Toward three-dimensional DNA industrial nanorobots.

Nanoscale industrial robots have potential as manufacturing platforms and are capable of automatically performing repetitive tasks to handle and produce nanomaterials with consistent precision and accuracy. We demonstrate a DNA industrial nanorobot that fabricates a three-dimensional (3D), optically active chiral structure from optically inactive parts. By making use of externally controlled temperature and ultraviolet (UV) light, our programmable robot, ~100 nanometers in size, grabs different parts, positions and aligns them so that they can be welded, releases the construct, and returns to its original configuration ready for its next operation. Our robot can also self-replicate its 3D structure and functions, surpassing single-step templating (restricted to two dimensions) by using folding to access the third dimension and more degrees of freedom. Our introduction of multiple-axis precise folding and positioning as a tool/technology for nanomanufacturing will open the door to more complex and useful nano- and microdevices.

Heterobimetallic Base Pair Programming in Designer 3D DNA Crystals.

Metal-mediated DNA (mmDNA) presents a pathway toward engineering bioinorganic and electronic behavior into DNA devices. Many chemical and biophysical forces drive the programmable chelation of metals between pyrimidine base pairs. Here, we developed a crystallographic method using the three-dimensional (3D) DNA tensegrity triangle motif to capture single- and multi-metal binding modes across granular changes to environmental pH using anomalous scattering. Leveraging this programmable crystal, we determined 28 biomolecular structures to capture mmDNA reactions. We found that silver(I) binds with increasing occupancy in T-T and U-U pairs at elevated pH levels, and we exploited this to capture silver(I) and mercury(II) within the same base pair and to isolate the titration points for homo- and heterometal base pair modes. We additionally determined the structure of a C-C pair with both silver(I) and mercury(II). Finally, we extend our paradigm to capture cadmium(II) in T-T pairs together with mercury(II) at high pH. The precision self-assembly of heterobimetallic DNA chemistry at the sub-nanometer scale will enable atomistic design frameworks for more elaborate mmDNA-based nanodevices and nanotechnologies.

Environmentally Controlled Oscillator with Triplex Guided Displacement of DNA Duplexes.

The use of DNA triplex association is advantageous for the reconfiguration of dynamic DNA nanostructures through pH alteration and can provide environmental control for both structural changes and molecular signaling. The combination of pH-induced triplex-forming oligonucleotide (TFOs) binding with toehold-mediated strand displacement has recently garnered significant attention in the field of structural DNA nanotechnology. While most previous studies use single-stranded DNA to displace or replace TFOs within the triplex, here we demonstrate that pH alteration allows a DNA duplex, with a toehold assistance, to displace TFOs from the components of another DNA duplex. We examined the dependence of this process on toehold length and show that the pH changes allow for cyclic oscillations between two molecular formations. We implemented the duplex/triplex design onto the surface of 2D DNA origami in the form outlining binary digits 0 or 1 and verified the oscillatory conformational changes between the two formations with atomic force microscopy.

Metal-Mediated DNA Nanotechnology in 3D: Structural Library by Templated Diffraction.

DNA double helices containing metal-mediated DNA (mmDNA) base pairs are constructed from Ag+ and Hg2+ ions between pyrimidine:pyrimidine pairs with the promise of nanoelectronics. Rational design of mmDNA nanomaterials is impractical without a complete lexical and structural description. Here, the programmability of structural DNA nanotechnology toward its founding mission of self-assembling a diffraction platform for biomolecular structure determination is explored. The tensegrity triangle is employed to build a comprehensive structural library of mmDNA pairs via X-ray diffraction and generalized design rules for mmDNA construction are elucidated. Two binding modes are uncovered: N3-dominant, centrosymmetric pairs and major groove binders driven by 5-position ring modifications. Energy gap calculations show additional levels in the lowest unoccupied molecular orbitals (LUMO) of mmDNA structures, rendering them attractive molecular electronic candidates.

Metal-Mediated DNA Nanotechnology in 3D: Structural Library by Templated Diffraction.

DNA double helices containing metal-mediated DNA (mmDNA) base pairs have been constructed from Ag+ and Hg2+ ions between pyrimidine:pyrimidine pairs with the promise of nanoelectronics. Rational design of mmDNA nanomaterials has been impractical without a complete lexical and structural description. Here, we explore the programmability of structural DNA nanotechnology toward its founding mission of self-assembling a diffraction platform for biomolecular structure determination. We employed the tensegrity triangle to build a comprehensive structural library of mmDNA pairs via X-ray diffraction and elucidated generalized design rules for mmDNA construction. We uncovered two binding modes: N3-dominant, centrosymmetric pairs and major groove binders driven by 5-position ring modifications. Energy gap calculations showed additional levels in the lowest unoccupied molecular orbitals (LUMO) of mmDNA structures, rendering them attractive molecular electronic candidates. This article is protected by copyright. All rights reserved.

Engineering DNA Crystals toward Studying DNA-Guest Molecule Interactions.

Sequence-selective recognition of DNA duplexes is important for a wide range of applications including regulating gene expression, drug development, and genome editing. Many small molecules can bind DNA duplexes with sequence selectivity. It remains as a challenge how to reliably and conveniently obtain the detailed structural information on DNA-molecule interactions because such information is critically needed for understanding the underlying rules of DNA-molecule interactions. If those rules were understood, we could design molecules to recognize DNA duplexes with a sequence preference and intervene in related biological processes, such as disease treatment. Here, we have demonstrated that DNA crystal engineering is a potential solution. A molecule-binding DNA sequence is engineered to self-assemble into highly ordered DNA crystals. An X-ray crystallographic study of molecule-DNA cocrystals reveals the structural details on how the molecule interacts with the DNA duplex. In this approach, the DNA will serve two functions: (1) being part of the molecule to be studied and (2) forming the crystal lattice. It is conceivable that this method will be a general method for studying drug/peptide-DNA interactions. The resulting DNA crystals may also find use as separation matrices, as hosts for catalysts, and as media for material storage.

The Formation and Displacement of Ordered DNA Triplexes in Self-Assembled Three-Dimensional DNA Crystals.

Reconfigurable structures engineered through DNA hybridization and self-assembly offer both structural and dynamic applications in nanotechnology. Here, we have demonstrated that strand displacement of triplex-forming oligonucleotides (TFOs) can be translated to a robust macroscopic DNA crystal by coloring the crystals with covalently attached fluorescent dyes. We show that three different types of triplex strand displacement are feasible within the DNA crystals and the bound TFOs can be removed and/or replaced by (a) changing the pH from 5 to 7, (b) the addition of the Watson-Crick complement to a TFO containing a short toehold, and (c) the addition of a longer TFO that uses the duplex edge as a toehold. We have also proved by X-ray diffraction that the structure of the crystals remains as designed in the presence of the TFOs.

Mesojunction-Based Design Paradigm of Structural DNA Nanotechnology.

Mesojunctions were introduced as a basic type of crossover configuration in the early development of structural DNA nanotechnology. However, the investigations of self-assembly from multiple mesojunction complexes have been overlooked in comparison to their counterparts based on regular junctions. In this work, we designed standardized component strands for the construction of complex mesojunction lattices. Three typical mesojunction configurations with three and four arms were showcased in the self-assembly of 1-, 2-, and 3-dimensional lattices constructed from both a scaffold-free tiling approach and a scaffolded origami approach.

The Rule of Thirds: Controlling Junction Chirality and Polarity in 3D DNA Tiles.

The successful self-assembly of tensegrity triangle DNA crystals heralded the ability to programmably construct macroscopic crystalline nanomaterials from rationally-designed, nanoscale components. This 3D DNA tile owes its "tensegrity" nature to its three rotationally stacked double helices locked together by the tensile winding of a center strand segmented into 7 base pair (bp) inter-junction regions, corresponding to two-thirds of a helical turn of DNA. All reported tensegrity triangles to date have employed ( Z + 2 / 3 ) \[\left( {Z{\bm{ + }}2{\bf /}3} \right)\] turn inter-junction segments, yielding right-handed, antiparallel, "J1" junctions. Here a minimal DNA triangle motif consisting of 3-bp inter-junction segments, or one-third of a helical turn is reported. It is found that the minimal motif exhibits a reversed morphology with a left-handed tertiary structure mediated by a locally-parallel Holliday junction-the "L1" junction. This parallel junction yields a predicted helical groove matching pattern that breaks the pseudosymmetry between tile faces, and the junction morphology further suggests a folding mechanism. A Rule of Thirds by which supramolecular chirality can be programmed through inter-junction DNA segment length is identified. These results underscore the role that global topological forces play in determining local DNA architecture and ultimately point to an under-explored class of self-assembling, chiral nanomaterials for topological processes in biological systems.

Highly Symmetric, Self-Assembling 3D DNA Crystals with Cubic and Trigonal Lattices.

The rational design of nanoscopic DNA tiles has yielded highly ordered crystalline matter in 2D and 3D. The most well-studied 3D tile is the DNA tensegrity triangle, which is known to self-assemble into macroscopic crystals. However, contemporary rational design parameters for 3D DNA crystals nearly universally invoke integer numbers of DNA helical turns and Watson-Crick (WC) base pairs. In this study, 24-bp edges are substituted into a previously 21-bp (two helical turns of DNA) tensegrity triangle motif to explore whether such unconventional motif can self-assemble into 3D crystals. The use of noncanonical base pairs in the sticky ends results in a cubic arrangement of tensegrity triangles with exceedingly high symmetry, assembling a lattice from winding helical axes and diamond-like tessellation patterns. Reverting this motif to sticky ends with Watson-Crick pairs results in a trigonal hexagonal arrangement, replicating this diamond arrangement in a hexagonal context. These results showcase that the authors can generate unexpected, highly complex, pathways for materials design by testing modifications to 3D tiles without prior knowledge of the ensuing symmetry. This study expands the rational design toolbox for DNA nanotechnology; and it further illustrates the existence of yet-unexplored arrangements of crystalline soft matter.

Programmable 3D Hexagonal Geometry of DNA Tensegrity Triangles.

Non-canonical interactions in DNA remain under-explored in DNA nanotechnology. Recently, many structures with non-canonical motifs have been discovered, notably a hexagonal arrangement of typically rhombohedral DNA tensegrity triangles that forms through non-canonical sticky end interactions. Here, we find a series of mechanisms to program a hexagonal arrangement using: the sticky end sequence; triangle edge torsional stress; and crystallization condition. We showcase cross-talking between Watson-Crick and non-canonical sticky ends in which the ratio between the two dictates segregation by crystal forms or combination into composite crystals. Finally, we develop a method for reconfiguring the long-range geometry of formed crystals from rhombohedral to hexagonal and vice versa. These data demonstrate fine control over non-canonical motifs and their topological self-assembly. This will vastly increase the programmability, functionality, and versatility of rationally designed DNA constructs.

Augmented DNA Nanoarchitectures: A Structural Library of 3D Self-Assembling Tensegrity Triangle Variants.

The DNA tensegrity triangle is known to reliably self-assemble into a 3D rhombohedral crystalline lattice via sticky-end cohesion. Here, the library of accessible motifs is expanded through covalent extensions of intertriangle regions and sticky-end-coordinated linkages of adjacent triangles with double helical segments using both geometrically symmetric and asymmetric configurations. The molecular structures of 18 self-assembled architectures at resolutions of 3.32-9.32 Å are reported; the observed cell dimensions, cavity sizes, and cross-sectional areas agree with theoretical expectations. These data demonstrate that fine control over triclinic and rhombohedral crystal parameters and the customizability of more complex 3D DNA lattices are attainable via rational design. It is anticipated that augmented DNA architectures may be fine-tuned for the self-assembly of designer nanocages, guest-host complexes, and proscriptive 3D nanomaterials, as originally envisioned. Finally, designer asymmetric crystalline building blocks can be seen as a first step toward controlling and encoding information in three dimensions.

Programming DNA Self-Assembly by Geometry†.

This manuscript introduces geometry as a means to program the tile-based DNA self-assembly in two and three dimensions. This strategy complements the sequence-focused programmable assembly. DNA crystal assembly critically relies on intermotif, sticky-end cohesion, which requires complementarity not only in sequence but also in geometry. For DNA motifs to assemble into crystals, they must be associated with each other in the proper geometry and orientation to ensure that geometric hindrance does not prevent sticky ends from associating. For DNA motifs with exactly the same pair of sticky-end sequences, by adjusting the length (thus, helical twisting phase) of the motif branches, it is possible to program the assembly of these distinct motifs to either mix with one another, to self-sort and consequently separate from one another, or to be alternatingly arranged. We demonstrate the ability to program homogeneous crystals, DNA "alloy" crystals, and definable grain boundaries through self-assembly. We believe that the integration of this strategy and conventional sequence-focused assembly strategy could further expand the programming versatility of DNA self-assembly.

Direct visualization of floppy two-dimensional DNA origami using cryogenic electron microscopy.

Two-dimensional (2D) DNA origami that is capable of self-assembling into complex 2D and 3D geometries pave the way for a bottom-up synthesis for various applications in nano/biotechnology. Here, we directly visualized the aqueous structure of 2D DNA origami cross-tiles and their assemblies using cryogenic electron microscopy. We uncovered flexible arms in cross-tile monomers and designated inter-tile folding. In addition, we observed the formation of clusters and stacks of DNA cross-tiles in solution, which could potentially affect the interaction and assembly of DNA origami. Finally, we quantitatively evaluated the flexibility of DNA origami in solution using finite element analysis. Our discovery has laid the foundation for investigating the dynamic structures of 2D DNA origami assemblies in solution, providing insights regarding the self-assembly and self-replication mechanisms of 2D DNA origami.

Regulation of 2D DNA Nanostructures by the Coupling of Intrinsic Tile Curvature and Arm Twist.

The overwinding and underwinding of DNA duplexes between junctions have been used in designing left- and right-handed DNA origami nanostructures, respectively. For DNA tubes obtained from self-assembled tiles, only a theoretical approach of the intrinsic curvature of the tiles has been previously used to explain their formation. Details regarding the quantitative and structural descriptions of the tile's intrinsic curvature in DNA nanostructures have so far never been addressed. In this work, we designed three types of tile cores built around a circular scaffold using three- and four-branched junctions. Joining the tile cores with arms having two kinds of inter-tile distances, an odd and an even number of DNA half-turns, tended to form planar 2D lattices and tubes, respectively. Streptavidin bound to biotin was used as a labeling technique to characterize the inside and outside surfaces of the tubes and thereby the tile conformation of dihedrals with addressable faces. DNA tubes with either right- or left-handed chirality were obtained by the coupling of the intrinsic curvature of the tiles with the arm twist. We were able to assign the chiral indices (n,m) to a tube with its structure resolved by AFM at the single-tile level and therefore to estimate the global curvature of the tube (or its component tile) using a regular polygon model that approximated its transverse section. A deeper understanding of the integrated actions of different types of twisting forces on DNA tubes will be extremely helpful in engineering more elaborate DNA nanostructures in the future.

Powering ≈50 µm Motion by a Molecular Event in DNA Crystals.

A major challenge in material design is to couple nanoscale molecular and supramolecular events into desired chemical, physical, and mechanical properties at the macroscopic scale. Here, a novel self-assembled DNA crystal actuator is reported, which has reversible, directional expansion and contraction for over 50 µm in response to versatile stimuli, including temperature, ionic strength, pH, and redox potential. The macroscopic actuation is powered by cooperative dissociation or cohesion of thousands of DNA sticky ends at the designed crystal contacts. The increase in crystal porosity and cavity in the expanded state dramatically enhances the crystal capability to accommodate/encapsulate nanoparticles/proteins, while the contraction enables a "sponge squeezing" motion for releasing nanoparticles. This crystal actuator is envisioned to be useful for a wide range of applications, including powering self-propelled robotics, sensing subtle environmental changes, constructing functional hybrid materials, and working in drug controlled-release systems.

Exciton Delocalization in a DNA-Templated Organic Semiconductor Dimer Assembly.

A chiral dimer of an organic semiconductor was assembled from octaniline (octamer of polyaniline) conjugated to DNA. Facile reconfiguration between the monomer and dimer of octaniline-DNA was achieved. The geometry of the dimer and the exciton coupling between octaniline molecules in the assembly was studied both experimentally and theoretically. The octaniline dimer was readily switched between different electronic states by protonic doping and exhibited a Davydov splitting comparable to those previously reported for DNA-dye systems employing dyes with strong transition dipoles. This approach provides a possible platform for studying the fundamental properties of organic semiconductors with DNA-templated assemblies, which serve as candidates for artificial light-harvesting systems and excitonic devices.

Orienting an Organic Semiconductor into DNA 3D Arrays by Covalent Bonds.

A quasi-one-dimensional organic semiconductor, hepta(p-phenylene vinylene) (HPV), was incorporated into a DNA tensegrity triangle motif using a covalent strategy. 3D arrays were self-assembled from an HPV-DNA pseudo-rhombohedron edge by rational design and characterized by X-ray diffraction. Templated by the DNA motif, HPV molecules exist as single-molecule fluorescence emitters at the concentration of 8 mM within the crystal lattice. The anisotropic fluorescence emission from HPV-DNA crystals indicates HPV molecules are well aligned in the macroscopic 3D DNA lattices. Sophisticated nanodevices and functional materials constructed from DNA can be developed from this strategy by addressing functional components with molecular accuracy.

3D Hexagonal Arrangement of DNA Tensegrity Triangles.

The tensegrity triangle motif utilizes Watson-Crick sticky end cohesion to self-assemble into a rhombohedral crystal lattice using complementary 5'-GA and 5'-TC sticky ends. Here, we report that using noncanonical 5'-AG and 5'-TC sticky ends in otherwise isomorphic tensegrity triangles results in crystal self-assembly in the P63 hexagonal space group as revealed by X-ray crystallography. In this structure, the DNA double helices bend at the crossover positions, a feature that was not observed in the original design. Instead of propagating linearly, the tilt between base pairs of each right-handed helix results in a left-handed superstructure along the screw axis, forming a microtubule-like structure composed of three double helices with an unbroken channel at the center. This hexagonal lattice has a cavity diameter of 11 nm and a unit cell volume of 886 000 Å3-far larger than the rhombohedral counterpart (5 nm, 330 000 Å3).

Reconfigurable Two-Dimensional DNA Lattices: Static and Dynamic Angle Control.

Branched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. In this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle-enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson-Crick base pairing or non-canonical nucleobase interactions (e.g., i-motif and G-quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely-ordered three-dimensional architectures.