Search

Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures 1-3 . By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates 4 conditional fluorophores derived from GFP. To extend previous structural studies 5,6 of fluorogenic RNAs, GFP and other fluorescent proteins 7 to DNA, we characterize Lettuce-fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen 8 in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding-including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA-and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Nanoscale structures demonstrate considerable potential utility in the construction of nanorobots, nanomachines, and many other devices. In this study, a hexagonal DNA origami ring was assembled and visualized via atomic force microscopy. The DNA origami shape could be programmed into either a hexagonal or linear shape with an open or folded pattern. The flexible origami was robust and switchable for dynamic pattern recognition. Its edges were folded by six bundles of DNA helices, which could be opened or folded in a honeycomb shape. Additionally, the edges were programmed into a concave-convex pattern, which enabled linkage between the origami and dipolymers. Furthermore, biotin-streptavidin labels were embedded at each edge for nanoscale calibration. The atomic force microscopy results demonstrated the stability and high-yield of the flexible DNA origami ring. The polymorphous nanostructure is useful for dynamic nano-construction and calibration of structural probes or sensors., (© 2021 IOP Publishing Ltd.)

DNA covers the genetic information in all living organisms. Numerous intrinsic and extrinsic factors may influence the local structure of the DNA molecule or compromise its integrity. Detailed understanding of structural modifications of DNA resulting from interactions with other molecules and surrounding environment is of central importance for the future development of medicine and pharmacology. In this paper, we review the recent achievements in research on DNA structure at nanoscale. In particular, we focused on the molecular structure of DNA revealed by high-resolution AFM (Atomic Force Microscopy) imaging at liquid/solid interfaces. Such detailed structural studies were driven by the technical developments made in SPM (Scanning Probe Microscopy) techniques. Therefore, we describe here the working principles of AFM modes allowing high-resolution visualization of DNA structure under native (liquid) environment. While AFM provides well-resolved structure of molecules at nanoscale, it does not reveal the chemical structure and composition of studied samples. The simultaneous information combining the structural and chemical details of studied analyte allows achieve a comprehensive picture of investigated phenomenon. Therefore, we also summarize recent molecular spectroscopy studies, including Tip-Enhanced Raman Spectroscopy (TERS), on the DNA structure and its structural rearrangements.

An increasing number of density maps of macromolecular structures, including proteins and DNA/RNA complexes, have been determined by cryo-electron microscopy (cryo-EM). Although lately maps at a near-atomic resolution are routinely reported, there are still substantial fractions of maps determined at intermediate or low resolutions, where extracting structure information is not trivial. Here, we report a new computational method, Emap2sec+, which identifies DNA or RNA as well as the secondary structures of proteins in cryo-EM maps of 5 to 10 Å resolution. Emap2sec+ employs the deep Residual convolutional neural network. Emap2sec+ assigns structural labels with associated probabilities at each voxel in a cryo-EM map, which will help structure modeling in an EM map. Emap2sec+ showed stable and high assignment accuracy for nucleotides in low resolution maps and improved performance for protein secondary structure assignments than its earlier version when tested on simulated and experimental maps.

Nucleic acid-binding proteins are traditionally divided into two categories: With the ability to bind DNA or RNA. In the light of new knowledge, such categorizing should be overcome because a large proportion of proteins can bind both DNA and RNA. Another even more important features of nucleic acid-binding proteins are so-called sequence or structure specificities. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs (zinc-fingers, leucine zipper, helix-turn-helix, or helix-loop-helix). In contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures (G-quadruplexes, i-motifs, triplexes, cruciforms, left-handed DNA/RNA form, and others). Finally, there are also proteins recognizing both sequence and local structural properties of nucleic acids (e.g., famous tumor suppressor p53). In this mini-review, we aim to summarize current knowledge about the amino acid composition of various types of nucleic acid-binding proteins with a special focus on significant enrichment and/or depletion in each category.

Since the pioneering work of Ned Seeman in the early 1980s, the use of the DNA molecule as a construction material experienced a rapid growth and led to the establishment of a new field of science, nowadays called structural DNA nanotechnology. Here, the self-recognition properties of DNA are employed to build micrometer-large molecular objects with nanometer-sized features, thus bridging the nano- to the microscopic world in a programmable fashion. Distinct design strategies and experimental procedures have been developed over the years, enabling the realization of extremely sophisticated structures with a level of control that approaches that of natural macromolecular assemblies. Nevertheless, our understanding of the building process, i.e., what defines the route that goes from the initial mixture of DNA strands to the final intertwined superstructure, is, in some cases, still limited. In this review, we describe the main structural and energetic features of DNA nanoconstructs, from the simple Holliday junction to more complicated DNA architectures, and present the theoretical frameworks that have been formulated until now to explain their self-assembly. Deeper insights into the underlying principles of DNA self-assembly may certainly help us to overcome current experimental challenges and foster the development of original strategies inspired to dissipative and evolutive assembly processes occurring in nature.

Beyond the consensus definition of G-quadruplex-forming motifs with tracts of continuous guanines, G-quadruplexes harboring bulges in the G-tetrad core are prevalent in the human genome. Here, we study the incorporation of a duplex hairpin within a bulge of a G-quadruplex. The NMR solution structure of a G-quadruplex containing a duplex bulge was resolved, revealing the structural details of the junction between the duplex bulge and the G-quadruplex. Unexpectedly, instead of an orthogonal connection the duplex stem was observed to stack below the G-quadruplex forming a unique quadruplex-duplex junction. Breaking up of the immediate base pair step at the junction, coupled with a narrowing of the duplex groove within the context of the bulge, led to a progressive transition between the quadruplex and duplex segments. This study revealed that a duplex bulge can be formed at various positions of a G-quadruplex scaffold. In contrast to a non-structured bulge, the stability of a G-quadruplex slightly increases with an increase in the duplex bulge size. A G-quadruplex structure containing a duplex bulge of up to 33 nt in size was shown to form, which was much larger than the previously reported 7-nt bulge. With G-quadruplexes containing duplex bulges representing new structural motifs with potential biological significance, our findings would broaden the definition of potential G-quadruplex-forming sequences., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A hybrid-type G-quadruplex is modified with LNA (locked nucleic acid) and 2'-F-riboguanosine in various combinations at the two syn positions of its third antiparallel G-tract. LNA substitution in the central tetrad causes a complete rearrangement to either a V-loop or antiparallel structure, depending on further modifications at the 5'-neighboring site. In the two distinct structural contexts, LNA-induced stabilization is most effective compared to modifications with other G surrogates, highlighting a potential use of LNA residues for designing not only parallel but various more complex G4 structures. For instance, the conventional V-loop is a structural element strongly favored by an LNA modification at the V-loop 3'-end in contrast with an alternative V-loop, clearly distinguishable by altered conformational properties and base-backbone interactions as shown in a detailed analysis of V-loop structures., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA nanotechnology is a rapidly advancing field, which increasingly attracts interest in many different disciplines, such as medicine, biotechnology, physics and biocomputing. The increasing complexity of novel applications requires significant computational support for the design, modelling and analysis of DNA nanostructures. However, current in silico design tools have not been developed in view of these new applications and their requirements. Here, we present Adenita, a novel software tool for the modelling of DNA nanostructures in a user-friendly environment. A data model supporting different DNA nanostructure concepts (multilayer DNA origami, wireframe DNA origami, DNA tiles etc.) has been developed allowing the creation of new and the import of existing DNA nanostructures. In addition, the nanostructures can be modified and analysed on-the-fly using an intuitive toolset. The possibility to combine and re-use existing nanostructures as building blocks for the creation of new superstructures, the integration of alternative molecules (e.g. proteins, aptamers) during the design process, and the export option for oxDNA simulations are outstanding features of Adenita, which spearheads a new generation of DNA nanostructure modelling software. We showcase Adenita by re-using a large nanorod to create a new nanostructure through user interactions that employ different editors to modify the original nanorod., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most of eukaryotic cellular DNA is packed in nucleosome core particles (NCPs), in which the DNA (DNA NCP ) is wrapped around histones. The influence of this organization on the intrinsic local dynamics of DNA is largely unknown, in particular because capturing such information from experiments remains notoriously challenging. Given the importance of dynamical properties in DNA functions, we addressed this issue using CHARMM36 MD simulations of a nucleosome containing the NCP positioning 601 sequence and four related free dodecamers. Comparison between DNA NCP and free DNA reveals a limited impact of the dense DNA-histone interface on correlated motions of dinucleotide constituents and on fluctuations of inter base pair parameters. A characteristic feature intimately associated with the DNA NCP super-helical path is a set of structural periodicities that includes a marked alternation of regions enriched in backbone BI and BII conformers. This observation led to uncover a convincing correspondence between the sequence effect on BI/BII propensities in both DNA NCP and free DNA, strengthening the idea that the histone preference for particular DNA sequences relies on those intrinsic structural properties. These results offer for the first time a detailed view of the DNA dynamical behavior within NCP. They show in particular that the DNA NCP dynamics is substantial enough to preserve the ability to structurally adjust to external proteins, for instance remodelers. Also, fresh structural arguments highlight the relevance of relationships between DNA sequence and structural properties for NCP formation. Overall, our work offers a more rational framework to approach the functional, biological roles of NCP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops-c-shapes or s-shapes-that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA cages can be joined together to make larger 3D nanostructures on which molecular electronic circuits and tiny containers are built for drug delivery. The mathematical models for these promising nanomaterials play important roles in clarifying their assembly mechanism and understanding their structures. In this study, we propose a mathematical and computer method to construct permissible topological structures with double-helical edges for a triangular bipyramid and pentangular bipyramid. Furthermore, we remove the same topological links, without eliminating the nonrepeated ones for a triangular bipyramid and pentangular bipyramid. By analyzing characteristics of these unique links, some self-assembly and statistic rules are discussed. This study may obtain some new insights into the DNA assembly from the viewpoint of mathematics, promoting the comprehending and design efficiency of DNA polyhedra with required topological structures., Competing Interests: The author declares that there are no conflicts of interest., (Copyright © 2020 Tao Deng.)

Long noncoding RNA molecules (lncRNAs) are estimated to account for the majority of eukaryotic genomic transcripts, and have been associated with multiple diseases in humans. However, our understanding of their structure-function relationships is scarce, with structural evidence coming mostly from indirect biochemical approaches or computational predictions. Here we describe direct visualization of the lncRNA HOTAIR (HOx Transcript AntIsense RNA) using atomic force microscopy (AFM) in nucleus-like conditions at 37°. Our observations reveal that HOTAIR has a discernible, although flexible, shape. Fast AFM scanning enabled the quantification of the motion of HOTAIR, and provided visual evidence of physical interactions with genomic DNA segments. Our report provides a biologically plausible description of the anatomy and intrinsic properties of HOTAIR, and presents a framework for studying the structural biology of lncRNAs., (© 2020 Spokoini-Stern et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

G-rich DNA sequences with tracts of three or more continuous guanines (G≥3) are known to have high propensity to adopt stable G-quadruplex (G4) structures. Bioinformatic analyses suggest high prevalence of G-rich sequences with short G-tracts (G≤2) in the human genome. However, due to limited structural studies, the folding principles of such sequences remain largely unexplored and hence poorly understood. Here, we present the solution NMR structure of a sequence named AT26 consisting of irregularly spaced G2 tracts and two isolated single guanines. The structure is a four-layered G4 featuring two bi-layered blocks, locked between themselves in an unprecedented fashion making it a stable scaffold. In addition to edgewise and propeller-type loops, AT26 also harbors two V-shaped loops: a 2-nt V-shaped loop spanning two G-tetrad layers and a 0-nt V-shaped loop spanning three G-tetrad layers, which are named as VS- and VR-loop respectively, based on their distinct structural features. The intra-lock motif can be a basis for extending the G-tetrad core and a very stable intra-locked G4 can be formed by a sequence with G-tracts of various lengths including several G2 tracts. Findings from this study will aid in understanding the folding of G4 topologies from sequences containing irregularly spaced multiple short G-tracts., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bending of double-stranded (ds) DNA plays a crucial role in many important biological processes and is relevant for nanotechnological applications. Among all the elements that have been studied in relation to dsDNA bending, A-tracts stand out as one of the most controversial. The "ApA wedge" theory was disproved when a series of linear polynucleotides containing phased 5'-A 4 T 4 -3' or 5'-T 4 A 4 -3' runs were shown to be bent or straight, respectively, and crystallographic evidence revealed that A-tracts are unbent. Furthermore, some of the smallest dsDNA minicircles described to date (~ 100 bp in size) lack A-tracts and are subjected to varying levels of torsional stress. Representative DNA sequences from this experimental background were modeled in atomic detail and their dynamic behavior was simulated over hundreds of nanoseconds using the AMBER force field ParmBSC1. Subsequent analysis of the resulting trajectories allowed us to (i) unambiguously establish the location of the bends in all cases; (ii) identify the structural elements that are directly responsible for the macroscopically detected curvature; and (iii) reveal the importance not only of coherently summing the effects of the bending elements when they are in synchrony with the natural repeat of the helix (i.e. separated by an integral number of helical turns) but also when alternated with a half-integral separation of opposite effects. We conclude that the major determinant of the macroscopically observed bending is the proper grouping and phasing of the positive roll imposed by pyrimidine-purine (YR) steps and the negative or null roll characteristic of RY steps and A-tracts, respectively. This conclusion is in very good agreement with the structural parameters experimentally derived for much smaller DNA molecules either alone or as found in DNA-protein complexes. We expect that this work will pave the way for future studies on drug-induced DNA bending, DNA shape readout by transcription factors, structure of circular extrachromosomal DNA, and custom design of curved DNA origami scaffolds.

HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves ('↓') to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded β sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the 'PD-D/EXK' superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5'-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Herein, we report self-assembled reconfigurable plasmonic diastereomers based on DNA nanotechnology. Up to three plasmonic chiral centers were organized by dynamic DNA origami platforms. Meanwhile, each chiral center could be individually controlled to switch between left-handed and right-handed states. Thus, complex and reconfigurable chiral plasmonic diastereomers with eight plasmonic stereoisomers were achieved, driven by programmed DNA reactions. With these plasmonic diastereomers, we demonstrated the existence of strong cross-talk near-field coupling among chiral centers, and the coupling of chiral centers could substantially contribute to the overall CD signals. Our work provides an important bottom-up approach for building complex and dynamic chiral plasmonics and for probing the interactions of plasmonic chiral centers.

Wireframe DNA origami has emerged as a powerful approach to fabricating nearly arbitrary 2D and 3D geometries at the nanometer-scale. Complex scaffold and staple routing needed to design wireframe DNA origami objects, however, render fully automated, geometry-based sequence design approaches essential for their synthesis. And wireframe DNA origami structural fidelity can be limited by wireframe edges that are composed only of one or two duplexes. Here we introduce a fully automated computational approach that programs 2D wireframe origami assemblies using honeycomb edges composed of six parallel duplexes. These wireframe assemblies show enhanced structural fidelity from electron microscopy-based measurement of programmed angles compared with identical geometries programmed using dual-duplex edges. Molecular dynamics provides additional theoretical support for the enhanced structural fidelity observed. Application of our top-down sequence design procedure to a variety of complex objects demonstrates its broad utility for programmable 2D nanoscale materials.

DNA origami tiles provide nanostructures for the spatial and temporal control of functional loads on the scaffolds. Here we introduce the active generation of nanoholes in the origami scaffolds using DNAzymes or light as triggers and present the programmed and switchable catalysis in the resulting nanocavities. We engineer "window" domains locked into the origami scaffolds by substrates of the Zn 2+ -ion- or Pb 2+ -ion-dependent DNAzymes. Using Zn 2+ ions and/or Pb 2+ ions, the programmed unlocking of the "window" domains is demonstrated. The tailored functionalization of the origami scaffolds allows the programmed operation of catalytic processes in the confined nanocavities. Also, the "window" domain is integrated into the origami scaffold using photoisomerizable azobenzene-modified locks. The cyclic photoisomerization of the locks between the cis and trans states leads to a reversible opening and closure of the nanoholes and to the cyclic light-induced switching of catalytic processes in the nanocavities.

We describe the triggered assembly of a bioinspired DNA origami meshwork on a lipid membrane. DNA triskelia, three-armed DNA origami nanostructures inspired by the membrane-modifying protein clathrin, are bound to lipid mono- and bilayers using cholesterol anchors. Polymerization of triskelia, triggered by the addition of DNA staples, links triskelion arms to form a mesh. Using transmission electron microscopy, we observe nanoscale local deformation of a lipid monolayer induced by triskelion polymerization that is reminiscent of the formation of clathrin-coated pits. We also show that the polymerization of triskelia bound to lipid bilayers modifies interactions between them, inhibiting the formation of a synapse between giant unilamellar vesicles and a supported lipid bilayer.

Much recent attention has been focused on small organic molecules binding to non-canonical structures of nucleic acids, especially, RNA. The Human Genome Project and the ENCODE (encyclopedia of DNA elements) project revealed that more than 75% of the human genome is transcribed into RNA, while only ∼3% of the human genome encodes a protein. These non-protein-coding RNAs are thought to play significant roles in many cellular processes and are promising targets for drug discovery. Emerging roles of the non-coding RNAs in a variety of diseases provides enormous opportunities for pharmaceutical research on developing drugs targeting undruggable and rare diseases. During the last two decades, our laboratory has focused attention on small molecules binding to non-canonical DNA and RNA structures, especially to mismatched base pairs. Mismatch binding ligands (MBLs) we have developed are synthetic molecules designed in silico based on the hypothesis of hydrogen-bonding and semi-intercalation to DNA and RNA. Most of MBLs consists of two heterocycles having hydrogen bonding surfaces fully or partially complementary to that of nucleotide bases. In our design, each heterocycle binds to one of the mismatched bases by hydrogen bonding to form pseudo-base pairs, which would be stacked with the adjacent base pairs. The hypothesis allows us in principle to design small molecules binding to any mismatched base pairs, but it turned out not to be the case in reality. However, we have so far succeeded in developing several MBLs binding to DNA and RNA motifs of biological significance. In this review, we shall describe the hypothesis of molecular design of MBLs and its outcome regarding RNA targeting., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DNA origami nano-objects are usually designed around generic single-stranded "scaffolds". Many properties of the target object are determined by details of those generic scaffold sequences. Here, we enable designers to fully specify the target structure not only in terms of desired 3D shape but also in terms of the sequences used. To this end, we built design tools to construct scaffold sequences de novo based on strand diagrams, and we developed scalable production methods for creating design-specific scaffold strands with fully user-defined sequences. We used 17 custom scaffolds having different lengths and sequence properties to study the influence of sequence redundancy and sequence composition on multilayer DNA origami assembly and to realize efficient one-pot assembly of multiscaffold DNA origami objects. Furthermore, as examples for functionalized scaffolds, we created a scaffold that enables direct, covalent cross-linking of DNA origami via UV irradiation, and we built DNAzyme-containing scaffolds that allow postfolding DNA origami domain separation.

We use the oxDNA coarse-grained model to provide a detailed characterization of the fundamental structural properties of DNA origami, focussing on archetypal 2D and 3D origami. The model reproduces well the characteristic pattern of helix bending in a 2D origami, showing that it stems from the intrinsic tendency of anti-parallel four-way junctions to splay apart, a tendency that is enhanced both by less screened electrostatic interactions and by increased thermal motion. We also compare to the structure of a 3D origami whose structure has been determined by cryo-electron microscopy. The oxDNA average structure has a root-mean-square deviation from the experimental structure of 8.4 Å, which is of the order of the experimental resolution. These results illustrate that the oxDNA model is capable of providing detailed and accurate insights into the structure of DNA origami, and has the potential to be used to routinely pre-screen putative origami designs and to investigate the molecular mechanisms that regulate the properties of DNA origami., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Self-assembled DNA origami nanostructures have a high degree of programmable spatial control that enables nanoscale molecular manipulations. A surface-tethered, flexible DNA nanomesh is reported herein which spontaneously undergoes sharp, dynamic conformational transitions under physiological conditions. The transitions occur between two major macrostates: a spread state dominated by the interaction between the DNA nanomesh and the BSA/streptavidin surface and a surface-avoiding contracted state. Due to a slow rate of stochastic transition events on the order of tens of minutes, the dynamic conformations of individual structures can be detected in situ with DNA PAINT microscopy. Time series localization data with automated imaging processing to track the dynamically changing radial distribution of structural markers are combined. Conformational distributions of tethered structures in buffers with elevated pH exhibit a calcium-dependent domination of the spread state. This is likely due to electrostatic interactions between the structures and immobilized surface proteins (BSA and streptavidin). An interaction is observed in solution under similar buffer conditions with dynamic light scattering. Exchanging between solutions that promote one or the other state leads to in situ sample-wide transitions between the states. The technique herein can be a useful tool for dynamic control and observation of nanoscale interactions and spatial relationships., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The self-organized formation of regular patterns is not only a fascinating topic encountered in a multitude of natural and artificial systems, but also presents a versatile and powerful route toward large-scale nanostructure assembly and materials synthesis. The hierarchical, interface-assisted assembly of DNA origami nanostructures into regular, 2D lattices represents a particularly promising example, as the resulting lattices may exhibit an astonishing degree of order and can be further utilized as masks in molecular lithography. Here, we thus investigate the development of order in such 2D DNA origami lattices assembled on mica surfaces by employing in situ high-speed atomic force microscopy imaging. DNA origami lattice formation is found to resemble thin-film growth in several aspects. In particular, the Na + /Mg 2+ ratio controls DNA origami adsorption, surface diffusion, and desorption, and is thus equivalent in its effects to substrate temperature which controls adatom dynamics in thin-film deposition. Consequently, we observe a pronounced dependence of lattice order on Na + concentration. At low Na + concentrations, lattice formation resembles random deposition and results in unordered monolayers, whereas very high Na + concentrations are accompanied by rapid diffusion and especially DNA origami desorption, which prevent lattice formation. At intermediate Na + concentrations, highly ordered DNA origami lattices are obtained that display an intricate symmetry, stemming from the complex shape of the employed Rothemund triangle. Nevertheless, even under such optimized conditions, the lattices display a considerable number of defects, including grain boundaries, point and line defects, and screw-like dislocations. By monitoring the dynamics of selected lattice defects, we identify mechanisms that limit the obtainable degree of lattice order. Possible routes toward further increasing lattice order by postassembly annealing are discussed.

Molecular knots represent one of the most extraordinary topological structures in biological polymers. Creating highly knotted nanostructures with well-defined and sophisticated geometries and topologies remains challenging. Here, we demonstrate a general strategy to design and construct highly knotted nucleic acid nanostructures, each weaved from a single-stranded DNA or RNA chain by hierarchical folding in a prescribed order. Sets of DNA and RNA knots of two- or three-dimensional shapes have been designed and constructed (ranging from 1700 to 7500 nucleotides), and they exhibit complex topological features, with high crossing numbers (from 9 up to 57). These single-stranded DNA/RNA knots can be replicated and amplified enzymatically in vitro and in vivo. This work establishes a general platform for constructing nucleic acid nanostructures with complex molecular topologies.

The i-motif represents a paradigmatic example of the wide structural versatility of nucleic acids. In remarkable contrast to duplex DNA, i-motifs are four-stranded DNA structures held together by hemi- protonated and intercalated cytosine base pairs (C:C+). First observed 25 years ago, and considered by many as a mere structural oddity, interest in and discussion on the biological role of i-motifs have grown dramatically in recent years. In this review we focus on structural aspects of i-motif formation, the factors leading to its stabilization and recent studies describing the possible role of i-motifs in fundamental biological processes.

Three-dimensional genome structures play a key role in gene regulation and cell functions. Characterization of genome structures necessitates single-cell measurements. This has been achieved for haploid cells but has remained a challenge for diploid cells. We developed a single-cell chromatin conformation capture method, termed Dip-C, that combines a transposon-based whole-genome amplification method to detect many chromatin contacts, called META (multiplex end-tagging amplification), and an algorithm to impute the two chromosome haplotypes linked by each contact. We reconstructed the genome structures of single diploid human cells from a lymphoblastoid cell line and from primary blood cells with high spatial resolution, locating specific single-nucleotide and copy number variations in the nucleus. The two alleles of imprinted loci and the two X chromosomes were structurally different. Cells of different types displayed statistically distinct genome structures. Such structural cell typing is crucial for understanding cell functions., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

DNA origami can be used to create a variety of complex and geometrically unique nanostructures that can be further modified to produce building blocks for applications such as in optical metamaterials. We describe a method for creating metal-coated nanostructures using DNA origami templates and a photochemical metallization technique. Triangular DNA origami forms were fabricated and coated with a thin metal layer by photochemical silver reduction while in solution or supported on a surface. The DNA origami template serves as a localized photosensitizer to facilitate reduction of silver ions directly from solution onto the DNA surface. The metallizing process is shown to result in a conformal metal coating, which grows in height to a self-limiting value with increasing photoreduction steps. Although this coating process results in a slight decrease in the triangle dimensions, the overall template shape is retained. Notably, this coating method exhibits characteristics of self-limiting and defect-filling growth, which results in a metal nanostructure that maps the shape of the original DNA template with a continuous and uniform metal layer and stops growing once all available DNA sites are exhausted.

Without losing its high resolution, high-speed atomic force microscope (HS-AFM) represents a perfect combinationof scanning speed and precision and allows real-time and in situ observation of the dynamic processes in a biological system atboth the cellular and molecular levels. By combining the extremely high temporal resolution with the spatial resolution andcoupling with other advanced technologies, HS-AFM shows promising prospects for applications in life sciences such as cellbiology. In this review, we summarize the latest progress of HS-AFM in the field of cell biology, and discuss the impact ofenvironmental factors on conformation dynamics of DNA, the binding processes between DNA and protein, the domainchanges of membrane proteins, motility of myosin, and surface structure changes of living cells.

Hydration interaction shapes biomolecules and is a dominant intermolecular force. Mapping the hydration patterns of biomolecules is therefore essential for understanding molecular processes in biology. Numerous studies have been devoted to this challenge, but current methods cannot map the hydration of single biomolecules, let alone do so under physiological conditions. Here, we show that frequency-modulation atomic force microscopy (FM-AFM) can fill this gap and generate 3D hydration maps of single DNA molecules under near-physiological conditions. Additionally, we present real-space images of DNA in which the double helix is resolved with unprecedented resolution, clearly revealing individual phosphate groups along the DNA backbone. FM-AFM therefore emerges as a powerful enabling tool in the study of individual biomolecules and their hydration under physiological conditions.

RNA polymerase III (Pol III) and transcription factor IIIB (TFIIIB) assemble together on different promoter types to initiate the transcription of small, structured RNAs. Here we present structures of Pol III preinitiation complexes, comprising the 17-subunit Pol III and the heterotrimeric transcription factor TFIIIB, bound to a natural promoter in different functional states. Electron cryo-microscopy reconstructions, varying from 3.7 Å to 5.5 Å resolution, include two early intermediates in which the DNA duplex is closed, an open DNA complex, and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight, multivalent interaction between TFIIIB and promoter DNA, and explain how TFIIIB recruits Pol III. Together, TFIIIB and Pol III subunit C37 activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate the melting of double-stranded DNA, in a mechanism similar to that of the Pol II system.

Hypochlorous acid (HOCl) is generated by activated phagocytes at the site of inflammation. Exposure of DNA to HOCl results in base and nucleotide modifications causing DNA damage, which is one of the leading causes of various pathological conditions including carcinogenesis. In the present work, various biophysical techniques were used to study HOCl induced structural and conformational changes in human placental DNA. The HOCl modified DNA showed hyperchromicity, reduced fluorescence and decrease in melting temperature. Circular dichorism (CD) and Fourier transform infra-red (FT-IR) studies exhibited conformational changes and shift in band positions of DNA, respectively, suggesting structural changes. Agarose gel electrophoresis and scanning electron microscopy showed strand breakage and decreased aggregation. These results suggest that HOCl causes conformational and structural perturbations in mammalian DNA, which may consequentially lead to DNA mutations resulting in perturbation of epigenetic signals leading to cancer and autoimmune diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Eukaryotic origins of replication are licensed upon loading of the MCM helicase motor onto DNA. ATP hydrolysis by MCM is required for loading and the post-catalytic MCM is an inactive double hexamer that encircles duplex DNA. Origin firing depends on MCM engagement of Cdc45 and GINS to form the CMG holo-helicase. CMG assembly requires several steps including MCM phosphorylation by DDK. To understand origin activation, here we have determined the cryo-EM structures of DNA-bound MCM, either unmodified or phosphorylated, and visualize a phospho-dependent MCM element likely important for Cdc45 recruitment. MCM pore loops touch both the Watson and Crick strands, constraining duplex DNA in a bent configuration. By comparing our new MCM-DNA structure with the structure of CMG-DNA, we suggest how the conformational transition from the loaded, post-catalytic MCM to CMG might promote DNA untwisting and melting at the onset of replication.

Natural biomolecular assemblies such as molecular 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 nanotechnology 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 numbers, positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nanometres in diameter and with atomic 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 nanometres in diameter. 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 equilibrium 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.

DNA origami makes it feasible to fabricate a tremendous number of DNA nanostructures with various geometries, dimensions, and functionalities. Moreover, an increasing amount of research on DNA nanostructures is focused on biological and biomedical applications. Here, the reversible regulation of microcosmic structural rigidity is accomplished using a DNA origami device in vitro. The designed DNA origami monomer is composed of an internal central axis and an external sliding tube. Due to the external tube sliding, the device transforms between flexible and rigid states. By transporting the device into the liposome, the conformational change of the origami device induces a structural change in the liposome. The results obtained demonstrate that the programmed DNA origami device can be applied to regulate the microcosmic structural rigidity of liposomes. Because microcosmic structural rigidity is important to cell proliferation and function, the results obtained potentially provide a foundation for the regulation of cell microcosmic structural rigidity using DNA nanostructures., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Designed DNA nanostructures of impressive size have been described, but designed structures of the size of protein enzymes that bind organic ligands with high specificity are rare. Here we report a four-helix motif consisting of three synthetic strands with 65 base pairs and 165 nucleotides in total that folds well. Furthermore, we show that in the interior of this small folded DNA nanostructure, cavities can be set up that bind pyrimidine nucleotides with micromolar affinity. Base-specific binding for both thymidine and cytidine derivatives is demonstrated. The binding affinity depends on the position in the structure, as expected for recognition beyond simple base pairing. The folding motif reported here can help to expand DNA nanotechnology into the realm of selective molecular recognition that is currently dominated by protein-based enzymes and receptors.

In living cells, DNA is highly confined in space with the help of condensing agents, DNA binding proteins and high levels of supercoiling. Due to challenges associated with experimentally studying DNA under confinement, little is known about the impact of spatial confinement on the local structure of the DNA. Here, we have used well characterized slits of different sizes to collect high resolution atomic force microscopy images of confined circular DNA with the aim of assessing the impact of the spatial confinement on global and local conformational properties of DNA. Our findings, supported by numerical simulations, indicate that confinement imposes a large mechanical stress on the DNA as evidenced by a pronounced anisotropy and tangent-tangent correlation function with respect to non-constrained DNA. For the strongest confinement we observed nanometer sized hairpins and interwound structures associated with the nicked sites in the DNA sequence. Based on these findings, we propose that spatial DNA confinement in vivo can promote the formation of localized defects at mechanically weak sites that could be co-opted for biological regulatory functions., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA based self-assembled nanostructures and DNA origami has proven useful for organizing nanomaterials with firm precision. However, for advanced applications like nanoelectronics and photonics, large-scale organization of self-assembled branched DNA (bDNA) into periodic lattices is desired. In this communication for the first time we report a facile method of self-assembly of Y-shaped bDNA nanostructures on the cationic surface of Aluminum (Al) foil to prepare periodic two dimensional (2D) bDNA lattice. Particularly those Y-shaped bDNA structures having smaller overhangs and unable to self-assemble in solution, they are easily assembled on the surface of Al foil in the absence of ligase. Field emission scanning electron microscopy (FESEM) analysis shows homogenous distribution of two-dimensional bDNA lattices across the Al foil. When the assembled bDNA structures were recovered from the Al foil and electrophoresed in nPAGE only higher order polymeric bDNA structures were observed without a trace of monomeric structures which confirms the stability and high yield of the bDNA lattices. Therefore, this enzyme-free economic and efficient strategy for developing bDNA lattices can be utilized in assembling various nanomaterials for functional molecular components towards development of DNA based self-assembled nanodevices., (Copyright © 2017 Elsevier Inc. All rights reserved.)

DNA encodes the genetic information of most living beings, except viruses that use RNA. Unlike other types of molecules, DNA is not usually described by its atomic structure being instead usually described by its base-pair sequence, i.e., the textual sequence of its subsidiary molecules known as nucleotides ( adenine (A), cytosine (C), guanine (G), and thymine (T)). The three-dimensional assembling of DNA molecules based on its base-pair sequence has been, for decades, a topic of interest for many research groups all over the world. In this paper, we survey the major methods found in the literature to assemble and visualize DNA molecules from their base-pair sequences. We divided these methods into three categories: predictive methods, adaptive methods, and thermodynamic methods . Predictive methods aim to predict a conformation of the DNA from its base pair sequence, while the goal of adaptive methods is to assemble DNA base-pairs sequences along previously known conformations, as needed in scenarios such as DNA Monte Carlo simulations. Unlike these two geometric methods, thermodynamic methods are energy-based and aim to predict secondary structural motifs of DNA in cases where hydrogen bonds between base pairs might be broken because of temperature changes. We also present the major software tools that implements predictive, adaptive, and thermodynamic methods.

Many hybrid devices integrate functional molecular or nanoparticle components with microstructures, as exemplified by the nanophotonic devices that couple emitters to optical resonators for potential use in single-molecule detection, precision magnetometry low threshold lasing and quantum information processing. These systems also illustrate a common difficulty for hybrid devices: although many proof-of-principle devices exist, practical applications face the challenge of how to incorporate large numbers of chemically diverse functional components into microfabricated resonators at precise locations. Here we show that the directed self-assembly of DNA origami onto lithographically patterned binding sites allows reliable and controllable coupling of molecular emitters to photonic crystal cavities (PCCs). The precision of this method is sufficient to enable us to visualize the local density of states within PCCs by simple wide-field microscopy and to resolve the antinodes of the cavity mode at a resolution of about one-tenth of a wavelength. By simply changing the number of binding sites, we program the delivery of up to seven DNA origami onto distinct antinodes within a single cavity and thereby digitally vary the intensity of the cavity emission. To demonstrate the scalability of our technique, we fabricate 65,536 independently programmed PCCs on a single chip. These features, in combination with the widely used modularity of DNA origami, suggest that our method is well suited for the rapid prototyping of a broad array of hybrid nanophotonic devices.

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Optical ruler methods employing multiple fluorescent labels offer great potential for correlating distances among several sites, but are generally limited to interlabel distances under 10 nm and suffer from complications due to spectral overlap. Here we demonstrate a multicolor surface energy transfer (McSET) technique able to triangulate multiple points on a biopolymer, allowing for analysis of global structure in complex biomolecules. McSET couples the competitive energy transfer pathways of Förster Resonance Energy Transfer (FRET) with gold-nanoparticle mediated Surface Energy Transfer (SET) in order to correlate systematically labeled points on the structure at distances greater than 10 nm and with reduced spectral overlap. To demonstrate the McSET method, the structures of a linear B-DNA and a more complex folded RNA ribozyme were analyzed within the McSET mathematical framework. The improved multicolor optical ruler method takes advantage of the broad spectral range and distances achievable when using a gold nanoparticle as the lowest energy acceptor. The ability to report distance information simultaneously across multiple length scales, short-range (10-50 Å), mid-range (50-150 Å), and long-range (150-350 Å), distinguishes this approach from other multicolor energy transfer methods.

The highly charged DNA chain may be either in an extended conformation, the coil, or condensed into a highly dense and ordered structure, the toroid. The transition, also called collapse of the chain, can be triggered in different ways, for example by changing the ionic conditions of the solution. We observe individual DNA molecules one by one, kept separated and confined inside a protein shell (the envelope of a bacterial virus, 80 nm in diameter). For subcritical concentrations of spermine (4+), part of the DNA is condensed and organized in a toroid and the other part of the chain remains uncondensed around. Two states coexist along the same DNA chain. These 'hairy' globules are imaged by cryo-electron microscopy. We describe the global conformation of the chain and the local ordering of DNA segments inside the toroid., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Rectangular DNA origami functionalized with thiols in each of the four corners immobilizes by self-assembly between lithographically patterned gold nanodots on a silicon oxide surface., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nucleic acid molecules can adopt a variety of structures and exhibit a large degree of conformational flexibility to fulfill their various functions in cells. Here we describe the use of Pulsed Electron-Electron Double Resonance (PELDOR or DEER) to investigate nucleic acid molecules where two cytosine analogs have been incorporated as spin probes. Because these new types of spin labels are rigid and incorporated into double stranded DNA and RNA molecules, there is no additional flexibility of the spin label itself present. Therefore the magnetic dipole-dipole interaction between both spin labels encodes for the distance as well as for the mutual orientation between the spin labels. All of this information can be extracted by multi-frequency/multi-field PELDOR experiments, which gives very precise and valuable information about the structure and conformational flexibility of the nucleic acid molecules. We describe in detail our procedure to obtain the conformational ensembles and show the accuracy and limitations with test examples and application to double-stranded DNA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Interaction between long DNA molecules and activated cisplatin is believed to be crucial to anticancer activity. However, the exact structural changes of long DNA molecules induced by cisplatin are still not very clear. In this study, structural changes of long linear double-stranded DNA (dsDNA) and short single-stranded DNA (ssDNA) induced by activated cisplatin have been investigated by atomic force microscopy (AFM). The results indicated that long DNA molecules gradually formed network structures, beads-on-string structures and their large aggregates. Electrostatic and coordination interactions were considered as the main driving forces producing these novel structures. An interesting finding in this study is the beads-on-string structures. Moreover, it is worth noting that the beads-on-string structures were linked into the networks, which can be ascribed to the strong DNA-DNA interactions. This study expands our knowledge of the interactions between DNA molecules and cisplatin., (Copyright © 2015 Elsevier Inc. All rights reserved.)