126hb: 20 nM scaffold + 220 nM staples; 42hb pentamer version I assembly setup: 10 nM scaffold + 100 nM staples; 42hb pentamer version II assembly setup: 10 nM scaffold + 200 nM staples; switch object and pointer object: 20 nM scaffold + 200 nM staples nM; 13hb variants: 40 nM scaffold + 200 nM staples). All reaction mixtures contained 5 mM Tris, 1 mM EDTA, 20 mM MgCl₂, and 5 mM NaCl (pH 8). To compare different scaffolds, 42hb monomers were assembled using a standardized folding screen (for details see the caption of Figure 1), which was also used to identify optimized temperature intervals for each DNA origami object. Hence, each reaction mixture was subjected to an individually optimized thermal annealing ramp using a TETRAD (MJ Research, now Biorad), which included a preincubation step at 65 °C for 15 min. Subsequent temperature ramps were 60–40 °C at 1 h/°C for the 94hb, the switch-object, the pointer object, and for all one-pot reaction mixtures with multiple objects; 60–40 °C at 3 h/°C for the 126 and 42hb oligomers and 60–40 °C at 15 min/°C for the 13hb variants. DNA origami objects were purified via PEG precipitation. (35,48) For the 13hb objects, a final PEG concentration of 10.7% (w/v), a final NaCl concentration of 535 mM, and a final MgCl₂ concentration of 14.7 mM were used for precipitation. After precipitation, all pellets were resuspended in a buffer containing 5 mM Tris, 1 mM EDTA, 5 mM MgCl₂, and 5 mM NaCl (pH 8).

Agarose Gel Electrophoresis

In general, scaffolds and assembled DNA origami objects were analyzed using 1–2% agarose gels in 0.5× TBE buffer (45 mM Tris base, 45 mM boric acid, 1 mM EDTA) including 5.5 mM MgCl₂ and 0.5 μg/mL ethidium bromide at 90 V for 1.5 h in a water bath. Agarose concentrations were 1.5% for the 94hb in Figure S9 and the 42hb oligomers in Figure 3 and 2% for all other gels. Gels were scanned using a Typhoon FLA 9500 laser scanner (GE) at a resolution of either 50 or 25 μm/px (EtBr-channel: excitation at 535 nm, emission >575 nm). Images were inverted and autoleveled (Adobe Photoshop CS6).

Transmission Electron Microscopy Imaging and Image Processing

Samples were diluted to final concentrations between 1 and 5 nM in a buffer containing 20 mM MgCl₂ and adsorbed for 30 s to 1 min on glow-discharged Formvar-supported carbon-coated Cu400 TEM grids (Science Services, Munich) and stained using a 2% aqueous uranyl formate solution containing 25 mM NaOH. Imaging was performed using a Philips CM100 EM operated at 100 kV and an AMT 4 megapixel CCD camera (magnification: 28500×) or using a Tecnai Spirit operated at 120 kV and a TVIPS F416 detector (Tietz Camera Systems) (magnification: 30000×). For reference-free class averaging, image libraries were created by individual particle picking and analyzed using Xmipp 3.0. (62) TEM micrographs shown were subjected to high-pass filtering and autoleveling (Adobe Photoshop CS6).

Sample Preparation of 126hb for the Cryo-EM Study

The 126hb sample was purified and enriched via ultrafiltration with centrifugation steps at 10000 rcf for 5 min at 20 °C (Amicon Ultra 0.5 mL 50 kDa cutoff filters, Millipore). The sample was diluted 4-fold with folding buffer (1× FOB = 1 mM Tris, 1 mM EDTA, and 5 mM NaCl) prior to the first run. The filters were rinsed with 1× FOB including 5 mM MgCl₂ (1× FOB5), filled with 500 μL sample each and subjected to a centrifugation step. After six washing steps consisting of removing of the flow-through, refilling of the filters to 500 μL with 1× FOB5, and a centrifugation step, the filters were placed upside down in fresh tubes and subjected to another centrifugation step. The recovered sample was pooled from the filter tubes and added to one fresh filter in steps of 500 μL each followed by a centrifugation step. The filter was placed upside down in a fresh tube and subjected to two final centrifugation steps. The final concentration of the recovered sample was measured to be 1.2 μM.

Cryo-EM: Acquisition and Processing of Data

The purified and enriched sample was applied to C-Flat 2/1 4C (Protochips) grids and plunge-frozen using a Vitrobot Mark V (FEI, now Thermo Scientific) at the following settings: temperature of 22 °C, humidity of 100%, 0 s wait time, 2 s blot time, −1 blot force, 0 s drain time. The data were acquired on a Titan Krios G2 electron microscope operated at 300 kV equipped with a Falcon 3 direct detector using the EPU software for automated data collection (FEI, now Thermo Scientific). Micrograph movies comprising 7 frames each were recorded at a calibrated magnification of 47000 and magnified pixel size of 1.39 Å, a total dose of ∼50 e ^–/Ų and defocus values from −1 to −2 μm. The image processing was performed in RELION 2.1 (63) and 3.0, (64) using MotionCor2 (65) and CTFFIND4.1 (66) for motion correction and contrast transfer function estimation, respectively. A total of 2118 particles were manually picked and subjected to reference-free 2D classification. Four of the best classes (as judged by visual inspection) were selected as templates for automated particle picking. The particles were subjected to multiple rounds of 2D and 3D classification using a 3D de novo initial model created inside RELION as a 3D reference. A total of 123433 particles assigned to classes showing the most features were selected for a 3D refinement and further motion-corrected using the Bayesian Polishing tool. A focused refinement of a subregion was performed using the Multibody Refinement tool. The refined maps were sharpened by automatically estimated B-factors and locally filtered using the post processing and local resolution tool, respectively.

Zn-Induced Excision of DNAzyme Cassettes and Postpurification Using EtOH

Cleavage reactions were performed by incubating 20 nM phagemid ssDNA or 20 nM switch object in a buffer containing 50 mM HEPES, 100 mM NaCl, and 2 mM ZnCl₂, pH 7.0 at 37 °C overnight. ssDNA cleavage products were purified by adding 0.3 vol of 3 M KOAc (pH5), 0.033 vol of 1 M MgCl₂, and 2 vol of isopropyl alcohol. After 5 h incubation at −20 °C, the sample was pelleted by centrifugation for 45 min at 16000 rcf. The pellet was washed with 75% ethanol to remove residual salt, centrifuged again, and dissolved in 1× TE buffer. Switch object cleavage products were purified via PEG purification and resuspended in 1× FOB5.

UV Irradiation and Buffer Exchange to PBS and Incubation at 40 °C

The pointer object was irradiated for 120 min in folding buffer (5 mM Tris, 1 mM EDTA, 30 mM MgCl₂, and 5 mM NaCl) with a 300 W xenon light source (MAX-303 from Asahi Spectra) with a high transmission band-pass filter centered around 310 nm (XAQA310 from Asahi Spectra). A light guide (Asahi Spectra) was placed directly on top of a 0.65 mL reaction tube to couple the light into the sample. For all samples (cross-linked and un-cross-linked control) buffer exchange to PBS was achieved via ultrafiltration (Amicon Ultra 0.5 mL 50 kDa cutoff filters, Millipore) with three centrifugation steps at 7000 rcf for 5 min at 20 °C (Eppendorf 5424R). Samples were then incubated at 40 °C, and aliquots were shock-frozen in liquid nitrogen after different time intervals.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b01025.

• Detailed notes on generating a design-specific scaffold sequence with the scaffold smith, overview of methods for phage-based production of ssDNA scaffolds, workflow for the design, cloning, and production of a custom scaffold, supplementary experimental data and design schematics (PDF)

• Sequence information for scaffolds and plasmids used in this work (XLSX)

• Tools used in this work to design and analyze scaffold sequences (ZIP)

• nn9b01025_si_001.pdf (12.78 MB)
• nn9b01025_si_002.xlsx (71.55 kb)
• nn9b01025_si_003.zip (1.07 MB)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

---

• • Hendrik Dietz - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany; Email: [email protected]

• • Floris A. S. Engelhardt - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Florian Praetorius - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany; http://orcid.org/0000-0002-0806-8101

  • Christian H. Wachauf - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Gereon Brüggenthies - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Fabian Kohler - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Benjamin Kick - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Karoline L. Kadletz - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Phuong Nhi Pham - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Karl L. Behler - Lehrstuhl für Bioverfahrenstechnik and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

  • Thomas Gerling - Physics Department and , Technical University of Munich, Am Coulombwall 4a, 85748 Garching bei München, Germany

• F.A.S.E. and F.P. contributed equally to this work. F.A.S.E., F.P., and C.H.W. performed the research, and H.D. designed the research. G.B., B.K., K.L.K., P.N.P., and K.L.B. performed auxiliary experiments. F.K. performed cryo-EM. T.G. designed the bisectable switch and performed auxiliary experiments. F.A.S.E., F.P., and H.D. prepared figures and wrote the manuscript.

• The authors declare no competing financial interest.
  

Acknowledgments

---

The authors thank Max Honemann for technical assistance, and Klaus Wagenbauer for auxiliary experiments. We thank Dirk Weuster-Botz for providing auxiliary resources. This project was supported by European Research Council consolidator Grant No. 724261, the Deutsche Forschungsgemeinschaft through grants provided within the Gottfried-Wilhelm-Leibniz Program, and the SFB863 TPA9.

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   A review. Unmethylated CpG motifs are prevalent in bacterial but not vertebrate genomic DNAs. Oligodeoxynucleotides (ODN) contg. CpG motifs activate host defense mechanisms leading to innate and acquired immune responses. The recognition of CpG motifs requires Toll-like receptor (TLR) 9, which triggers alterations in cellular redox balance and the induction of cell signaling pathways including the mitogen activated protein kinases (MAPKs) and NFκB. Cells that express TLR-9, which include plasmacytoid dendritic cells (PDCs) and B cells, produce Th1-like proinflammatory cytokines, interferons, and chemokines. Certain CpG motifs (CpG-A) are esp. potent at activating NK cells and inducing IFN-α prodn. by PDCs, while other motifs (CpG-B) are esp. potent B cell activators. CpG-induced activation of innate immunity protects against lethal challenge with a wide variety of pathogens, and has therapeutic activity in murine models of cancer and allergy. CpG ODN also enhance the development of acquired immune responses for prophylactic and therapeutic vaccination.

   https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtlWgtbw%253D&md5=e5bafa2173f07799a9584f4ba37b86b3

8. 8

   Strauss, M. T. ; Schueder, F. ; Haas, D. ; Nickels, P. C. ; Jungmann, R. Quantifying Absolute Addressability in DNA Origami with Molecular Resolution. Nat. Commun. 2018, 9 , 1600,  DOI: 10.1038/s41467-018-04031-z

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   8

   Quantifying absolute addressability in DNA origami with molecular resolution

   Strauss Maximilian T; Schueder Florian; Haas Daniel; Nickels Philipp C; Jungmann Ralf; Strauss Maximilian T; Schueder Florian; Haas Daniel; Nickels Philipp C; Jungmann Ralf

   Nature communications (2018), 9 (1), 1600 ISSN:.

   Self-assembled DNA nanostructures feature an unprecedented addressability with sub-nanometer precision and accuracy. This addressability relies on the ability to attach functional entities to single DNA strands in these structures. The efficiency of this attachment depends on two factors: incorporation of the strand of interest and accessibility of this strand for downstream modification. Here we use DNA-PAINT super-resolution microscopy to quantify both incorporation and accessibility of all individual strands in DNA origami with molecular resolution. We find that strand incorporation strongly correlates with the position in the structure, ranging from a minimum of 48% on the edges to a maximum of 95% in the center. Our method offers a direct feedback for the rational refinement of the design and assembly process of DNA nanostructures and provides a long sought-after quantitative explanation for efficiencies of DNA-based nanomachines.

   https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mjlslyksw%253D%253D&md5=b9a83f6cace855a6ebf73db91ef12e5c

9. 9

   Gerling, T. ; Kube, M. ; Kick, B. ; Dietz, H. Sequence-Programmable Covalent Bonding of Designed DNA Assemblies. Sci. Adv. 2018, 4 , eaau1157  DOI: 10.1126/sciadv.aau1157

10. 10

    Ducani, C. ; Kaul, C. ; Moche, M. ; Shih, W. M. ; Hogberg, B. Enzymatic Production of 'Monoclonal Stoichiometric' Single-Stranded DNA Oligonucleotides. Nat. Methods 2013, 10 , 647– 652,  DOI: 10.1038/nmeth.2503

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    10

    Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Hoegberg, Bjoern

    Nature Methods (2013), 10 (7), 647-652CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

    Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnol. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biol. systems produce. The no. of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated prodn. of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepd. 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXosFWqu7g%253D&md5=6bccb58b098229e83692815cbae97a4e

11. 11

    Ducani, C. ; Bernardinelli, G. ; Hogberg, B. Rolling Circle Replication Requires Single-Stranded DNA Binding Protein to Avoid Termination and Production of Double-Stranded DNA. Nucleic Acids Res. 2014, 42 , 10596– 10604,  DOI: 10.1093/nar/gku737

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    11

    Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

    Ducani, Cosimo; Bernardinelli, Giulio; Hoegberg, Bjoern

    Nucleic Acids Research (2014), 42 (16), 10596-10604CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

    In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3'- ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amts. of double stranded DNA and that the prodn. of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the obsd. data. From this, we also provide an est. value of the switching rate const.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKmur7K&md5=ca083d91a90ba2ef122f3a3ec0b9c950

12. 12

    Schmidt, T. L. ; Beliveau, B. J. ; Uca, Y. O. ; Theilmann, M. ; Da Cruz, F. ; Wu, C. T. ; Shih, W. M. Scalable Amplification of Strand Subsets from Chip-Synthesized Oligonucleotide Libraries. Nat. Commun. 2015, 6 , 8634,  DOI: 10.1038/ncomms9634

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    12

    Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

    Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

    Nature Communications (2015), 6 (), 8634CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

    Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnol., genetics and synthetic biol., which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amts. of single-stranded oligonucleotides per mL reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nmol total oligonucleotides produced) and is dominated by the use of com. enzymes.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVOmtrjF&md5=636d4a1470bf21e088b662bf73499707

13. 13

    Elbaz, J. ; Yin, P. ; Voigt, C. A. Genetic Encoding of DNA Nanostructures and Their Self-Assembly in Living Bacteria. Nat. Commun. 2016, 7 , 11179,  DOI: 10.1038/ncomms11179

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    13

    Genetic encoding of DNA nanostructures and their self-assembly in living bacteria

    Elbaz, Johann; Yin, Peng; Voigt, Christopher A.

    Nature Communications (2016), 7 (), 11179CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

    The field of DNA nanotechnol. has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32-205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA contg. a 3'-hairpin (HTBS). HTBS recruits HIV reverse transcriptase, which nucleates DNA synthesis and is aided in elongation by murine leukemia reverse transcriptase. Purified ssDNA that is produced in vivo is used to assemble large 1D wires (300 nm) and 2D sheets (5.8 μm2) in vitro. Intracellular assembly is demonstrated using a four-ssDNA crossover nanostructure that recruits split YFP when properly assembled. Genetically encoding DNA nanostructures provides a route for their prodn. as well as applications in living cells.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsFyhtrY%253D&md5=4ff7a1705be9bd9f0daee732ddf4243a

14. 14

    Krieg, E. ; Shih, W. M. Selective Nascent Polymer Catch-and-Release Enables Scalable Isolation of Multi-Kilobase Single-Stranded DNA. Angew. Chem., Int. Ed. 2018, 57 , 714– 718,  DOI: 10.1002/anie.201710469

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    14

    Selective Nascent Polymer Catch-and-Release Enables Scalable Isolation of Multi-Kilobase Single-Stranded DNA

    Krieg, Elisha; Shih, William M.

    Angewandte Chemie, International Edition (2018), 57 (3), 714-718CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    Scalable methods currently are lacking for isolation of long ssDNA, an important material for numerous biotechnol. applications. Conventional biomol. purifn. strategies achieve target capture using solid supports, which are limited in scale and susceptible to contamination owing to nonspecific adsorption and desorption on the substrate surface. The authors herein disclose selective nascent polymer catch and release (SNAPCAR), a method that utilizes the reactivity of growing poly(acrylamide-coacrylate) chains to capture acrylamide-labeled mols. in free soln. The copolymer acts as a stimuli-responsive anchor that can be pptd. on demand to pull down the target from soln. SNAPCAR enabled scalable isolation of multikilobase ssDNA with high purity and 50-70% yield. The ssDNA products were used to fold various DNA origami. SNAPCAR-produced ssDNA will expand the scope of applications in nanotechnol., gene editing, and DNA library construction.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvF2ls7bM&md5=bc16a7a7e0d6aa32a4a007c565512ddb

15. 15

    Veneziano, R. ; Shepherd, T. R. ; Ratanalert, S. ; Bellou, L. ; Tao, C. ; Bathe, M. In Vitro Synthesis of Gene-Length Single-Stranded DNA. Sci. Rep. 2018, 8 , 24677,  DOI: 10.1038/s41598-018-24677-5

16. 16

    Marchi, A. N. ; Saaem, I. ; Vogen, B. N. ; Brown, S. ; LaBean, T. H. Toward Larger DNA Origami. Nano Lett. 2014, 14 , 5740– 5747,  DOI: 10.1021/nl502626s

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    16

    Toward Larger DNA Origami

    Marchi, Alexandria N.; Saaem, Ishtiaq; Vogen, Briana N.; Brown, Stanley; LaBean, Thomas H.

    Nano Letters (2014), 14 (10), 5740-5747CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)

    Structural DNA nanotechnol., and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Addnl., increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) prodn. of the largest to-date biol. derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have exptl. demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addn., we have demonstrated two-dimensional, asym. origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by at. force microscopy.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVCqtbfE&md5=4d56d2b1b71049f42e6619d671bb74e2

17. 17

    Brown, S. ; Majikes, J. ; Martínez, A. ; Girón, T. ; Fennell, H. ; Samano, E. ; LaBean, T. An Easy-to-Prepare Mini-Scaffold for DNA Origami. Nanoscale 2015, 7 , 16621– 16624,  DOI: 10.1039/C5NR04921K

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    17

    An easy-to-prepare mini-scaffold for DNA origami

    Brown, S.; Majikes, J.; Martinez, A.; Giron, T. M.; Fennell, H.; Samano, E. C.; LaBean, T. H.

    Nanoscale (2015), 7 (40), 16621-16624CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)

    The DNA origami strategy for assembling designed supramol. complexes requires ssDNA as a scaffold strand. A system is described that was designed approx. one third the length of the M13 bacteriophage genome for ease of ssDNA prodn. Folding of the 2404-base ssDNA scaffold into a variety of origami shapes with high assembly yields is demonstrated.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFWhur%252FL&md5=174f5d3e273e50fb79b08f9f68fd081d

18. 18

    Kick, B. ; Praetorius, F. ; Dietz, H. ; Weuster-Botz, D. Efficient Production of Single-Stranded Phage DNA as Scaffolds for DNA Origami. Nano Lett. 2015, 15 , 4672,  DOI: 10.1021/acs.nanolett.5b01461

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    18

    Efficient Production of Single-Stranded Phage DNA as Scaffolds for DNA Origami

    Kick, Benjamin; Praetorius, Florian; Dietz, Hendrik; Weuster-Botz, Dirk

    Nano Letters (2015), 15 (7), 4672-4676CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)

    Scaffolded DNA origami enables the fabrication of a variety of complex nanostructures that promise utility in diverse fields of application, ranging from biosensing over advanced therapeutics to metamaterials. The broad applicability of DNA origami as a material beyond the level of proof-of-concept studies critically depends, among other factors, on the availability of large amts. of pure single-stranded scaffold DNA. Here, we present a method for the efficient prodn. of M13 bacteriophage-derived genomic DNA using high-cell-d. fermn. of Escherichia coli in stirred-tank bioreactors. We achieve phage titers of up to 1.6 × 1014 plaque-forming units per mL. Downstream processing yields up to 410 mg of high-quality single-stranded DNA per one liter reaction vol., thus upgrading DNA origami-based nanotechnol. from the milligram to the gram scale.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptF2ntLc%253D&md5=06d07cc77d7eaffd64bfe0a1d1c5e138

19. 19

    Niekamp, S. ; Blumer, K. ; Nafisi, P. M. ; Tsui, K. ; Garbutt, J. ; Douglas, S. M. Folding Complex DNA Nanostructures from Limited Sets of Reusable Sequences. Nucleic Acids Res. 2016, 44 , e102  DOI: 10.1093/nar/gkw208

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    19

    Folding complex DNA nanostructures from limited sets of reusable sequences

    Niekamp, Stefan; Blumer, Katy; Nafisi, Parsa M.; Tsui, Kathy; Garbutt, John; Douglas, Shawn M.

    Nucleic Acids Research (2016), 44 (11), e102/1-e102/6CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

    Scalable prodn. of DNA nanostructures remains a substantial obstacle to realizing new applications of DNA nanotechnol. Typical DNA nanostructures comprise hundreds of DNA oligonucleotide strands, where each unique strand requires a sep. synthesis step. New design methods that reduce the strand count for a given shape while maintaining overall size and complexity would be highly beneficial for efficiently producing DNA nanostructures. Here, we report a method for folding a custom template strand by binding individual staple sequences to multiple locations on the template. We built several nanostructures for well-controlled testing of various design rules, and demonstrate folding of a 6-kb template by as few as 10 unique strand sequences binding to 10 ± 2 locations on the template strand.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsF2jsbfI&md5=e6604bc54e9b8b688be13a73605848a6

20. 20

    Kick, B. ; Hensler, S. ; Praetorius, F. ; Dietz, H. ; Weuster-Botz, D. Specific Growth Rate and Multiplicity of Infection Affect High-Cell-Density Fermentation with Bacteriophage M13 for Ssdna Production. Biotechnol. Bioeng. 2017, 114 , 777– 784,  DOI: 10.1002/bit.26200

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    20

    Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production

    Kick, Benjamin; Hensler, Samantha; Praetorius, Florian; Dietz, Hendrik; Weuster-Botz, Dirk

    Biotechnology and Bioengineering (2017), 114 (4), 777-784CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)

    The bacteriophage M13 has found frequent applications in nanobiotechnol. due to its chem. and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Addnl., its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream prodn. methods for phage and scaffold ssDNA are underexamd. with respect to future industrial usage. Here, the high-cell-d. phage prodn. with Escherichia coli as host organism was studied in respect of medium compn., infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concn. of produced ssDNA on the other hand. Using a growth rate of 0.15 h-1 and a multiplicity of infection of 0.05 pfu cfu-1 in the fed-batch prodn. process, the concn. of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L-1. Thus, our results help enabling M13 prodn. for industrial uses in nanobiotechnol. Biotechnol. Bioeng. 2016;9999: 1-8. © 2016 Wiley Periodicals, Inc.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslCrs7zN&md5=ff429c307988309455756899b4f42302

21. 21

    Praetorius, F. ; Kick, B. ; Behler, K. L. ; Honemann, M. N. ; Weuster-Botz, D. ; Dietz, H. Biotechnological Mass Production of DNA Origami. Nature 2017, 552 , 84– 87,  DOI: 10.1038/nature24650

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    21

    Biotechnological mass production of DNA origami

    Praetorius, Florian; Kick, Benjamin; Behler, Karl L.; Honemann, Maximilian N.; Weuster-Botz, Dirk; Dietz, Hendrik

    Nature (London, United Kingdom) (2017), 552 (7683), 84-87CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

    DNA nanotechnol., in particular DNA origami, enables the bottom-up self-assembly of micrometer-scale, three-dimensional structures with nanometer-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behavior. Their use has been limited to applications that require only small amts. of material (of the order of micrograms), owing to the limitations of current prodn. methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold mol. held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold mols. are amenable to scalable and efficient mass prodn.; the shorter staple strands are obtained through costly solid-phase synthesis or enzymic processes. Here the authors show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. The authors produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end prodn. of macroscopic amts. of a DNA origami nanorod in a liter-scale stirred-tank bioreactor. The method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the prodn. and purifn. steps amenable to scaling, the authors expect that the method will expand the scope of DNA nanotechnol. in many areas of science and technol.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurjO&md5=8773cd37db2a75a049ae65ff28921da7

22. 22

    Douglas, S. M. ; Marblestone, A. H. ; Teerapittayanon, S. ; Vazquez, A. ; Church, G. M. ; Shih, W. M. Rapid Prototyping of 3d DNA-Origami Shapes with Cadnano. Nucleic Acids Res. 2009, 37 , 5001– 5006,  DOI: 10.1093/nar/gkp436

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    22

    Rapid prototyping of 3D DNA-origami shapes with caDNAno

    Douglas, Shawn M.; Marblestone, Adam H.; Teerapittayanon, Surat; Vazquez, Alejandro; Church, George M.; Shih, William M.

    Nucleic Acids Research (2009), 37 (15), 5001-5006CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

    DNA nanotechnol. exploits the programmable specificity afforded by base-pairing to produce self-assembling macromol. objects of custom shape. For building megadalton-scale DNA nanostructures, a long scaffold' strand can be employed to template the assembly of hundreds of oligonucleotide staple' strands into a planar antiparallel array of cross-linked helixes. The authors recently adapted this scaffolded DNA origami' method to producing 3-dimensional shapes formed as pleated layers of double helixes constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here the authors present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3-dimensional honeycomb-pleated shapes rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3-dimensional DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKntbzE&md5=aa99732c1666373a70e9b7b4de6e6d5d

23. 23

    Veneziano, R. ; Ratanalert, S. ; Zhang, K. ; Zhang, F. ; Yan, H. ; Chiu, W. ; Bathe, M. Designer Nanoscale DNA Assemblies Programmed from the Top Down. Science 2016, 352 , 1534,  DOI: 10.1126/science.aaf4388

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    23

    Designer nanoscale DNA assemblies programmed from the top down

    Veneziano, Remi; Ratanalert, Sakul; Zhang, Kaiming; Zhang, Fei; Yan, Hao; Chiu, Wah; Bathe, Mark

    Science (Washington, DC, United States) (2016), 352 (6293), 1534CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

    Scaffolded DNA origami is a versatile means of synthesizing complex mol. architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, the authors report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asym. polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biol. as well as nonbiol. applications.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVaitL3I&md5=3772f7806345ae3b655fa3c1b147d752

24. 24

    Jun, H. ; Zhang, F. ; Shepherd, T. ; Ratanalert, S. ; Qi, X. ; Yan, H. ; Bathe, M. Autonomously Designed Free-Form 2d DNA Origami. Sci. Adv. 2019, 5 , eaav0655  DOI: 10.1126/sciadv.aav0655

25. 25

    Jun, H. ; Shepherd, T. R. ; Zhang, K. ; Bricker, W. P. ; Li, S. ; Chiu, W. ; Bathe, M. Automated Sequence Design of 3d Polyhedral Wireframe DNA Origami with Honeycomb Edges. ACS Nano 2019,  DOI: 10.1021/acsnano.8b08671

26. 26

    Benson, E. ; Mohammed, A. ; Gardell, J. ; Masich, S. ; Czeizler, E. ; Orponen, P. ; Hogberg, B. DNA Rendering of Polyhedral Meshes at the Nanoscale. Nature 2015, 523 , 441– 444,  DOI: 10.1038/nature14586

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    26

    DNA rendering of polyhedral meshes at the nanoscale

    Benson, Erik; Mohammed, Abdulmelik; Gardell, Johan; Masich, Sergej; Czeizler, Eugen; Orponen, Pekka; Hoegberg, Bjoern

    Nature (London, United Kingdom) (2015), 523 (7561), 441-444CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

    It was suggested more than thirty years ago that Watson-Crick base pairing might be used for the rational design of nanometer-scale structures from nucleic acids. Since then, and esp. since the introduction of the origami technique, DNA nanotechnol. has enabled increasingly more complex structures. But although general approaches for creating DNA origami polygonal meshes and design software are available, there are still important constraints arising from DNA geometry and sense/antisense pairing, necessitating some manual adjustment during the design process. Here we present a general method of folding arbitrary polygonal digital meshes in DNA that readily produces structures that would be very difficult to realize using previous approaches. The design process is highly automated, using a routing algorithm based on graph theory and a relaxation simulation that traces scaffold strands through the target structures. Moreover, unlike conventional origami designs built from close-packed helixes, our structures have a more open conformation with one helix per edge and are therefore stable under the ionic conditions usually used in biol. assays.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1WksrfJ&md5=38be5240fa4b9bdac057fdbbbb6915c5

27. 27

    Bellot, G. ; McClintock, M. A. ; Chou, J. J. ; Shih, W. M. DNA Nanotubes for Nmr Structure Determination of Membrane Proteins. Nat. Protoc. 2013, 8 , 755– 770,  DOI: 10.1038/nprot.2013.037

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    DNA nanotubes for NMR structure determination of membrane proteins

    Bellot, Gaetan; McClintock, Mark A.; Chou, James J.; Shih, William M.

    Nature Protocols (2013), 8 (4), 755-770, 16 pp.CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)

    Finding a way to det. the structures of integral membrane proteins using soln. NMR (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dil. liq. crystals for application as weak-alignment media in soln. NMR structure detn. of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable pos. charge as well as modulation of mol. alignment, toward collection of linearly independent restraints, can be introduced by reducing the neg. charge of DNA nanotubes using counter ions and small DNA-binding mols. This detergent-resistant liq.-crystal medium offers a no. of properties conducive for membrane protein alignment, including high-yield prodn., thermal stability, buffer compatibility and structural programmability. Prodn. of sufficient nanotubes for four or five NMR expts. can be completed in 1 wk by a single individual.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmtlShsbo%253D&md5=21847aedab3e0bcecdca6028ac8c92de

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    Vieira, J. ; Messing, J. Production of Single-Stranded Plasmid DNA. Methods Enzymol. 1987, 153 , 3– 11,  DOI: 10.1016/0076-6879(87)53044-0

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    Production of single-stranded plasmid DNA

    Vieira, Jeffrey; Messing, Joachim

    Methods in Enzymology (1987), 153 (Recomb. DNA, Pt. D), 3-11CODEN: MENZAU; ISSN:0076-6879.

    A helper phage, M13KO7, was constructed that preferentially packages plasmid DNA over phage DNA. This then produces single-stranded plasmid DNA.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXht1GgtLw%253D&md5=dbbde887802d2c81f887633d5017a94a

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    Chasteen, L. ; Ayriss, J. ; Pavlik, P. ; Bradbury, A. R. Eliminating Helper Phage from Phage Display. Nucleic Acids Res. 2006, 34 , e145  DOI: 10.1093/nar/gkl772

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    Eliminating helper phage from phage display

    Chasteen, L.; Ayriss, J.; Pavlik, P.; Bradbury, A. R. M.

    Nucleic Acids Research (2006), 34 (21), e145/1-e145/11CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

    Phage display technol. involves the display of proteins or peptides, as coat protein fusions, on the surface of a phage or phagemid particles. Using std. technol., helper phage are essential for the replication and assembly of phagemid particles, during library prodn. and biopanning. We have eliminated the need to add helper phage by using 'bacterial packaging cell lines' that provide the same functions. These cell lines contain M13-based helper plasmids that express phage packaging proteins which assemble phagemid particles as efficiently as helper phage, but without helper phage contamination. This results in genetically pure phagemid particle prepns. Furthermore, by using constructs differing in the form of gene 3 that they contain, we have shown that the display, from a single library, can be modulated between monovalent (phagemid-like) and multivalent display (phage-like) without any further engineering. These packaging cells eliminate the use of helper phage from phagemid-based selection protocols; reducing the amt. of tech. prepn., facilitating automation, optimizing selections by matching display levels to diversity, and effectively using the packaged phagemid particles as means to transfer genetic information at an efficiency approaching 100%.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlCru7fO&md5=4850daf57dd2b0d00509f4639d7c7ee0

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    Specthrie, L. ; Bullitt, E. ; Horiuchi, K. ; Model, P. ; Russel, M. ; Makowski, L. Construction of a Microphage Variant of Filamentous Bacteriophage. J. Mol. Biol. 1992, 228 , 720– 724,  DOI: 10.1016/0022-2836(92)90858-H

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    Construction of a microphage variant of filamentous bacteriophage

    Specthrie, Leon; Bullitt, Esther; Horiuchi, Kensuke; Model, Peter; Russel, Marjorie; Makowski, Lee

    Journal of Molecular Biology (1992), 228 (3), 720-4CODEN: JMOBAK; ISSN:0022-2836.

    The intergenic region in the genome of the Ff class of filamentous phage (comprising strains f1, fd and M13) genome constitutes 8% of the viral genome, and has essential functions in DNA replication and phage morphogenesis. The functional domains of this region may be inserted into sep. sites of a plasmid to function independently. Here, the authors demonstrate the construction of a plasmid contg., sequentially, the origin of (+)-strand synthesis, the packaging signal, and a terminator of (+)-strand synthesis. When host cells harboring this plasmid (pLS7) are infected with helper phage they produce a microphage particle contg. all the structural elements of the mature, native phage. The microphage is 65 Å in diam. and ∼500 Å long. It contains a 221-base single-stranded circle of DNA coated by ∼95 copies of the major coat protein (gene 8 protein).

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlsFOktA%253D%253D&md5=ae1abdd9ef5054a869a0ac85607707f7

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    Nafisi, P. M. ; Aksel, T. ; Douglas, S. M. Construction of a Novel Phagemid to Produce Custom DNA Origami Scaffolds. Synth. Biol. 2018,  DOI: 10.1093/synbio/ysy015

32. 32

    Said, H. ; Schuller, V. J. ; Eber, F. J. ; Wege, C. ; Liedl, T. ; Richert, C. M1.3--a Small Scaffold for DNA Origami. Nanoscale 2013, 5 , 284– 290,  DOI: 10.1039/C2NR32393A

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    M1.3 - a small scaffold for DNA origami

    Said, Hassan; Schueller, Verena J.; Eber, Fabian J.; Wege, Christina; Liedl, Tim; Richert, Clemens

    Nanoscale (2013), 5 (1), 284-290CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)

    The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient prepn. of a 704 nt fragment dubbed "M1.3" as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepd. and were shown to give higher UV-m.ps. than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chem., phys., and biol. expts.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVSktb3O&md5=309c7bcfa7a4f04d8e25a9cb0af39c4b

33. 33

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    Sobczak, J. P. ; Martin, T. G. ; Gerling, T. ; Dietz, H. Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature. Science 2012, 338 , 1458– 1461,  DOI: 10.1126/science.1229919

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    Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature

    Sobczak, Jean-Philippe J.; Martin, Thomas G.; Gerling, Thomas; Dietz, Hendrik

    Science (Washington, DC, United States) (2012), 338 (6113), 1458-1461CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

    At const. temp., hundreds of DNA strands can cooperatively fold a long template DNA strand within minutes into complex nanoscale objects. Folding occurred out of equil. along nucleation-driven pathways at temps. that could be influenced by the choice of sequences, strand lengths, and chain topol. Unfolding occurred in apparent equil. at higher temps. than those for folding. Folding at optimized const. temps. enabled the rapid prodn. of three-dimensional DNA objects with yields that approached 100%. The results point to similarities with protein folding in spite of chem. and structural differences. The possibility for rapid and high-yield assembly will enable DNA nanotechnol. for practical applications.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVSktLfJ&md5=718307e01a75a76aab5d04f4667e191d

35. 35

    Wagenbauer, K. F. ; Engelhardt, F. A. ; Stahl, E. K. ; Hechtl, V. K. ; Stömmer, P. ; Seebacher, F. ; Meregalli, L. ; Ketterer, P. ; Gerling, T. ; Dietz, H. How We Make DNA Origami. ChemBioChem 2017, 18 , 1873,  DOI: 10.1002/cbic.201700377

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    How We Make DNA Origami

    Wagenbauer, Klaus F.; Engelhardt, Floris A. S.; Stahl, Evi; Hechtl, Vera K.; Stoemmer, Pierre; Seebacher, Fabian; Meregalli, Letizia; Ketterer, Philip; Gerling, Thomas; Dietz, Hendrik

    ChemBioChem (2017), 18 (19), 1873-1885CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)

    DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for prepg. them at appropriate concns. with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnol. may emerge. To foster faster progress, we share in this article the experience in making and prepg. DNA origami that we have accumulated over recent years. We discuss design solns. for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addn., we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free prepn. of DNA origami. These methods are agarose-gel purifn., filtration through mol. cut-off membranes, PEG pptn., size-exclusion chromatog., and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their exptl. characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami prodn. workflow.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht12ltbzE&md5=ea439ce860cf1988018b6a223421a4b7

36. 36

    SantaLucia, J., Jr. ; Hicks, D. The Thermodynamics of DNA Structural Motifs. Annu. Rev. Biophys. Biomol. Struct. 2004, 33 , 415– 440,  DOI: 10.1146/annurev.biophys.32.110601.141800

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    The thermodynamics of DNA structural motifs

    SantaLucia, John, Jr.; Hicks, Donald

    Annual Review of Biophysics and Biomolecular Structure (2004), 33 (), 415-440, 2 platesCODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)

    A review. DNA secondary structure plays an important role in biol., genotyping diagnostics, a variety of mol. biol. techniques, in vitro-selected DNA catalysts, nanotechnol., and DNA-based computing. Accurate prediction of DNA secondary structure and hybridization using dynamic programming algorithms requires a database of thermodn. parameters for several motifs including Watson-Crick base pairs, internal mismatches, terminal mismatches, terminal dangling ends, hairpins, bulges, internal loops, and multibranched loops. To make the database useful for predictions under a variety of salt conditions, empirical equations for monovalent and Mg2+ dependence of thermodn. have been developed. Bimol. hybridization is often inhibited by competing unimol. folding of a target or probe DNA. Powerful numerical methods have been developed to solve multistate-coupled equil. in bimol. and higher-order complexes. This review presents the current parameter set available for making accurate DNA structure predictions and also points to future directions for improvement.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltlKkt7k%253D&md5=4079be22e69c61a888673d54a1ccf309

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    Berardi, M. J. ; Shih, W. M. ; Harrison, S. C. ; Chou, J. J. Mitochondrial Uncoupling Protein 2 Structure Determined by Nmr Molecular Fragment Searching. Nature 2011, 476 , 109– 113,  DOI: 10.1038/nature10257

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    Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

    Berardi, Marcelo J.; Shih, William M.; Harrison, Stephen C.; Chou, James J.

    Nature (London, United Kingdom) (2011), 476 (7358), 109-113CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

    Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small mols. across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochem. potential. A fraction of this electrochem. potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or uncoupling' of the proton electrochem. potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a soln.-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges assocd. with membrane-protein structure detn., combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquant. distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were detd. by piecing together mol. fragments from the Protein Data Bank that best fit exptl. RDCs from samples weakly aligned in a DNA nanotube liq. crystal. The RDCs also det. the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labeled GDP binds inside the channel and seems to be closer to transmembrane helixes 1-4. We believe that this biophys. approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure detn. but also to characterize various conformational states of these proteins linked to substrate transport.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptlalu74%253D&md5=7ecd0be8d83ab63eb09baef8cc70b8ae

38. 38

    Gerling, T. ; Wagenbauer, K. F. ; Neuner, A. M. ; Dietz, H. Dynamic DNA Devices and Assemblies Formed by Shape-Complementary, Non-Base Pairing 3d Components. Science 2015, 347 , 1446– 1452,  DOI: 10.1126/science.aaa5372

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    38

    Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components

    Gerling, Thomas; Wagenbauer, Klaus F.; Neuner, Andrea M.; Dietz, Hendrik

    Science (Washington, DC, United States) (2015), 347 (6229), 1446-1452CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

    The authors demonstrate that discrete three-dimensional (3D) DNA components can specifically self-assemble in soln. on the basis of shape-complementarity and without base pairing. Using this principle, homo- and heteromultimeric objects were produced, including micrometer-scale one- and two-stranded filaments and lattices, as well as reconfigurable devices, including an actuator, a switchable gear, an unfoldable nanobook, and a nanorobot. These multidomain assemblies were stabilized via short-ranged nucleobase stacking bonds that compete against electrostatic repulsion between the components' interfaces. Using imaging by electron microscopy, ensemble and single-mol. fluorescence resonance energy transfer spectroscopy, and electrophoretic mobility anal., it was shown that the balance between attractive and repulsive interactions, and thus the conformation of the assemblies, may be finely controlled by global parameters such as cation concn. or temp. and by an allosteric mechanism based on strand-displacement reactions.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvFCnu74%253D&md5=78481da1dd3700df6cca848328bbd77b

39. 39

    Ketterer, P. ; Willner, E. M. ; Dietz, H. Nanoscale Rotary Apparatus Formed from Tight-Fitting 3d DNA Components. Sci. Adv. 2016, 2 , e1501209  DOI: 10.1126/sciadv.1501209

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    Nanoscale rotary apparatus formed from tight-fitting 3D DNA components

    Ketterer, Philip; Willner, Elena M.; Dietz, Hendrik

    Science Advances (2016), 2 (2), e1501209/1-e1501209/9CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)

    We report a nanoscale rotary mechanism that reproduces some of the dynamic properties of biol. rotary motors in the absence of an energy source, such as random walks on a circle with dwells at docking sites. Our mechanism is built modularly from tight-fitting components that were self-assembled using multilayer DNA origami. The app. has greater structural complexity than previous mech. interlocked objects and features a well-defined angular degree of freedom without restricting the range of rotation. We studied the dynamics of our mechanism using single-particle expts. analogous to those performed previously with actin-labeled ATP synthases. In our mechanism, rotor mobility, the no. of docking sites, and the dwell times at these sites may be controlled through rational design. Our prototype thus realizes a working platform toward creating synthetic nanoscale rotary motors. Our methods will support creating other complex nanoscale mechanisms based on tightly fitting, sterically constrained, but mobile, DNA components.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlvVWku7w%253D&md5=b84b80d00436c4f1c95556819bc2f6dd

40. 40

    Tikhomirov, G. ; Petersen, P. ; Qian, L. Fractal Assembly of Micrometre-Scale DNA Origami Arrays with Arbitrary Patterns. Nature 2017, 552 , 67– 71,  DOI: 10.1038/nature24655

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    Fractal assembly of micrometre-scale DNA origami arrays with arbitrary patterns

    Tikhomirov, Grigory; Petersen, Philip; Qian, Lulu

    Nature (London, United Kingdom) (2017), 552 (7683), 67-71CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

    Self-assembled DNA nanostructures enable nanometer-precise patterning that can be used to create programmable mol. machines and arrays of functional materials. DNA origami is particularly versatile in this context because each DNA strand in the origami nanostructure occupies a unique position and can serve as a uniquely addressable pixel. However, the scale of such structures has been limited to about 0.05 square micrometres, hindering applications that demand a larger layout and integration with more conventional patterning methods. Hierarchical multistage assembly of simple sets of tiles can in principle overcome this limitation, but so far has not been sufficiently robust to enable successful implementation of larger structures using DNA origami tiles. Here we show that by using simple local assembly rules that are modified and applied recursively throughout a hierarchical, multistage assembly process, a small and const. set of unique DNA strands can be used to create DNA origami arrays of increasing size and with arbitrary patterns. We illustrate this method, which we term 'fractal assembly', by producing DNA origami arrays with sizes of up to 0.5 square micrometres and with up to 8,704 pixels, allowing us to render images such as the Mona Lisa and a rooster. We find that self-assembly of the tiles into arrays is unaffected by changes in surface patterns on the tiles, and that the yield of the fractal assembly process corresponds to about 0.95m - 1 for arrays contg. m tiles. When used in conjunction with a software tool that we developed that converts an arbitrary pattern into DNA sequences and exptl. protocols, our assembly method is readily accessible and will facilitate the construction of sophisticated materials and devices with sizes similar to that of a bacterium using DNA nanostructures.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurjN&md5=a69eb157ae84c6ea4105a9f41da0680f

41. 41

    Wagenbauer, K. F. ; Sigl, C. ; Dietz, H. Gigadalton-Scale Shape-Programmable DNA Assemblies. Nature 2017, 552 , 78– 83,  DOI: 10.1038/nature24651

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    Gigadalton-scale shape-programmable DNA assemblies

    Wagenbauer, Klaus F.; Sigl, Christian; Dietz, Hendrik

    Nature (London, United Kingdom) (2017), 552 (7683), 78-83CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

    Natural biomol. assemblies such as mol. motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnol. aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy nos., positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nm in diam. and with at. masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nm in diam. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equil. to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurjM&md5=3872a1f31d5dd833f9b3ee7a9123de21

42. 42

    Ong, L. L. ; Hanikel, N. ; Yaghi, O. K. ; Grun, C. ; Strauss, M. T. ; Bron, P. ; Lai-Kee-Him, J. ; Schueder, F. ; Wang, B. ; Wang, P. ; Kishi, J. Y. ; Myhrvold, C. ; Zhu, A. ; Jungmann, R. ; Bellot, G. ; Ke, Y. ; Yin, P. Programmable Self-Assembly of Three-Dimensional Nanostructures from 10,000 Unique Components. Nature 2017, 552 , 72– 77,  DOI: 10.1038/nature24648

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    Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components

    Ong, Luvena L.; Hanikel, Nikita; Yaghi, Omar K.; Grun, Casey; Strauss, Maximilian T.; Bron, Patrick; Lai-Kee-Him, Josephine; Schueder, Florian; Wang, Bei; Wang, Pengfei; Kishi, Jocelyn Y.; Myhrvold, Cameron; Zhu, Allen; Jungmann, Ralf; Bellot, Gaetan; Ke, Yonggang; Yin, Peng

    Nature (London, United Kingdom) (2017), 552 (7683), 72-77CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

    Nucleic acids (DNA and RNA) are widely used to construct nanometer-scale structures with ever increasing complexity, with possible application in fields such as structural biol., biophysics, synthetic biol. and photonics. The nanostructures are formed through one-pot self-assembly, with early kDa-scale examples contg. typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufg. and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid contg. about 30,000 unique bricks and a 1-gigadalton rotationally sym. tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair 'voxels' that serves as a mol. canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this mol. canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurvE&md5=96106fc32d528653317c767e96b078b2

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    Chen, X. ; Wang, Q. ; Peng, J. ; Long, Q. ; Yu, H. ; Li, Z. Self-Assembly of Large DNA Origami with Custom-Designed Scaffolds. ACS Appl. Mater. Interfaces 2018, 10 , 24344– 24348,  DOI: 10.1021/acsami.8b09222

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    Self-Assembly of Large DNA Origami with Custom-Designed Scaffolds

    Chen, Xiaoxing; Wang, Qian; Peng, Jin; Long, Qipeng; Yu, Hanyang; Li, Zhe

    ACS Applied Materials & Interfaces (2018), 10 (29), 24344-24348CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)

    As a milestone in DNA self-assembly, DNA origami has demonstrated powerful applications in many fields. However, the scarce availability of long single-stranded DNA (ssDNA) limits the size and sequences of DNA origami nanostructures, which in turn impedes the further development. In this study, the authors present a robust strategy to produce long circular ssDNA scaffold strands with custom-tailored lengths and sequences. These ssDNA products were then used as scaffolds for constructing various DNA origami nanostructures. This scalable method produces ssDNA at low cost with high purity and high yield, which can enable prodn. of custom-designed DNA origami for various applications.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht12mtbzP&md5=39f0c69906cbbc88d91a8babd4ab2819

44. 44

    Iinuma, R. ; Ke, Y. ; Jungmann, R. ; Schlichthaerle, T. ; Woehrstein, J. B. ; Yin, P. Polyhedra Self-Assembled from DNA Tripods and Characterized with 3d DNA-Paint. Science 2014, 344 , 65– 69,  DOI: 10.1126/science.1250944

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    Polyhedra Self-Assembled from DNA Tripods and Characterized with 3D DNA-PAINT

    Iinuma, Ryosuke; Ke, Yonggang; Jungmann, Ralf; Schlichthaerle, Thomas; Woehrstein, Johannes B.; Yin, Peng

    Science (Washington, DC, United States) (2014), 344 (6179), 65-69CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

    DNA self-assembly has produced diverse synthetic three-dimensional polyhedra. These structures typically have a mol. wt. no greater than 5 megadaltons. We report a simple, general strategy for one-step self-assembly of wireframe DNA polyhedra that are more massive than most previous structures. A stiff three-arm-junction DNA origami tile motif with precisely controlled angles and arm lengths was used for hierarchical assembly of polyhedra. We exptl. constructed a tetrahedron (20 megadaltons), a triangular prism (30 megadaltons), a cube (40 megadaltons), a pentagonal prism (50 megadaltons), and a hexagonal prism (60 megadaltons) with edge widths of 100 nm. The structures were visualized by means of transmission electron microscopy and three-dimensional DNA-PAINT super-resoln. fluorescent microscopy of single mols. in soln.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlt1eht7o%253D&md5=b6a44fdd0a45451b120729ab0849849b

45. 45

    He, Y. ; Ye, T. ; Su, M. ; Zhang, C. ; Ribbe, A. E. ; Jiang, W. ; Mao, C. Hierarchical Self-Assembly of DNA into Symmetric Supramolecular Polyhedra. Nature 2008, 452 , 198– 201,  DOI: 10.1038/nature06597

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    Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra

    He, Yu; Ye, Tao; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, Chengde

    Nature (London, United Kingdom) (2008), 452 (7184), 198-201CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

    DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute mol. computations. Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large nos. (hundreds) of unique DNA strands poses a challenging design problem. Here, the authors demonstrate a simple soln. to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger 3-dimensional structures. The authors test this hierarchical self-assembly concept with DNA mols. that form 3-point-star motifs, or tiles. By controlling the flexibility and concn. of the tiles, the one-pot assembly yields tetrahedra, dodecahedra or buckyballs that are tens of nanometers in size and comprised of four, twenty or sixty individual tiles, resp. The authors expect that this assembly strategy can be adapted to allow the fabrication of a range of relatively complex 3-dimensional structures.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjt1Gmsr4%253D&md5=25396f1220643c9ff865175aec2cfe81

46. 46

    Zhang, F. ; Jiang, S. ; Wu, S. ; Li, Y. ; Mao, C. ; Liu, Y. ; Yan, H. Complex Wireframe DNA Origami Nanostructures with Multi-Arm Junction Vertices. Nat. Nanotechnol. 2015, 10 , 779– 784,  DOI: 10.1038/nnano.2015.162

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    46

    Complex wireframe DNA origami nanostructures with multi-arm junction vertices

    Zhang, Fei; Jiang, Shuoxing; Wu, Siyu; Li, Yulin; Mao, Chengde; Liu, Yan; Yan, Hao

    Nature Nanotechnology (2015), 10 (9), 779-784CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)

    Structural DNA nanotechnol. and the DNA origami technique, in particular, have provided a range of spatially addressable two- and three-dimensional nanostructures. These structures are, however, typically formed of tightly packed parallel helixes. The development of wireframe structures should allow the creation of novel designs with unique functionalities, but engineering complex wireframe architectures with arbitrarily designed connections between selected vertices in three-dimensional space remains a challenge. Here, we report a design strategy for fabricating finite-size wireframe DNA nanostructures with high complexity and programmability. In our approach, the vertices are represented by n × 4 multi-arm junctions (n = 2-10) with controlled angles, and the lines are represented by antiparallel DNA crossover tiles of variable lengths. Scaffold strands are used to integrate the vertices and lines into fully assembled structures displaying intricate architectures. To demonstrate the versatility of the technique, a series of two-dimensional designs including quasi-cryst. patterns and curvilinear arrays or variable curvatures, and three-dimensional designs including a complex snub cube and a reconfigurable Archimedean solid were constructed.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1ajtrrF&md5=cf327ed66012b1a7acafd73a4b078cf0

47. 47

    Bruetzel, L. K. ; Walker, P. U. ; Gerling, T. ; Dietz, H. ; Lipfert, J. Time-Resolved Small-Angle X-Ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch. Nano Lett. 2018, 18 , 2672– 2676,  DOI: 10.1021/acs.nanolett.8b00592

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    47

    Time-Resolved Small-Angle X-ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch

    Bruetzel, Linda K.; Walker, Philipp U.; Gerling, Thomas; Dietz, Hendrik; Lipfert, Jan

    Nano Letters (2018), 18 (4), 2672-2676CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)

    Self-assembled DNA structures enable creation of specific shapes at the nanometer-micrometer scale with mol. resoln. The construction of functional DNA assemblies will likely require dynamic structures that can undergo controllable conformational changes. DNA devices based on shape complementary stacking interactions have been demonstrated to undergo reversible conformational changes triggered by changes in ionic environment or temp. An exptl. unexplored aspect is how quickly conformational transitions of large synthetic DNA origami structures can actually occur. Here, the authors use time-resolved small-angle x-ray scattering to monitor large-scale conformational transitions of a two-state DNA origami switch in free soln. The DNA device switches from its open to its closed conformation upon addn. of MgCl2 in milliseconds, which is close to the theor. diffusive speed limit. In contrast, measurements of the dimerization of DNA origami bricks reveal much slower and concn.-dependent assembly kinetics. DNA brick dimerization occurs on a time scale of minutes to hours suggesting that the kinetics depend on local concn. and mol. alignment.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltFehtLk%253D&md5=1ddad808bab8e9232e53f61296ee8565

48. 48

    Stahl, E. ; Martin, T. G. ; Praetorius, F. ; Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angew. Chem., Int. Ed. 2014, 53 , 12735– 12740,  DOI: 10.1002/anie.201405991

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    48

    Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions

    Stahl, Evi; Martin, Thomas G.; Praetorius, Florian; Dietz, Hendrik

    Angewandte Chemie, International Edition (2014), 53 (47), 12735-12740CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    DNA has become a prime material for assembling complex three-dimensional objects that promise utility in various areas of application. However, achieving user-defined goals with DNA objects has been hampered by the difficulty to prep. them at arbitrary concns. and in user-defined soln. conditions. Here, we describe a method that solves this problem. The method is based on poly(ethylene glycol)-induced depletion of species with high mol. wt. We demonstrate that our method is applicable to a wide spectrum of DNA shapes and that it achieves excellent recovery yields of target objects up to 97 %, while providing efficient sepn. from non-integrated DNA strands. DNA objects may be prepd. at concns. up to the limit of soly., including the possibility for bringing DNA objects into a solid phase. Due to the fidelity and simplicity of our method we anticipate that it will help to catalyze the development of new types of applications that use self-assembled DNA objects.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvV2mtrrM&md5=5d749b225ce5405de5c89eeb1a5b1897

49. 49

    Bai, X. C. ; Martin, T. G. ; Scheres, S. H. ; Dietz, H. Cryo-Em Structure of a 3d DNA-Origami Object. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 , 20012– 20017,  DOI: 10.1073/pnas.1215713109

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    49

    Cryo-EM structure of a 3D DNA-origami object

    Bai, Xiao-chen; Martin, Thomas G.; Scheres, Sjors H. W.; Dietz, Hendrik

    Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (49), 20012-20017, S20012/1-S20012/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    A key goal for nanotechnol. is to design synthetic objects that may ultimately achieve functionalities known today only from natural macromol. complexes. Mol. self-assembly with DNA has shown potential for creating user-defined 3D scaffolds, but the level of attainable positional accuracy has been unclear. Here we report the cryo-EM structure and a full pseudoat. model of a discrete DNA object that is almost twice the size of a prokaryotic ribosome. The structure provides a variety of stable, previously undescribed DNA topologies for future use in nanotechnol. and exptl. evidence that discrete 3D DNA scaffolds allow the positioning of user-defined structural motifs with an accuracy that is similar to that obsd. in natural macromols. Thereby, our results indicate an attractive route to fabricate nanoscale devices that achieve complex functionalities by DNA-templated design steered by structural feedback.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslKnsQ%253D%253D&md5=cfcc06e1fdc335ffd88f92768825ce70

50. 50

    Engler, C. ; Kandzia, R. ; Marillonnet, S. A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS One 2008, 3 , e3647  DOI: 10.1371/journal.pone.0003647

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    A one pot, one step, precision cloning method with high throughput capability

    Engler Carola; Kandzia Romy; Marillonnet Sylvestre

    PloS one (2008), 3 (11), e3647 ISSN:.

    Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1cjhvV2ltA%253D%253D&md5=8910183a67aa28a0cc69522dcf7050b0

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    List, J. ; Falgenhauer, E. ; Kopperger, E. ; Pardatscher, G. ; Simmel, F. C. Long-Range Movement of Large Mechanically Interlocked DNA Nanostructures. Nat. Commun. 2016, 7 , 12414,  DOI: 10.1038/ncomms12414

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    Long-range movement of large mechanically interlocked DNA nanostructures

    List, Jonathan; Falgenhauer, Elisabeth; Kopperger, Enzo; Pardatscher, Guenther; Simmel, Friedrich C.

    Nature Communications (2016), 7 (), 12414CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

    Interlocked mols. such as catenanes and rotaxanes, connected only via mech. bonds have the ability to perform large-scale sliding and rotational movements, making them attractive components for the construction of artificial mol. machines and motors. We here demonstrate the realization of large, rigid rotaxane structures composed of DNA origami subunits. The structures can be easily modified to carry a mol. cargo or nanoparticles. By using multiple axle modules, rotaxane constructs are realized with axle lengths of up to 355 nm and a fuel/anti-fuel mechanism is employed to switch the rotaxanes between a mobile and a fixed state. We also create extended pseudo-rotaxanes, in which origami rings can slide along supramol. DNA filaments over several hundreds of nanometers. The rings can be actively moved and tracked using at. force microscopy.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlalsLvO&md5=c5392b3f9015e2cf1c4817a9cb38a047

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    Plesa, C. ; van Loo, N. ; Ketterer, P. ; Dietz, H. ; Dekker, C. Velocity of DNA During Translocation through a Solid-State Nanopore. Nano Lett. 2015, 15 , 732– 737,  DOI: 10.1021/nl504375c

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    Velocity of DNA during Translocation through a Solid-State Nanopore

    Plesa, Calin; van Loo, Nick; Ketterer, Philip; Dietz, Hendrik; Dekker, Cees

    Nano Letters (2015), 15 (1), 732-737CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)

    While understanding translocation of DNA through a solid-state nanopore is vital for exploiting its potential for sensing and sequencing at the single-mol. level, surprisingly little is known about the dynamics of the propagation of DNA through the nanopore. Here we use linear double-stranded DNA mols., assembled by the DNA origami technique, with markers at known positions in order to det. for the first time the local velocity of different segments along the length of the mol. We observe large intramol. velocity fluctuations, likely related to changes in the drag force as the DNA blob unfolds. Furthermore, we observe an increase in the local translocation velocity toward the end of the translocation process, consistent with a speeding up due to unfolding of the last part of the DNA blob. We use the velocity profile to est. the uncertainty in detg. the position of a feature along the DNA given its temporal location and demonstrate the error introduced by assuming a const. translocation velocity.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitV2jurfI&md5=14c812b7f31b188a6a190d87a1b1b763

53. 53

    Bell, N. A. ; Keyser, U. F. Digitally Encoded DNA Nanostructures for Multiplexed, Single-Molecule Protein Sensing with Nanopores. Nat. Nanotechnol. 2016, 11 , 645– 651,  DOI: 10.1038/nnano.2016.50

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    Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

    Bell, Nicholas A. W.; Keyser, Ulrich F.

    Nature Nanotechnology (2016), 11 (7), 645-651CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)

    The simultaneous detection of a large no. of different analytes is important in bionanotechnol. research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, highly multiplexed sensing of single mols. can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, the authors designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signaled by the presence or absence of multiple DNA dumbbell hairpins. A 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows the authors to simultaneously detect four different antibodies of the same isotype at nanomolar concn. levels.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xltlyisbo%253D&md5=e3de2085bf984a84e6208a2f41b1503f

54. 54

    Schickinger, M. ; Zacharias, M. ; Dietz, H. Tethered Multifluorophore Motion Reveals Equilibrium Transition Kinetics of Single DNA Double Helices. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 , E7512,  DOI: 10.1073/pnas.1800585115

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    54

    Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices

    Schickinger, Matthias; Zacharias, Martin; Dietz, Hendrik

    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (32), E7512-E7521CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimol. reaction kinetics on the single-mol. level. Mol. binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 h. We applied the system to study in equil. reversible hybridization and dissocn. of complementary DNA single strands as a function of tether length, cation concn., and sequence. We obsd. up to hundreds of hybridization and dissocn. events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from mol. to mol., which enabled sepg. data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chem. truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissocn., is affected by cation concn., in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodn. stability, but different sequences, and obsd. strongly differing hybridization kinetics. Complementary full-atom mol.-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, resp., the fraction of strand collisions leading to successful nucleation events for duplex formation.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVanu7%252FK&md5=3b189b9257392137d9a6534ac6b4f5aa

55. 55

    Jungmann, R. ; Avendaño, M. S. ; Woehrstein, J. B. ; Dai, M. ; Shih, W. M. ; Yin, P. Multiplexed 3d Cellular Super-Resolution Imaging with DNA-Paint and Exchange-Paint. Nat. Methods 2014, 11 , 313,  DOI: 10.1038/nmeth.2835

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    Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT

    Jungmann, Ralf; Avendano, Maier S.; Woehrstein, Johannes B.; Dai, Mingjie; Shih, William M.; Yin, Peng

    Nature Methods (2014), 11 (3), 313-318CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

    Super-resoln. fluorescence microscopy is a powerful tool for biol. research, but obtaining multiplexed images for a large no. of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topog.) for simple and easy-to-implement multiplexed super-resoln. imaging that achieves sub-10-nm spatial resoln. in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We exptl. demonstrate ten-color super-resoln. imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Slu74%253D&md5=dbefcad33ee566c8f640258d28169420

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    Gibson, D. G. ; Young, L. ; Chuang, R.-Y. ; Venter, J. C. ; Hutchison, C. A., III ; Smith, H. O. Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases. Nat. Methods 2009, 6 , 343,  DOI: 10.1038/nmeth.1318

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    Enzymatic assembly of DNA molecules up to several hundred kilobases

    Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.

    Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

    The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVemsbw%253D&md5=46284924c7d73c47cfb490983338e480

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    Luke, J. ; Carnes, A. E. ; Hodgson, C. P. ; Williams, J. A. Improved Antibiotic-Free DNA Vaccine Vectors Utilizing a Novel Rna Based Plasmid Selection System. Vaccine 2009, 27 , 6454– 6459,  DOI: 10.1016/j.vaccine.2009.06.017

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    Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

    Luke, Jeremy; Carnes, Aaron E.; Hodgson, Clague P.; Williams, James A.

    Vaccine (2009), 27 (46), 6454-6459CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)

    To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermn. manufg. (>1 g/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlSqsr3K&md5=d91ccc3df965744d66956dd5905e8164

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    Douglas, S. M. ; Chou, J. J. ; Shih, W. M. DNA-Nanotube-Induced Alignment of Membrane Proteins for Nmr Structure Determination. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 , 6644– 6648,  DOI: 10.1073/pnas.0700930104

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    58

    DNA-nanotube-induced alignment of membrane proteins for NMR structure determination

    Douglas, Shawn M.; Chou, James J.; Shih, William M.

    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (16), 6644-6648CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    Membrane proteins are encoded by 20-35% of genes but represent <1% of known protein structures to date. Thus, improved methods for membrane-protein structure detn. are of crit. importance. Residual dipolar couplings (RDCs), commonly measured for biol. macromols. weakly aligned by liq.-cryst. media, are important global angular restraints for NMR structure detn. For α-helical membrane proteins >15 kDa in size, Nuclear-Overhauser effect-derived distance restraints are difficult to obtain, and RDCs could serve as the main reliable source of NMR structural information. In many of these cases, RDCs would enable full structure detn. that otherwise would be impossible. However, none of the existing liq.-cryst. media used to align water-sol. proteins are compatible with the detergents required to solubilize membrane proteins. The authors report the design and construction of a detergent-resistant liq. crystal of 0.8μm-long DNA-nanotubes that can be used to induce weak alignment of membrane proteins. The nanotubes are heterodimers of 0.4μm-long six-helix bundles each self-assembled from a 7.3-kb scaffold strand and >170 short oligonucleotide staple strands. The authors show that the DNA-nanotube liq. crystal enables the accurate measurement of backbone NH and CαHα RDCs for the detergent-reconstituted ζ-ζ transmembrane domain of the T cell receptor. The measured RDCs validate the high-resoln. structure of this transmembrane dimer. The authors anticipate that this medium will extend the advantages of weak alignment to NMR structure detn. of a broad range of detergent-solubilized membrane proteins.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkvFWjs7k%253D&md5=ad5031d791858f4435c5dd65ae72beb9

59. 59

    Castro, C. E. ; Kilchherr, F. ; Kim, D. N. ; Shiao, E. L. ; Wauer, T. ; Wortmann, P. ; Bathe, M. ; Dietz, H. A Primer to Scaffolded DNA Origami. Nat. Methods 2011, 8 , 221– 229,  DOI: 10.1038/nmeth.1570

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    A primer to scaffolded DNA origami

    Castro, Carlos Ernesto; Kilchherr, Fabian; Kim, Do-Nyun; Shiao, Enrique Lin; Wauer, Tobias; Wortmann, Philipp; Bathe, Mark; Dietz, Hendrik

    Nature Methods (2011), 8 (3), 221-229CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

    A review. Mol. self-assembly with scaffolded DNA origami enables building custom-shaped nanometer-scale objects with mol. wts. in the megadalton regime. Here the authors provide a practical guide for design and assembly of scaffolded DNA origami objects. The authors also introduce a computational tool for predicting the structure of DNA origami objects and provide information on the conditions under which DNA origami objects can be expected to maintain their structure.

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    DNA nanotechnol. enables the programmed synthesis of intricate nanometer-scale structures for diverse applications in materials and biol. science. Precise control over the 3D soln. shape and mech. flexibility of target designs is important to achieve desired functionality. Because exptl. validation of designed nanostructures is time-consuming and cost-intensive, predictive phys. models of nanostructure shape and flexibility have the capacity to enhance dramatically the design process. Here, the authors significantly extend and exptl. validate a computational modeling framework for DNA origami previously presented as CanDo A primer to scaffolded DNA origami. 3D soln. shape and flexibility are predicted from basepair connectivity maps now accounting for nicks in the DNA double helix, entropic elasticity of single-stranded DNA, and distant crossovers required to model wireframe structures, in addn. to previous modeling that accounted only for the canonical twist, bend and stretch stiffness of double-helical DNA domains. Systematic exptl. validation of nanostructure flexibility mediated by internal crossover d. probed using a 32-helix DNA bundle demonstrates for the first time that our model not only predicts the 3D soln. shape of complex DNA nanostructures but also their mech. flexibility. Thus, our model represents an important advance in the quant. understanding of DNA-based nanostructure shape and flexibility, and we anticipate that this model will increase significantly the no. and variety of synthetic nanostructures designed using nucleic acids.

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    Angewandte Chemie, International Edition (2013), 52 (30), 7766-7771CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    This paper developed DNA helix bundles as linker system for single-mol. mech. assays.

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    Nature Protocols (2008), 3 (6), 977-990CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)

    The authors describe a collection of standardized image processing protocols for electron microscopy single-particle anal. using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through max.-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with min. efforts in learning about the details of this comprehensive package. Examples of applications are presented for a neg. stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.

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    By reaching near-at. resoln. for a wide range of specimens, single-particle cryo-EM structure detn. is transforming structural biol. However, the necessary calcns. come at large computational costs, which has introduced a bottleneck that is currently limiting throughput and the development of new methods. Here, we present an implementation of the RELION image processing software that uses graphics processors (GPUs) to address the most computationally intensive steps of its cryo-EM structure detn. workflow. Both image classification and high-resoln. refinement have been accelerated more than an order-of-magnitude, and template-based particle selection has been accelerated well over two orders-of-magnitude on desktop hardware. Memory requirements on GPUs have been reduced to fit widely available hardware, and we show that the use of single precision arithmetic does not adversely affect results. This enables high-resoln. cryo-EM structure detn. in a matter of days on a single workstation.

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    eLife (2018), 7 (), e42166/1-e42166/22CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)

    Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addn. to GPU support, which provides flexibility in use of resources and avoids memory limitations. Ref.-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2Dclassification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estd. beam tilt provides higher resoln. reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resoln. for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beaminduced motion correction it leads to resoln. improvements of 0.2-0.7 Å compared to previous RELION versions.

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    Nature Methods (2017), 14 (4), 331-332CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

    A review on anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Here we describe MotionCor2, a software tool for anisotropic correction of beam-induced motion. Overall, MotionCor2 is extremely robust and sufficiently accurate at correcting local motions so that the very time-consuming and computationally intensive particle polishing in RELION can be skipped, importantly, it also works on a wide range of data sets, including cryo tomog. tilt series.

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    Accurate determination of local defocus and specimen tilt in electron microscopy

    Mindell Joseph A; Grigorieff Nikolaus

    Journal of structural biology (2003), 142 (3), 334-47 ISSN:1047-8477.

    Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10nm, whereas tilt axis and tilt angle are determined with an error of about 2 degrees and 1 degrees, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6 degrees, respectively.

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• Figures
• References
• Support Info

• Abstract

  

  Figure 1

  

  Figure 1. Design-specific scaffold sequences in minimum-constraint vectors for making fully user-defined DNA origami. (A) Schematic diagram of input for the scaffold smith used for creating custom scaffold sequences: exemplary caDNAno design diagram with scaffold strand indicated in blue and staple strands in multiple colors (I), user-specific constraints (II), and weighting factors for a stochastic base distribution (III). (B) Illustration of scaffold production with helper-plasmid system using phagemids with a split-ori approach (top) and a modified split-ori approach where the backbone sequence is flanked by self-cleaving DNAzymes (bottom). Zn²⁺ addition leads to excision of the backbone and linearization. Black, constant parts for each type of scaffold; gray, user-definable parts; light green, backbone present only in the double-stranded plasmid and not in the single-stranded product; red, self-cleaving DNAzymes.

  Figure 2

  

  Figure 2. Influence of base composition and sequence redundancy of custom scaffolds on DNA origami self-assembly. Blue indicates M13-based scaffolds; orange, magenta, red, cyan, and green indicate custom scaffolds. (A) Schematic representations of six different 42-helix bundles folded using the six different scaffolds. SC1, M13-based scaffold; SC2, reduced backbone phagemid scaffold with CpG-free de Bruijn insert sequence; SC3, conventional phagemid with high duplicity fragment and de Bruijn insert sequence; SC4, conventional phagemid with de Bruijn insert sequence; SC5 and SC6, split-ori based scaffold with de Bruijn sequence; L, length; GC, GC content of the corresponding scaffold. (B) Electrophoretic mobility analysis of self-assembly reactions of the 42-helix bundles shown in (A) at different temperatures and salt concentrations. SC, scaffold reference; C50 and C20, assembly reactions containing 50 nM (C50) or 20 nM (C20) scaffold, 200 nM staples, and 20 mM MgCl₂ that were subjected to an annealing ramp from 60 to 44 °C (1 h per °C); temperature screen, assembly mixtures as in C50 but subjected to annealing ramps covering the temperature intervals indicated above each lane (1 h per °C); magnesium screen, assembly reactions containing 50 nM scaffold, 200 nM staples, and MgCl₂ concentrations between 5 mM (M5) and 30 mM (M30). P, pocket; F, folded 42-helix bundle. All samples were loaded onto the gel at an approximate scaffold concentration of 20 nM. All temperature ramps contained an initial denaturation step at 65 °C for 15 min. Laser scanned fluorescent images of the electrophoretic analysis were autoleveled. (C) Statistics of sequence duplicates of different scaffold variants as a function of fragment length. Colors as in (A). (D) Experimentally observed optimal folding temperature intervals of the 42-helix bundles plotted against total NN energy of corresponding scaffold variant. Total NN energy was calculated using nearest-neighbor free energy parameters, (36) ignoring edge effects. Dots in red indicate upper, and dots in blue indicate lower limit of the highest folding temperature interval where the sample appeared fully folded. Solid lines represent linear fits.

  Figure 3

  

  Figure 3. DNA origami objects with sizes ranging between 1024 bp (633 kDa) and 37800 bp (23.4 MDa) can be assembled using mini-scaffolds or in one-pot assembly reactions containing multiple scaffolds. Blue indicates M13-based scaffolds; orange, green, cyan, and red indicate custom scaffolds. (A) Schematic representation of a circular DNA single strand (top left) that, in the presence of Zn²⁺, cleaves itself to yield four copies of a short, linear scaffold (top right) that can subsequently be used to assemble a small DNA origami object (bottom). (B) Schematic representation (top) and average TEM images of 13-helix bundle (13hb) variants assembled from linear mini-scaffolds comprising 1024 (I), 1536 (II), or 2048 bases (III). Scale bar: 20 nm. (C) Electrophoretic mobility analysis of mini-scaffolds and 13-helix bundle variants described in (B). (D) Schematic representations, single TEM images, and average TEM images (from top to bottom) of a 42-helix bundle assembled with five scaffolds in one-pot reactions. Scale bar: 50 nm. (E) Schematic representations, single TEM images, and average TEM images (from top to bottom) of an improved 42-helix bundle design with five interlocked scaffolds. Scale bar: 50 nm. (F) Electrophoretic mobility analysis of the two 42-helix bundle versions shown in (D,E). (G) Schematic representation (top), average TEM images with corresponding model views (left), and gel electrophoretic analysis (right) of a 126-helix bundle (126hb) assembled with two interlocked scaffolds. Scale bar: 50 nm. (H) Overlay of a cryo-EM density map fragment and the corresponding scaffold routing diagram. Blue and orange paths indicate the two orthogonal scaffolds. Laser scanned fluorescent images of the electrophoretic analyses were autoleveled. P, pocket; sta, staples.

  Figure 4

  

  Figure 4. Self-cleaving DNA origami. (A) Schematic representations of circular scaffolds containing two self-excising DNAzyme cassettes (top left) that can be cleaved into two linear scaffolds (bottom left) or assembled into a switch object (top right). Individual switch arms (bottom right) can be obtained by cleavage of assembled switch objects or assembly using cleaved linear scaffolds. (B) Electrophoretic analysis of reaction kinetics of scaffold cleavage. Controls: cleaved scaffold (lane 1), undigested sample (lane 2), and switch arms assembled separately (lane 7) using cleaved scaffold. (C) Field-of-view TEM images of uncleaved (left) and cleaved (right) switch objects. (D) Electrophoretic analysis of cleavage reactions containing unpurified (lanes 1 and 5) and PEG-purified (lanes 2–4, 6–8) switch objects at 1.4, 4, 10, or 20 mM MgCl₂. Laser scanned fluorescent images of the electrophoretic analysis were autoleveled, and the highlighted region was autoleveled individually. P, pocket; U, undigested species; D, digested species. Scale bar: 100 nm.

  Figure 5

  

  Figure 5. UV point-welding of DNA origami with a custom scaffold. (A) Section of a multilayer DNA origami strand diagram with a customized scaffold featuring AA motifs every 8 base pairs, which results in adjacent Thymidines in separate staple strands that may be UV-cross-linked. Blue lines, scaffold strand; gray lines, staple strands. (B) Schematic representation (left) and average TEM images of the pointer object assembled with the welding scaffold. Average images of the pointer as obtained in the presence of 30 mM MgCl₂ before irradiation (I), after irradiation for 2 h at 310 nm (II) in the presence of 30 mM MgCl₂, and after irradiation for 2 h at 310 nm and 48 h long incubation in low ionic strength phosphate-buffered saline (PBS) at 40 °C (III). (C) Electrophoretic analysis of nonirradiated and irradiated pointer objects incubated over time in PBS at 40 °C. L, 1kB Ladder; NI, not irradiated; RT, room temperature; P, pocket; F, folded species; sta, staples. Scale bar: 50 nm.

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      Kick, Benjamin; Praetorius, Florian; Dietz, Hendrik; Weuster-Botz, Dirk

      Nano Letters (2015), 15 (7), 4672-4676CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)

      Scaffolded DNA origami enables the fabrication of a variety of complex nanostructures that promise utility in diverse fields of application, ranging from biosensing over advanced therapeutics to metamaterials. The broad applicability of DNA origami as a material beyond the level of proof-of-concept studies critically depends, among other factors, on the availability of large amts. of pure single-stranded scaffold DNA. Here, we present a method for the efficient prodn. of M13 bacteriophage-derived genomic DNA using high-cell-d. fermn. of Escherichia coli in stirred-tank bioreactors. We achieve phage titers of up to 1.6 × 1014 plaque-forming units per mL. Downstream processing yields up to 410 mg of high-quality single-stranded DNA per one liter reaction vol., thus upgrading DNA origami-based nanotechnol. from the milligram to the gram scale.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptF2ntLc%253D&md5=06d07cc77d7eaffd64bfe0a1d1c5e138

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      Niekamp, S. ; Blumer, K. ; Nafisi, P. M. ; Tsui, K. ; Garbutt, J. ; Douglas, S. M. Folding Complex DNA Nanostructures from Limited Sets of Reusable Sequences. Nucleic Acids Res. 2016, 44 , e102  DOI: 10.1093/nar/gkw208

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      19

      Folding complex DNA nanostructures from limited sets of reusable sequences

      Niekamp, Stefan; Blumer, Katy; Nafisi, Parsa M.; Tsui, Kathy; Garbutt, John; Douglas, Shawn M.

      Nucleic Acids Research (2016), 44 (11), e102/1-e102/6CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

      Scalable prodn. of DNA nanostructures remains a substantial obstacle to realizing new applications of DNA nanotechnol. Typical DNA nanostructures comprise hundreds of DNA oligonucleotide strands, where each unique strand requires a sep. synthesis step. New design methods that reduce the strand count for a given shape while maintaining overall size and complexity would be highly beneficial for efficiently producing DNA nanostructures. Here, we report a method for folding a custom template strand by binding individual staple sequences to multiple locations on the template. We built several nanostructures for well-controlled testing of various design rules, and demonstrate folding of a 6-kb template by as few as 10 unique strand sequences binding to 10 ± 2 locations on the template strand.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsF2jsbfI&md5=e6604bc54e9b8b688be13a73605848a6

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      Kick, B. ; Hensler, S. ; Praetorius, F. ; Dietz, H. ; Weuster-Botz, D. Specific Growth Rate and Multiplicity of Infection Affect High-Cell-Density Fermentation with Bacteriophage M13 for Ssdna Production. Biotechnol. Bioeng. 2017, 114 , 777– 784,  DOI: 10.1002/bit.26200

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      20

      Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production

      Kick, Benjamin; Hensler, Samantha; Praetorius, Florian; Dietz, Hendrik; Weuster-Botz, Dirk

      Biotechnology and Bioengineering (2017), 114 (4), 777-784CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)

      The bacteriophage M13 has found frequent applications in nanobiotechnol. due to its chem. and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Addnl., its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream prodn. methods for phage and scaffold ssDNA are underexamd. with respect to future industrial usage. Here, the high-cell-d. phage prodn. with Escherichia coli as host organism was studied in respect of medium compn., infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concn. of produced ssDNA on the other hand. Using a growth rate of 0.15 h-1 and a multiplicity of infection of 0.05 pfu cfu-1 in the fed-batch prodn. process, the concn. of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L-1. Thus, our results help enabling M13 prodn. for industrial uses in nanobiotechnol. Biotechnol. Bioeng. 2016;9999: 1-8. © 2016 Wiley Periodicals, Inc.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslCrs7zN&md5=ff429c307988309455756899b4f42302

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      Praetorius, F. ; Kick, B. ; Behler, K. L. ; Honemann, M. N. ; Weuster-Botz, D. ; Dietz, H. Biotechnological Mass Production of DNA Origami. Nature 2017, 552 , 84– 87,  DOI: 10.1038/nature24650

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      21

      Biotechnological mass production of DNA origami

      Praetorius, Florian; Kick, Benjamin; Behler, Karl L.; Honemann, Maximilian N.; Weuster-Botz, Dirk; Dietz, Hendrik

      Nature (London, United Kingdom) (2017), 552 (7683), 84-87CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

      DNA nanotechnol., in particular DNA origami, enables the bottom-up self-assembly of micrometer-scale, three-dimensional structures with nanometer-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behavior. Their use has been limited to applications that require only small amts. of material (of the order of micrograms), owing to the limitations of current prodn. methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold mol. held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold mols. are amenable to scalable and efficient mass prodn.; the shorter staple strands are obtained through costly solid-phase synthesis or enzymic processes. Here the authors show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. The authors produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end prodn. of macroscopic amts. of a DNA origami nanorod in a liter-scale stirred-tank bioreactor. The method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the prodn. and purifn. steps amenable to scaling, the authors expect that the method will expand the scope of DNA nanotechnol. in many areas of science and technol.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurjO&md5=8773cd37db2a75a049ae65ff28921da7

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      Douglas, S. M. ; Marblestone, A. H. ; Teerapittayanon, S. ; Vazquez, A. ; Church, G. M. ; Shih, W. M. Rapid Prototyping of 3d DNA-Origami Shapes with Cadnano. Nucleic Acids Res. 2009, 37 , 5001– 5006,  DOI: 10.1093/nar/gkp436

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      22

      Rapid prototyping of 3D DNA-origami shapes with caDNAno

      Douglas, Shawn M.; Marblestone, Adam H.; Teerapittayanon, Surat; Vazquez, Alejandro; Church, George M.; Shih, William M.

      Nucleic Acids Research (2009), 37 (15), 5001-5006CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

      DNA nanotechnol. exploits the programmable specificity afforded by base-pairing to produce self-assembling macromol. objects of custom shape. For building megadalton-scale DNA nanostructures, a long scaffold' strand can be employed to template the assembly of hundreds of oligonucleotide staple' strands into a planar antiparallel array of cross-linked helixes. The authors recently adapted this scaffolded DNA origami' method to producing 3-dimensional shapes formed as pleated layers of double helixes constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here the authors present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3-dimensional honeycomb-pleated shapes rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3-dimensional DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKntbzE&md5=aa99732c1666373a70e9b7b4de6e6d5d

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      Veneziano, R. ; Ratanalert, S. ; Zhang, K. ; Zhang, F. ; Yan, H. ; Chiu, W. ; Bathe, M. Designer Nanoscale DNA Assemblies Programmed from the Top Down. Science 2016, 352 , 1534,  DOI: 10.1126/science.aaf4388

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      Designer nanoscale DNA assemblies programmed from the top down

      Veneziano, Remi; Ratanalert, Sakul; Zhang, Kaiming; Zhang, Fei; Yan, Hao; Chiu, Wah; Bathe, Mark

      Science (Washington, DC, United States) (2016), 352 (6293), 1534CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

      Scaffolded DNA origami is a versatile means of synthesizing complex mol. architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, the authors report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asym. polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biol. as well as nonbiol. applications.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVaitL3I&md5=3772f7806345ae3b655fa3c1b147d752

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      Jun, H. ; Zhang, F. ; Shepherd, T. ; Ratanalert, S. ; Qi, X. ; Yan, H. ; Bathe, M. Autonomously Designed Free-Form 2d DNA Origami. Sci. Adv. 2019, 5 , eaav0655  DOI: 10.1126/sciadv.aav0655

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      Jun, H. ; Shepherd, T. R. ; Zhang, K. ; Bricker, W. P. ; Li, S. ; Chiu, W. ; Bathe, M. Automated Sequence Design of 3d Polyhedral Wireframe DNA Origami with Honeycomb Edges. ACS Nano 2019,  DOI: 10.1021/acsnano.8b08671

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      Benson, E. ; Mohammed, A. ; Gardell, J. ; Masich, S. ; Czeizler, E. ; Orponen, P. ; Hogberg, B. DNA Rendering of Polyhedral Meshes at the Nanoscale. Nature 2015, 523 , 441– 444,  DOI: 10.1038/nature14586

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      DNA rendering of polyhedral meshes at the nanoscale

      Benson, Erik; Mohammed, Abdulmelik; Gardell, Johan; Masich, Sergej; Czeizler, Eugen; Orponen, Pekka; Hoegberg, Bjoern

      Nature (London, United Kingdom) (2015), 523 (7561), 441-444CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

      It was suggested more than thirty years ago that Watson-Crick base pairing might be used for the rational design of nanometer-scale structures from nucleic acids. Since then, and esp. since the introduction of the origami technique, DNA nanotechnol. has enabled increasingly more complex structures. But although general approaches for creating DNA origami polygonal meshes and design software are available, there are still important constraints arising from DNA geometry and sense/antisense pairing, necessitating some manual adjustment during the design process. Here we present a general method of folding arbitrary polygonal digital meshes in DNA that readily produces structures that would be very difficult to realize using previous approaches. The design process is highly automated, using a routing algorithm based on graph theory and a relaxation simulation that traces scaffold strands through the target structures. Moreover, unlike conventional origami designs built from close-packed helixes, our structures have a more open conformation with one helix per edge and are therefore stable under the ionic conditions usually used in biol. assays.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1WksrfJ&md5=38be5240fa4b9bdac057fdbbbb6915c5

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      Bellot, G. ; McClintock, M. A. ; Chou, J. J. ; Shih, W. M. DNA Nanotubes for Nmr Structure Determination of Membrane Proteins. Nat. Protoc. 2013, 8 , 755– 770,  DOI: 10.1038/nprot.2013.037

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      DNA nanotubes for NMR structure determination of membrane proteins

      Bellot, Gaetan; McClintock, Mark A.; Chou, James J.; Shih, William M.

      Nature Protocols (2013), 8 (4), 755-770, 16 pp.CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)

      Finding a way to det. the structures of integral membrane proteins using soln. NMR (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dil. liq. crystals for application as weak-alignment media in soln. NMR structure detn. of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable pos. charge as well as modulation of mol. alignment, toward collection of linearly independent restraints, can be introduced by reducing the neg. charge of DNA nanotubes using counter ions and small DNA-binding mols. This detergent-resistant liq.-crystal medium offers a no. of properties conducive for membrane protein alignment, including high-yield prodn., thermal stability, buffer compatibility and structural programmability. Prodn. of sufficient nanotubes for four or five NMR expts. can be completed in 1 wk by a single individual.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmtlShsbo%253D&md5=21847aedab3e0bcecdca6028ac8c92de

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      Vieira, J. ; Messing, J. Production of Single-Stranded Plasmid DNA. Methods Enzymol. 1987, 153 , 3– 11,  DOI: 10.1016/0076-6879(87)53044-0

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      Production of single-stranded plasmid DNA

      Vieira, Jeffrey; Messing, Joachim

      Methods in Enzymology (1987), 153 (Recomb. DNA, Pt. D), 3-11CODEN: MENZAU; ISSN:0076-6879.

      A helper phage, M13KO7, was constructed that preferentially packages plasmid DNA over phage DNA. This then produces single-stranded plasmid DNA.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXht1GgtLw%253D&md5=dbbde887802d2c81f887633d5017a94a

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      Chasteen, L. ; Ayriss, J. ; Pavlik, P. ; Bradbury, A. R. Eliminating Helper Phage from Phage Display. Nucleic Acids Res. 2006, 34 , e145  DOI: 10.1093/nar/gkl772

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      Eliminating helper phage from phage display

      Chasteen, L.; Ayriss, J.; Pavlik, P.; Bradbury, A. R. M.

      Nucleic Acids Research (2006), 34 (21), e145/1-e145/11CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

      Phage display technol. involves the display of proteins or peptides, as coat protein fusions, on the surface of a phage or phagemid particles. Using std. technol., helper phage are essential for the replication and assembly of phagemid particles, during library prodn. and biopanning. We have eliminated the need to add helper phage by using 'bacterial packaging cell lines' that provide the same functions. These cell lines contain M13-based helper plasmids that express phage packaging proteins which assemble phagemid particles as efficiently as helper phage, but without helper phage contamination. This results in genetically pure phagemid particle prepns. Furthermore, by using constructs differing in the form of gene 3 that they contain, we have shown that the display, from a single library, can be modulated between monovalent (phagemid-like) and multivalent display (phage-like) without any further engineering. These packaging cells eliminate the use of helper phage from phagemid-based selection protocols; reducing the amt. of tech. prepn., facilitating automation, optimizing selections by matching display levels to diversity, and effectively using the packaged phagemid particles as means to transfer genetic information at an efficiency approaching 100%.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlCru7fO&md5=4850daf57dd2b0d00509f4639d7c7ee0

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      Specthrie, L. ; Bullitt, E. ; Horiuchi, K. ; Model, P. ; Russel, M. ; Makowski, L. Construction of a Microphage Variant of Filamentous Bacteriophage. J. Mol. Biol. 1992, 228 , 720– 724,  DOI: 10.1016/0022-2836(92)90858-H

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      Construction of a microphage variant of filamentous bacteriophage

      Specthrie, Leon; Bullitt, Esther; Horiuchi, Kensuke; Model, Peter; Russel, Marjorie; Makowski, Lee

      Journal of Molecular Biology (1992), 228 (3), 720-4CODEN: JMOBAK; ISSN:0022-2836.

      The intergenic region in the genome of the Ff class of filamentous phage (comprising strains f1, fd and M13) genome constitutes 8% of the viral genome, and has essential functions in DNA replication and phage morphogenesis. The functional domains of this region may be inserted into sep. sites of a plasmid to function independently. Here, the authors demonstrate the construction of a plasmid contg., sequentially, the origin of (+)-strand synthesis, the packaging signal, and a terminator of (+)-strand synthesis. When host cells harboring this plasmid (pLS7) are infected with helper phage they produce a microphage particle contg. all the structural elements of the mature, native phage. The microphage is 65 Å in diam. and ∼500 Å long. It contains a 221-base single-stranded circle of DNA coated by ∼95 copies of the major coat protein (gene 8 protein).

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlsFOktA%253D%253D&md5=ae1abdd9ef5054a869a0ac85607707f7

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      Nafisi, P. M. ; Aksel, T. ; Douglas, S. M. Construction of a Novel Phagemid to Produce Custom DNA Origami Scaffolds. Synth. Biol. 2018,  DOI: 10.1093/synbio/ysy015

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      Said, H. ; Schuller, V. J. ; Eber, F. J. ; Wege, C. ; Liedl, T. ; Richert, C. M1.3--a Small Scaffold for DNA Origami. Nanoscale 2013, 5 , 284– 290,  DOI: 10.1039/C2NR32393A

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      M1.3 - a small scaffold for DNA origami

      Said, Hassan; Schueller, Verena J.; Eber, Fabian J.; Wege, Christina; Liedl, Tim; Richert, Clemens

      Nanoscale (2013), 5 (1), 284-290CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)

      The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient prepn. of a 704 nt fragment dubbed "M1.3" as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepd. and were shown to give higher UV-m.ps. than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chem., phys., and biol. expts.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVSktb3O&md5=309c7bcfa7a4f04d8e25a9cb0af39c4b

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      Sobczak, J. P. ; Martin, T. G. ; Gerling, T. ; Dietz, H. Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature. Science 2012, 338 , 1458– 1461,  DOI: 10.1126/science.1229919

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      Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature

      Sobczak, Jean-Philippe J.; Martin, Thomas G.; Gerling, Thomas; Dietz, Hendrik

      Science (Washington, DC, United States) (2012), 338 (6113), 1458-1461CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

      At const. temp., hundreds of DNA strands can cooperatively fold a long template DNA strand within minutes into complex nanoscale objects. Folding occurred out of equil. along nucleation-driven pathways at temps. that could be influenced by the choice of sequences, strand lengths, and chain topol. Unfolding occurred in apparent equil. at higher temps. than those for folding. Folding at optimized const. temps. enabled the rapid prodn. of three-dimensional DNA objects with yields that approached 100%. The results point to similarities with protein folding in spite of chem. and structural differences. The possibility for rapid and high-yield assembly will enable DNA nanotechnol. for practical applications.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVSktLfJ&md5=718307e01a75a76aab5d04f4667e191d

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      Wagenbauer, K. F. ; Engelhardt, F. A. ; Stahl, E. K. ; Hechtl, V. K. ; Stömmer, P. ; Seebacher, F. ; Meregalli, L. ; Ketterer, P. ; Gerling, T. ; Dietz, H. How We Make DNA Origami. ChemBioChem 2017, 18 , 1873,  DOI: 10.1002/cbic.201700377

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      How We Make DNA Origami

      Wagenbauer, Klaus F.; Engelhardt, Floris A. S.; Stahl, Evi; Hechtl, Vera K.; Stoemmer, Pierre; Seebacher, Fabian; Meregalli, Letizia; Ketterer, Philip; Gerling, Thomas; Dietz, Hendrik

      ChemBioChem (2017), 18 (19), 1873-1885CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)

      DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for prepg. them at appropriate concns. with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnol. may emerge. To foster faster progress, we share in this article the experience in making and prepg. DNA origami that we have accumulated over recent years. We discuss design solns. for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addn., we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free prepn. of DNA origami. These methods are agarose-gel purifn., filtration through mol. cut-off membranes, PEG pptn., size-exclusion chromatog., and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their exptl. characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami prodn. workflow.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht12ltbzE&md5=ea439ce860cf1988018b6a223421a4b7

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      SantaLucia, J., Jr. ; Hicks, D. The Thermodynamics of DNA Structural Motifs. Annu. Rev. Biophys. Biomol. Struct. 2004, 33 , 415– 440,  DOI: 10.1146/annurev.biophys.32.110601.141800

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      The thermodynamics of DNA structural motifs

      SantaLucia, John, Jr.; Hicks, Donald

      Annual Review of Biophysics and Biomolecular Structure (2004), 33 (), 415-440, 2 platesCODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)

      A review. DNA secondary structure plays an important role in biol., genotyping diagnostics, a variety of mol. biol. techniques, in vitro-selected DNA catalysts, nanotechnol., and DNA-based computing. Accurate prediction of DNA secondary structure and hybridization using dynamic programming algorithms requires a database of thermodn. parameters for several motifs including Watson-Crick base pairs, internal mismatches, terminal mismatches, terminal dangling ends, hairpins, bulges, internal loops, and multibranched loops. To make the database useful for predictions under a variety of salt conditions, empirical equations for monovalent and Mg2+ dependence of thermodn. have been developed. Bimol. hybridization is often inhibited by competing unimol. folding of a target or probe DNA. Powerful numerical methods have been developed to solve multistate-coupled equil. in bimol. and higher-order complexes. This review presents the current parameter set available for making accurate DNA structure predictions and also points to future directions for improvement.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltlKkt7k%253D&md5=4079be22e69c61a888673d54a1ccf309

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      Berardi, M. J. ; Shih, W. M. ; Harrison, S. C. ; Chou, J. J. Mitochondrial Uncoupling Protein 2 Structure Determined by Nmr Molecular Fragment Searching. Nature 2011, 476 , 109– 113,  DOI: 10.1038/nature10257

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      Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

      Berardi, Marcelo J.; Shih, William M.; Harrison, Stephen C.; Chou, James J.

      Nature (London, United Kingdom) (2011), 476 (7358), 109-113CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

      Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small mols. across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochem. potential. A fraction of this electrochem. potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or uncoupling' of the proton electrochem. potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a soln.-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges assocd. with membrane-protein structure detn., combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquant. distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were detd. by piecing together mol. fragments from the Protein Data Bank that best fit exptl. RDCs from samples weakly aligned in a DNA nanotube liq. crystal. The RDCs also det. the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labeled GDP binds inside the channel and seems to be closer to transmembrane helixes 1-4. We believe that this biophys. approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure detn. but also to characterize various conformational states of these proteins linked to substrate transport.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptlalu74%253D&md5=7ecd0be8d83ab63eb09baef8cc70b8ae

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      Gerling, T. ; Wagenbauer, K. F. ; Neuner, A. M. ; Dietz, H. Dynamic DNA Devices and Assemblies Formed by Shape-Complementary, Non-Base Pairing 3d Components. Science 2015, 347 , 1446– 1452,  DOI: 10.1126/science.aaa5372

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      38

      Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components

      Gerling, Thomas; Wagenbauer, Klaus F.; Neuner, Andrea M.; Dietz, Hendrik

      Science (Washington, DC, United States) (2015), 347 (6229), 1446-1452CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

      The authors demonstrate that discrete three-dimensional (3D) DNA components can specifically self-assemble in soln. on the basis of shape-complementarity and without base pairing. Using this principle, homo- and heteromultimeric objects were produced, including micrometer-scale one- and two-stranded filaments and lattices, as well as reconfigurable devices, including an actuator, a switchable gear, an unfoldable nanobook, and a nanorobot. These multidomain assemblies were stabilized via short-ranged nucleobase stacking bonds that compete against electrostatic repulsion between the components' interfaces. Using imaging by electron microscopy, ensemble and single-mol. fluorescence resonance energy transfer spectroscopy, and electrophoretic mobility anal., it was shown that the balance between attractive and repulsive interactions, and thus the conformation of the assemblies, may be finely controlled by global parameters such as cation concn. or temp. and by an allosteric mechanism based on strand-displacement reactions.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvFCnu74%253D&md5=78481da1dd3700df6cca848328bbd77b

  39. 39

      Ketterer, P. ; Willner, E. M. ; Dietz, H. Nanoscale Rotary Apparatus Formed from Tight-Fitting 3d DNA Components. Sci. Adv. 2016, 2 , e1501209  DOI: 10.1126/sciadv.1501209

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      Nanoscale rotary apparatus formed from tight-fitting 3D DNA components

      Ketterer, Philip; Willner, Elena M.; Dietz, Hendrik

      Science Advances (2016), 2 (2), e1501209/1-e1501209/9CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)

      We report a nanoscale rotary mechanism that reproduces some of the dynamic properties of biol. rotary motors in the absence of an energy source, such as random walks on a circle with dwells at docking sites. Our mechanism is built modularly from tight-fitting components that were self-assembled using multilayer DNA origami. The app. has greater structural complexity than previous mech. interlocked objects and features a well-defined angular degree of freedom without restricting the range of rotation. We studied the dynamics of our mechanism using single-particle expts. analogous to those performed previously with actin-labeled ATP synthases. In our mechanism, rotor mobility, the no. of docking sites, and the dwell times at these sites may be controlled through rational design. Our prototype thus realizes a working platform toward creating synthetic nanoscale rotary motors. Our methods will support creating other complex nanoscale mechanisms based on tightly fitting, sterically constrained, but mobile, DNA components.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlvVWku7w%253D&md5=b84b80d00436c4f1c95556819bc2f6dd

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      Tikhomirov, G. ; Petersen, P. ; Qian, L. Fractal Assembly of Micrometre-Scale DNA Origami Arrays with Arbitrary Patterns. Nature 2017, 552 , 67– 71,  DOI: 10.1038/nature24655

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      Fractal assembly of micrometre-scale DNA origami arrays with arbitrary patterns

      Tikhomirov, Grigory; Petersen, Philip; Qian, Lulu

      Nature (London, United Kingdom) (2017), 552 (7683), 67-71CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

      Self-assembled DNA nanostructures enable nanometer-precise patterning that can be used to create programmable mol. machines and arrays of functional materials. DNA origami is particularly versatile in this context because each DNA strand in the origami nanostructure occupies a unique position and can serve as a uniquely addressable pixel. However, the scale of such structures has been limited to about 0.05 square micrometres, hindering applications that demand a larger layout and integration with more conventional patterning methods. Hierarchical multistage assembly of simple sets of tiles can in principle overcome this limitation, but so far has not been sufficiently robust to enable successful implementation of larger structures using DNA origami tiles. Here we show that by using simple local assembly rules that are modified and applied recursively throughout a hierarchical, multistage assembly process, a small and const. set of unique DNA strands can be used to create DNA origami arrays of increasing size and with arbitrary patterns. We illustrate this method, which we term 'fractal assembly', by producing DNA origami arrays with sizes of up to 0.5 square micrometres and with up to 8,704 pixels, allowing us to render images such as the Mona Lisa and a rooster. We find that self-assembly of the tiles into arrays is unaffected by changes in surface patterns on the tiles, and that the yield of the fractal assembly process corresponds to about 0.95m - 1 for arrays contg. m tiles. When used in conjunction with a software tool that we developed that converts an arbitrary pattern into DNA sequences and exptl. protocols, our assembly method is readily accessible and will facilitate the construction of sophisticated materials and devices with sizes similar to that of a bacterium using DNA nanostructures.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurjN&md5=a69eb157ae84c6ea4105a9f41da0680f

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      Wagenbauer, K. F. ; Sigl, C. ; Dietz, H. Gigadalton-Scale Shape-Programmable DNA Assemblies. Nature 2017, 552 , 78– 83,  DOI: 10.1038/nature24651

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      Gigadalton-scale shape-programmable DNA assemblies

      Wagenbauer, Klaus F.; Sigl, Christian; Dietz, Hendrik

      Nature (London, United Kingdom) (2017), 552 (7683), 78-83CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

      Natural biomol. assemblies such as mol. motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnol. aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy nos., positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nm in diam. and with at. masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nm in diam. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equil. to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurjM&md5=3872a1f31d5dd833f9b3ee7a9123de21

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      Ong, L. L. ; Hanikel, N. ; Yaghi, O. K. ; Grun, C. ; Strauss, M. T. ; Bron, P. ; Lai-Kee-Him, J. ; Schueder, F. ; Wang, B. ; Wang, P. ; Kishi, J. Y. ; Myhrvold, C. ; Zhu, A. ; Jungmann, R. ; Bellot, G. ; Ke, Y. ; Yin, P. Programmable Self-Assembly of Three-Dimensional Nanostructures from 10,000 Unique Components. Nature 2017, 552 , 72– 77,  DOI: 10.1038/nature24648

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      Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components

      Ong, Luvena L.; Hanikel, Nikita; Yaghi, Omar K.; Grun, Casey; Strauss, Maximilian T.; Bron, Patrick; Lai-Kee-Him, Josephine; Schueder, Florian; Wang, Bei; Wang, Pengfei; Kishi, Jocelyn Y.; Myhrvold, Cameron; Zhu, Allen; Jungmann, Ralf; Bellot, Gaetan; Ke, Yonggang; Yin, Peng

      Nature (London, United Kingdom) (2017), 552 (7683), 72-77CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

      Nucleic acids (DNA and RNA) are widely used to construct nanometer-scale structures with ever increasing complexity, with possible application in fields such as structural biol., biophysics, synthetic biol. and photonics. The nanostructures are formed through one-pot self-assembly, with early kDa-scale examples contg. typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufg. and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid contg. about 30,000 unique bricks and a 1-gigadalton rotationally sym. tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair 'voxels' that serves as a mol. canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this mol. canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFCjurvE&md5=96106fc32d528653317c767e96b078b2

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      Chen, X. ; Wang, Q. ; Peng, J. ; Long, Q. ; Yu, H. ; Li, Z. Self-Assembly of Large DNA Origami with Custom-Designed Scaffolds. ACS Appl. Mater. Interfaces 2018, 10 , 24344– 24348,  DOI: 10.1021/acsami.8b09222

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      Self-Assembly of Large DNA Origami with Custom-Designed Scaffolds

      Chen, Xiaoxing; Wang, Qian; Peng, Jin; Long, Qipeng; Yu, Hanyang; Li, Zhe

      ACS Applied Materials & Interfaces (2018), 10 (29), 24344-24348CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)

      As a milestone in DNA self-assembly, DNA origami has demonstrated powerful applications in many fields. However, the scarce availability of long single-stranded DNA (ssDNA) limits the size and sequences of DNA origami nanostructures, which in turn impedes the further development. In this study, the authors present a robust strategy to produce long circular ssDNA scaffold strands with custom-tailored lengths and sequences. These ssDNA products were then used as scaffolds for constructing various DNA origami nanostructures. This scalable method produces ssDNA at low cost with high purity and high yield, which can enable prodn. of custom-designed DNA origami for various applications.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht12mtbzP&md5=39f0c69906cbbc88d91a8babd4ab2819

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      Iinuma, R. ; Ke, Y. ; Jungmann, R. ; Schlichthaerle, T. ; Woehrstein, J. B. ; Yin, P. Polyhedra Self-Assembled from DNA Tripods and Characterized with 3d DNA-Paint. Science 2014, 344 , 65– 69,  DOI: 10.1126/science.1250944

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      Polyhedra Self-Assembled from DNA Tripods and Characterized with 3D DNA-PAINT

      Iinuma, Ryosuke; Ke, Yonggang; Jungmann, Ralf; Schlichthaerle, Thomas; Woehrstein, Johannes B.; Yin, Peng

      Science (Washington, DC, United States) (2014), 344 (6179), 65-69CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

      DNA self-assembly has produced diverse synthetic three-dimensional polyhedra. These structures typically have a mol. wt. no greater than 5 megadaltons. We report a simple, general strategy for one-step self-assembly of wireframe DNA polyhedra that are more massive than most previous structures. A stiff three-arm-junction DNA origami tile motif with precisely controlled angles and arm lengths was used for hierarchical assembly of polyhedra. We exptl. constructed a tetrahedron (20 megadaltons), a triangular prism (30 megadaltons), a cube (40 megadaltons), a pentagonal prism (50 megadaltons), and a hexagonal prism (60 megadaltons) with edge widths of 100 nm. The structures were visualized by means of transmission electron microscopy and three-dimensional DNA-PAINT super-resoln. fluorescent microscopy of single mols. in soln.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlt1eht7o%253D&md5=b6a44fdd0a45451b120729ab0849849b

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      He, Y. ; Ye, T. ; Su, M. ; Zhang, C. ; Ribbe, A. E. ; Jiang, W. ; Mao, C. Hierarchical Self-Assembly of DNA into Symmetric Supramolecular Polyhedra. Nature 2008, 452 , 198– 201,  DOI: 10.1038/nature06597

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      Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra

      He, Yu; Ye, Tao; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, Chengde

      Nature (London, United Kingdom) (2008), 452 (7184), 198-201CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

      DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute mol. computations. Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large nos. (hundreds) of unique DNA strands poses a challenging design problem. Here, the authors demonstrate a simple soln. to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger 3-dimensional structures. The authors test this hierarchical self-assembly concept with DNA mols. that form 3-point-star motifs, or tiles. By controlling the flexibility and concn. of the tiles, the one-pot assembly yields tetrahedra, dodecahedra or buckyballs that are tens of nanometers in size and comprised of four, twenty or sixty individual tiles, resp. The authors expect that this assembly strategy can be adapted to allow the fabrication of a range of relatively complex 3-dimensional structures.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjt1Gmsr4%253D&md5=25396f1220643c9ff865175aec2cfe81

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      Zhang, F. ; Jiang, S. ; Wu, S. ; Li, Y. ; Mao, C. ; Liu, Y. ; Yan, H. Complex Wireframe DNA Origami Nanostructures with Multi-Arm Junction Vertices. Nat. Nanotechnol. 2015, 10 , 779– 784,  DOI: 10.1038/nnano.2015.162

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      Complex wireframe DNA origami nanostructures with multi-arm junction vertices

      Zhang, Fei; Jiang, Shuoxing; Wu, Siyu; Li, Yulin; Mao, Chengde; Liu, Yan; Yan, Hao

      Nature Nanotechnology (2015), 10 (9), 779-784CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)

      Structural DNA nanotechnol. and the DNA origami technique, in particular, have provided a range of spatially addressable two- and three-dimensional nanostructures. These structures are, however, typically formed of tightly packed parallel helixes. The development of wireframe structures should allow the creation of novel designs with unique functionalities, but engineering complex wireframe architectures with arbitrarily designed connections between selected vertices in three-dimensional space remains a challenge. Here, we report a design strategy for fabricating finite-size wireframe DNA nanostructures with high complexity and programmability. In our approach, the vertices are represented by n × 4 multi-arm junctions (n = 2-10) with controlled angles, and the lines are represented by antiparallel DNA crossover tiles of variable lengths. Scaffold strands are used to integrate the vertices and lines into fully assembled structures displaying intricate architectures. To demonstrate the versatility of the technique, a series of two-dimensional designs including quasi-cryst. patterns and curvilinear arrays or variable curvatures, and three-dimensional designs including a complex snub cube and a reconfigurable Archimedean solid were constructed.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1ajtrrF&md5=cf327ed66012b1a7acafd73a4b078cf0

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      Bruetzel, L. K. ; Walker, P. U. ; Gerling, T. ; Dietz, H. ; Lipfert, J. Time-Resolved Small-Angle X-Ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch. Nano Lett. 2018, 18 , 2672– 2676,  DOI: 10.1021/acs.nanolett.8b00592

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      47

      Time-Resolved Small-Angle X-ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch

      Bruetzel, Linda K.; Walker, Philipp U.; Gerling, Thomas; Dietz, Hendrik; Lipfert, Jan

      Nano Letters (2018), 18 (4), 2672-2676CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)

      Self-assembled DNA structures enable creation of specific shapes at the nanometer-micrometer scale with mol. resoln. The construction of functional DNA assemblies will likely require dynamic structures that can undergo controllable conformational changes. DNA devices based on shape complementary stacking interactions have been demonstrated to undergo reversible conformational changes triggered by changes in ionic environment or temp. An exptl. unexplored aspect is how quickly conformational transitions of large synthetic DNA origami structures can actually occur. Here, the authors use time-resolved small-angle x-ray scattering to monitor large-scale conformational transitions of a two-state DNA origami switch in free soln. The DNA device switches from its open to its closed conformation upon addn. of MgCl2 in milliseconds, which is close to the theor. diffusive speed limit. In contrast, measurements of the dimerization of DNA origami bricks reveal much slower and concn.-dependent assembly kinetics. DNA brick dimerization occurs on a time scale of minutes to hours suggesting that the kinetics depend on local concn. and mol. alignment.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltFehtLk%253D&md5=1ddad808bab8e9232e53f61296ee8565

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      Stahl, E. ; Martin, T. G. ; Praetorius, F. ; Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angew. Chem., Int. Ed. 2014, 53 , 12735– 12740,  DOI: 10.1002/anie.201405991

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      Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions

      Stahl, Evi; Martin, Thomas G.; Praetorius, Florian; Dietz, Hendrik

      Angewandte Chemie, International Edition (2014), 53 (47), 12735-12740CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

      DNA has become a prime material for assembling complex three-dimensional objects that promise utility in various areas of application. However, achieving user-defined goals with DNA objects has been hampered by the difficulty to prep. them at arbitrary concns. and in user-defined soln. conditions. Here, we describe a method that solves this problem. The method is based on poly(ethylene glycol)-induced depletion of species with high mol. wt. We demonstrate that our method is applicable to a wide spectrum of DNA shapes and that it achieves excellent recovery yields of target objects up to 97 %, while providing efficient sepn. from non-integrated DNA strands. DNA objects may be prepd. at concns. up to the limit of soly., including the possibility for bringing DNA objects into a solid phase. Due to the fidelity and simplicity of our method we anticipate that it will help to catalyze the development of new types of applications that use self-assembled DNA objects.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvV2mtrrM&md5=5d749b225ce5405de5c89eeb1a5b1897

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      Bai, X. C. ; Martin, T. G. ; Scheres, S. H. ; Dietz, H. Cryo-Em Structure of a 3d DNA-Origami Object. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 , 20012– 20017,  DOI: 10.1073/pnas.1215713109

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      Cryo-EM structure of a 3D DNA-origami object

      Bai, Xiao-chen; Martin, Thomas G.; Scheres, Sjors H. W.; Dietz, Hendrik

      Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (49), 20012-20017, S20012/1-S20012/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

      A key goal for nanotechnol. is to design synthetic objects that may ultimately achieve functionalities known today only from natural macromol. complexes. Mol. self-assembly with DNA has shown potential for creating user-defined 3D scaffolds, but the level of attainable positional accuracy has been unclear. Here we report the cryo-EM structure and a full pseudoat. model of a discrete DNA object that is almost twice the size of a prokaryotic ribosome. The structure provides a variety of stable, previously undescribed DNA topologies for future use in nanotechnol. and exptl. evidence that discrete 3D DNA scaffolds allow the positioning of user-defined structural motifs with an accuracy that is similar to that obsd. in natural macromols. Thereby, our results indicate an attractive route to fabricate nanoscale devices that achieve complex functionalities by DNA-templated design steered by structural feedback.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslKnsQ%253D%253D&md5=cfcc06e1fdc335ffd88f92768825ce70

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      Engler, C. ; Kandzia, R. ; Marillonnet, S. A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS One 2008, 3 , e3647  DOI: 10.1371/journal.pone.0003647

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      A one pot, one step, precision cloning method with high throughput capability

      Engler Carola; Kandzia Romy; Marillonnet Sylvestre

      PloS one (2008), 3 (11), e3647 ISSN:.

      Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1cjhvV2ltA%253D%253D&md5=8910183a67aa28a0cc69522dcf7050b0

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      List, J. ; Falgenhauer, E. ; Kopperger, E. ; Pardatscher, G. ; Simmel, F. C. Long-Range Movement of Large Mechanically Interlocked DNA Nanostructures. Nat. Commun. 2016, 7 , 12414,  DOI: 10.1038/ncomms12414

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      Long-range movement of large mechanically interlocked DNA nanostructures

      List, Jonathan; Falgenhauer, Elisabeth; Kopperger, Enzo; Pardatscher, Guenther; Simmel, Friedrich C.

      Nature Communications (2016), 7 (), 12414CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

      Interlocked mols. such as catenanes and rotaxanes, connected only via mech. bonds have the ability to perform large-scale sliding and rotational movements, making them attractive components for the construction of artificial mol. machines and motors. We here demonstrate the realization of large, rigid rotaxane structures composed of DNA origami subunits. The structures can be easily modified to carry a mol. cargo or nanoparticles. By using multiple axle modules, rotaxane constructs are realized with axle lengths of up to 355 nm and a fuel/anti-fuel mechanism is employed to switch the rotaxanes between a mobile and a fixed state. We also create extended pseudo-rotaxanes, in which origami rings can slide along supramol. DNA filaments over several hundreds of nanometers. The rings can be actively moved and tracked using at. force microscopy.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlalsLvO&md5=c5392b3f9015e2cf1c4817a9cb38a047

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      Plesa, C. ; van Loo, N. ; Ketterer, P. ; Dietz, H. ; Dekker, C. Velocity of DNA During Translocation through a Solid-State Nanopore. Nano Lett. 2015, 15 , 732– 737,  DOI: 10.1021/nl504375c

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      Velocity of DNA during Translocation through a Solid-State Nanopore

      Plesa, Calin; van Loo, Nick; Ketterer, Philip; Dietz, Hendrik; Dekker, Cees

      Nano Letters (2015), 15 (1), 732-737CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)

      While understanding translocation of DNA through a solid-state nanopore is vital for exploiting its potential for sensing and sequencing at the single-mol. level, surprisingly little is known about the dynamics of the propagation of DNA through the nanopore. Here we use linear double-stranded DNA mols., assembled by the DNA origami technique, with markers at known positions in order to det. for the first time the local velocity of different segments along the length of the mol. We observe large intramol. velocity fluctuations, likely related to changes in the drag force as the DNA blob unfolds. Furthermore, we observe an increase in the local translocation velocity toward the end of the translocation process, consistent with a speeding up due to unfolding of the last part of the DNA blob. We use the velocity profile to est. the uncertainty in detg. the position of a feature along the DNA given its temporal location and demonstrate the error introduced by assuming a const. translocation velocity.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitV2jurfI&md5=14c812b7f31b188a6a190d87a1b1b763

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      Bell, N. A. ; Keyser, U. F. Digitally Encoded DNA Nanostructures for Multiplexed, Single-Molecule Protein Sensing with Nanopores. Nat. Nanotechnol. 2016, 11 , 645– 651,  DOI: 10.1038/nnano.2016.50

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      Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

      Bell, Nicholas A. W.; Keyser, Ulrich F.

      Nature Nanotechnology (2016), 11 (7), 645-651CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)

      The simultaneous detection of a large no. of different analytes is important in bionanotechnol. research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, highly multiplexed sensing of single mols. can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, the authors designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signaled by the presence or absence of multiple DNA dumbbell hairpins. A 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows the authors to simultaneously detect four different antibodies of the same isotype at nanomolar concn. levels.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xltlyisbo%253D&md5=e3de2085bf984a84e6208a2f41b1503f

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      Schickinger, M. ; Zacharias, M. ; Dietz, H. Tethered Multifluorophore Motion Reveals Equilibrium Transition Kinetics of Single DNA Double Helices. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 , E7512,  DOI: 10.1073/pnas.1800585115

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      Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices

      Schickinger, Matthias; Zacharias, Martin; Dietz, Hendrik

      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (32), E7512-E7521CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

      We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimol. reaction kinetics on the single-mol. level. Mol. binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 h. We applied the system to study in equil. reversible hybridization and dissocn. of complementary DNA single strands as a function of tether length, cation concn., and sequence. We obsd. up to hundreds of hybridization and dissocn. events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from mol. to mol., which enabled sepg. data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chem. truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissocn., is affected by cation concn., in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodn. stability, but different sequences, and obsd. strongly differing hybridization kinetics. Complementary full-atom mol.-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, resp., the fraction of strand collisions leading to successful nucleation events for duplex formation.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVanu7%252FK&md5=3b189b9257392137d9a6534ac6b4f5aa

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      Jungmann, R. ; Avendaño, M. S. ; Woehrstein, J. B. ; Dai, M. ; Shih, W. M. ; Yin, P. Multiplexed 3d Cellular Super-Resolution Imaging with DNA-Paint and Exchange-Paint. Nat. Methods 2014, 11 , 313,  DOI: 10.1038/nmeth.2835

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      Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT

      Jungmann, Ralf; Avendano, Maier S.; Woehrstein, Johannes B.; Dai, Mingjie; Shih, William M.; Yin, Peng

      Nature Methods (2014), 11 (3), 313-318CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

      Super-resoln. fluorescence microscopy is a powerful tool for biol. research, but obtaining multiplexed images for a large no. of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topog.) for simple and easy-to-implement multiplexed super-resoln. imaging that achieves sub-10-nm spatial resoln. in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We exptl. demonstrate ten-color super-resoln. imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Slu74%253D&md5=dbefcad33ee566c8f640258d28169420

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      Gibson, D. G. ; Young, L. ; Chuang, R.-Y. ; Venter, J. C. ; Hutchison, C. A., III ; Smith, H. O. Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases. Nat. Methods 2009, 6 , 343,  DOI: 10.1038/nmeth.1318

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      Enzymatic assembly of DNA molecules up to several hundred kilobases

      Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.

      Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

      The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVemsbw%253D&md5=46284924c7d73c47cfb490983338e480

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      Luke, J. ; Carnes, A. E. ; Hodgson, C. P. ; Williams, J. A. Improved Antibiotic-Free DNA Vaccine Vectors Utilizing a Novel Rna Based Plasmid Selection System. Vaccine 2009, 27 , 6454– 6459,  DOI: 10.1016/j.vaccine.2009.06.017

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      Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

      Luke, Jeremy; Carnes, Aaron E.; Hodgson, Clague P.; Williams, James A.

      Vaccine (2009), 27 (46), 6454-6459CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)

      To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermn. manufg. (>1 g/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlSqsr3K&md5=d91ccc3df965744d66956dd5905e8164

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      Douglas, S. M. ; Chou, J. J. ; Shih, W. M. DNA-Nanotube-Induced Alignment of Membrane Proteins for Nmr Structure Determination. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 , 6644– 6648,  DOI: 10.1073/pnas.0700930104

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      DNA-nanotube-induced alignment of membrane proteins for NMR structure determination

      Douglas, Shawn M.; Chou, James J.; Shih, William M.

      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (16), 6644-6648CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

      Membrane proteins are encoded by 20-35% of genes but represent <1% of known protein structures to date. Thus, improved methods for membrane-protein structure detn. are of crit. importance. Residual dipolar couplings (RDCs), commonly measured for biol. macromols. weakly aligned by liq.-cryst. media, are important global angular restraints for NMR structure detn. For α-helical membrane proteins >15 kDa in size, Nuclear-Overhauser effect-derived distance restraints are difficult to obtain, and RDCs could serve as the main reliable source of NMR structural information. In many of these cases, RDCs would enable full structure detn. that otherwise would be impossible. However, none of the existing liq.-cryst. media used to align water-sol. proteins are compatible with the detergents required to solubilize membrane proteins. The authors report the design and construction of a detergent-resistant liq. crystal of 0.8μm-long DNA-nanotubes that can be used to induce weak alignment of membrane proteins. The nanotubes are heterodimers of 0.4μm-long six-helix bundles each self-assembled from a 7.3-kb scaffold strand and >170 short oligonucleotide staple strands. The authors show that the DNA-nanotube liq. crystal enables the accurate measurement of backbone NH and CαHα RDCs for the detergent-reconstituted ζ-ζ transmembrane domain of the T cell receptor. The measured RDCs validate the high-resoln. structure of this transmembrane dimer. The authors anticipate that this medium will extend the advantages of weak alignment to NMR structure detn. of a broad range of detergent-solubilized membrane proteins.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkvFWjs7k%253D&md5=ad5031d791858f4435c5dd65ae72beb9

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      Castro, C. E. ; Kilchherr, F. ; Kim, D. N. ; Shiao, E. L. ; Wauer, T. ; Wortmann, P. ; Bathe, M. ; Dietz, H. A Primer to Scaffolded DNA Origami. Nat. Methods 2011, 8 , 221– 229,  DOI: 10.1038/nmeth.1570

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      A primer to scaffolded DNA origami

      Castro, Carlos Ernesto; Kilchherr, Fabian; Kim, Do-Nyun; Shiao, Enrique Lin; Wauer, Tobias; Wortmann, Philipp; Bathe, Mark; Dietz, Hendrik

      Nature Methods (2011), 8 (3), 221-229CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

      A review. Mol. self-assembly with scaffolded DNA origami enables building custom-shaped nanometer-scale objects with mol. wts. in the megadalton regime. Here the authors provide a practical guide for design and assembly of scaffolded DNA origami objects. The authors also introduce a computational tool for predicting the structure of DNA origami objects and provide information on the conditions under which DNA origami objects can be expected to maintain their structure.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXisFentb0%253D&md5=623326f738b8ea373dd5cb4906994fcc

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      Kim, D. N. ; Kilchherr, F. ; Dietz, H. ; Bathe, M. Quantitative Prediction of 3d Solution Shape and Flexibility of Nucleic Acid Nanostructures. Nucleic Acids Res. 2012, 40 , 2862– 2868,  DOI: 10.1093/nar/gkr1173

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      Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructures

      Kim, Do-Nyun; Kilchherr, Fabian; Dietz, Hendrik; Bathe, Mark

      Nucleic Acids Research (2012), 40 (7), 2862-2868CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

      DNA nanotechnol. enables the programmed synthesis of intricate nanometer-scale structures for diverse applications in materials and biol. science. Precise control over the 3D soln. shape and mech. flexibility of target designs is important to achieve desired functionality. Because exptl. validation of designed nanostructures is time-consuming and cost-intensive, predictive phys. models of nanostructure shape and flexibility have the capacity to enhance dramatically the design process. Here, the authors significantly extend and exptl. validate a computational modeling framework for DNA origami previously presented as CanDo A primer to scaffolded DNA origami. 3D soln. shape and flexibility are predicted from basepair connectivity maps now accounting for nicks in the DNA double helix, entropic elasticity of single-stranded DNA, and distant crossovers required to model wireframe structures, in addn. to previous modeling that accounted only for the canonical twist, bend and stretch stiffness of double-helical DNA domains. Systematic exptl. validation of nanostructure flexibility mediated by internal crossover d. probed using a 32-helix DNA bundle demonstrates for the first time that our model not only predicts the 3D soln. shape of complex DNA nanostructures but also their mech. flexibility. Thus, our model represents an important advance in the quant. understanding of DNA-based nanostructure shape and flexibility, and we anticipate that this model will increase significantly the no. and variety of synthetic nanostructures designed using nucleic acids.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xls1Snu7k%253D&md5=5570389892b346fe6198edd9361e21e1

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      Pfitzner, E. ; Wachauf, C. ; Kilchherr, F. ; Pelz, B. ; Shih, W. M. ; Rief, M. ; Dietz, H. Rigid DNA Beams for High-Resolution Single-Molecule Mechanics. Angew. Chem., Int. Ed. 2013, 52 , 7766– 7771,  DOI: 10.1002/anie.201302727

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      Rigid DNA Beams for High-Resolution Single-Molecule Mechanics

      Pfitzner, Emanuel; Wachauf, Christian; Kilchherr, Fabian; Pelz, Benjamin; Shih, William M.; Rief, Matthias; Dietz, Hendrik

      Angewandte Chemie, International Edition (2013), 52 (30), 7766-7771CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

      This paper developed DNA helix bundles as linker system for single-mol. mech. assays.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVersr0%253D&md5=cdb2d328b617ae95fe26569f506650a2

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      Scheres, S. H. ; Nunez-Ramirez, R. ; Sorzano, C. O. ; Carazo, J. M. ; Marabini, R. Image Processing for Electron Microscopy Single-Particle Analysis Using Xmipp. Nat. Protoc. 2008, 3 , 977– 990,  DOI: 10.1038/nprot.2008.62

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      Image processing for electron microscopy single-particle analysis using XMIPP

      Scheres, Sjors H. W.; Nunez-Ramirez, Rafael; Sorzano, Carlos O. S.; Carazo, Jose Maria; Marabini, Roberto

      Nature Protocols (2008), 3 (6), 977-990CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)

      The authors describe a collection of standardized image processing protocols for electron microscopy single-particle anal. using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through max.-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with min. efforts in learning about the details of this comprehensive package. Examples of applications are presented for a neg. stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvVemtrY%253D&md5=4528b58686ad6c8bea731b9b31e1e976

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      Kimanius, D. ; Forsberg, B. O. ; Scheres, S. H. ; Lindahl, E. Accelerated Cryo-Em Structure Determination with Parallelisation Using Gpus in Relion-2. eLife 2016, 5 , 18722,  DOI: 10.7554/eLife.18722

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      Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2

      Kimanius, Dari; Forsberg, Bjoern O.; Scheres, Sjors H. W.; Lindahl, Erik

      eLife (2016), 5 (), e18722/1-e18722/21CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)

      By reaching near-at. resoln. for a wide range of specimens, single-particle cryo-EM structure detn. is transforming structural biol. However, the necessary calcns. come at large computational costs, which has introduced a bottleneck that is currently limiting throughput and the development of new methods. Here, we present an implementation of the RELION image processing software that uses graphics processors (GPUs) to address the most computationally intensive steps of its cryo-EM structure detn. workflow. Both image classification and high-resoln. refinement have been accelerated more than an order-of-magnitude, and template-based particle selection has been accelerated well over two orders-of-magnitude on desktop hardware. Memory requirements on GPUs have been reduced to fit widely available hardware, and we show that the use of single precision arithmetic does not adversely affect results. This enables high-resoln. cryo-EM structure detn. in a matter of days on a single workstation.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVahtr3I&md5=93c053b864a278979f74bac0a0313321

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      Zivanov, J. ; Nakane, T. ; Forsberg, B. O. ; Kimanius, D. ; Hagen, W. J. ; Lindahl, E. ; Scheres, S. H. New Tools for Automated High-Resolution Cryo-Em Structure Determination in Relion-3. eLife 2018, 7 , 42166,  DOI: 10.7554/eLife.42166

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      New tools for automated high-resolution cryo-EM structure determination in RELION-3

      Zivanov, Jasenko; Nakane, Takanori; Forsberg, Bjoern O.; Kimanius, Dari; Hagen, Wim Jh; Lindahl, Erik; Scheres, Sjors Hw

      eLife (2018), 7 (), e42166/1-e42166/22CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)

      Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addn. to GPU support, which provides flexibility in use of resources and avoids memory limitations. Ref.-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2Dclassification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estd. beam tilt provides higher resoln. reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resoln. for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beaminduced motion correction it leads to resoln. improvements of 0.2-0.7 Å compared to previous RELION versions.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlyqsrbL&md5=6ad79861243c29459be9b515e21c4b0e

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      Zheng, S. Q. ; Palovcak, E. ; Armache, J. P. ; Verba, K. A. ; Cheng, Y. ; Agard, D. A. Motioncor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy. Nat. Methods 2017, 14 , 331– 332,  DOI: 10.1038/nmeth.4193

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      MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

      Zheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul; Verba, Kliment A.; Cheng, Yifan; Agard, David A.

      Nature Methods (2017), 14 (4), 331-332CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)

      A review on anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Here we describe MotionCor2, a software tool for anisotropic correction of beam-induced motion. Overall, MotionCor2 is extremely robust and sufficiently accurate at correcting local motions so that the very time-consuming and computationally intensive particle polishing in RELION can be skipped, importantly, it also works on a wide range of data sets, including cryo tomog. tilt series.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjt1ags7g%253D&md5=5f4e225ef8123dacd8475d526175e1d2

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      Mindell, J. A. ; Grigorieff, N. Accurate Determination of Local Defocus and Specimen Tilt in Electron Microscopy. J. Struct. Biol. 2003, 142 , 334– 347,  DOI: 10.1016/S1047-8477(03)00069-8

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      Accurate determination of local defocus and specimen tilt in electron microscopy

      Mindell Joseph A; Grigorieff Nikolaus

      Journal of structural biology (2003), 142 (3), 334-47 ISSN:1047-8477.

      Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10nm, whereas tilt axis and tilt angle are determined with an error of about 2 degrees and 1 degrees, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6 degrees, respectively.

      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3s3lslenug%253D%253D&md5=4def54fed3d1c3f984432ab128d4f953

• Supporting Information

  Supporting Information

  ---

  The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b01025.

  • Detailed notes on generating a design-specific scaffold sequence with the scaffold smith, overview of methods for phage-based production of ssDNA scaffolds, workflow for the design, cloning, and production of a custom scaffold, supplementary experimental data and design schematics (PDF)

  • Sequence information for scaffolds and plasmids used in this work (XLSX)

  • Tools used in this work to design and analyze scaffold sequences (ZIP)

  • nn9b01025_si_001.pdf (12.78 MB)
  • nn9b01025_si_002.xlsx (71.55 kb)
  • nn9b01025_si_003.zip (1.07 MB)

  
  

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Autonomously Designed Free Form 2d Dna Origami

Source: https://pubs.acs.org/doi/10.1021/acsnano.9b01025

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