Six-Letter DNA Nanotechnology: Incorporation of Z-P Base Pairs into Self-Assembling 3D Crystals.

Artificially expanded genetic information systems (AEGIS) were developed to expand the diversity and functionality of biological systems. Recent experiments have shown that these expanded DNA molecular systems are robust platforms for information storage and retrieval as well as useful for basic biotechnologies. In tandem, nucleic acid nanotechnology has seen the use of information-based "semantomorphic" encoding to drive the self-assembly of a vast array of supramolecular devices. To establish the effectiveness of AEGIS toward nanotechnological applications, we investigated the ability of a six-letter alphabet composed of A:T, G:C and synthetic Z:P (Z, 6-amino-3-(1'-ß-d-2'-deoxy ribofuranosyl)-5-nitro-(1H)-pyridin-2-one; P, 2-amino-8-(1'-ß-d-2'-deoxyribofuranosyl)-imidazo-[1,2a]-1,3,5-triazin-(8H)-4-one) base pairs to engage in 3D self-assembly. We found that crystals could be programmably assembled from AEGIS oligomers. We conclude that unnatural base pairs can be used for the topological self-assembly of crystals. We anticipate the expansion of AEGIS-based nucleic acid nanotechnologies to enable the development of novel nanomaterials, high-fidelity signal cascades, and dynamic nanoscale devices.

Engineering tertiary chirality in helical biopolymers.

Tertiary chirality describes the handedness of supramolecular assemblies and relies not only on the primary and secondary structures of the building blocks but also on topological driving forces that have been sparsely characterized. Helical biopolymers, especially DNA, have been extensively investigated as they possess intrinsic chirality that determines the optical, mechanical, and physical properties of the ensuing material. Here, we employ the DNA tensegrity triangle as a model system to locate the tipping points in chirality inversion at the tertiary level by X-ray diffraction. We engineer tensegrity triangle crystals with incremental rotational steps between immobile junctions from 3 to 28 base pairs (bp). We construct a mathematical model that accurately predicts and explains the molecular configurations in both this work and previous studies. Our design framework is extendable to other supramolecular assemblies of helical biopolymers and can be used in the design of chiral nanomaterials, optically active molecules, and mesoporous frameworks, all of which are of interest to physical, biological, and chemical nanoscience.

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.

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.

The wending rhombus: Self-assembling 3D DNA crystals.

In this perspective, we provide a summary of recent developments in self-assembling three-dimensional (3D) DNA crystals. Starting from the inception of this subfield, we describe the various advancements in structure that have led to an increase in the diversity of macromolecular crystal motifs formed through self-assembly, and we further comment on the future directions of the field, which exploit noncanonical base pairing interactions beyond Watson-Crick. We then survey the current applications of self-assembling 3D DNA crystals in reversibly active nanodevices and materials engineering and provide an outlook on the direction researchers are taking these structures. Finally, we compare 3D DNA crystals with DNA origami and suggest how these distinct subfields might work together to enhance biomolecule structure solution, nanotechnological motifs, and their applications.

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.

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.

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.

Designing Higher Resolution Self-Assembled 3D DNA Crystals via Strand Terminus Modifications.

DNA tensegrity triangles self-assemble into rhombohedral three-dimensional crystals via sticky ended cohesion. Crystals containing two-nucleotide (nt) sticky ends (GA:TC) have been reported previously, and those crystals diffracted to 4.9 Å at beamline NSLS-I-X25. Here, we analyze the effect of varying sticky end lengths and sequences as well as the impact of 5'- and 3'-phosphates on crystal formation and resolution. Tensegrity triangle motifs having 1-, 2-, 3-, and 4-nt sticky ends all form crystals. X-ray diffraction data from the same beamline reveal that the crystal resolution for a 1-nt sticky end (G:C) and a 3-nt sticky end (GAT:ATC) were 3.4 and 4.2 Å, respectively. Resolutions were determined from complete data sets in each case. We also conducted trials that examined every possible combination of 1-nucleotide and 2-nucleotide sticky-ended phosphorylated strands and successfully crystallized all 16 possible combinations of strands. We observed the position of the 5'-phosphate on either the crossover (1), helical (2), or central strand (3) affected the resolution of the self-assembled crystals for the 2-turn monomer (3.0 Å for 1-2P-3P) and 2-turn dimer sticky ended (4.1 Å for 1-2-3P) systems. We have also examined the impact of the identity of the base flanking the sticky ends as well as the use of 3'-phosphate. We conclude that crystal resolution is not a simple consequence of the thermodynamics of the direct nucleotide pairing interactions involved in molecular cohesion in this system.

Paranemic Crossover DNA: There and Back Again.

Over the past 35 years, DNA has been used to produce various nanometer-scale constructs, nanomechanical devices, and walkers. Construction of complex DNA nanostructures relies on the creation of rigid DNA motifs. Paranemic crossover (PX) DNA is one such motif that has played many roles in DNA nanotechnology. Specifically, PX cohesion has been used to connect topologically closed molecules, to assemble a three-dimensional object, and to create two-dimensional DNA crystals. Additionally, a sequence-dependent nanodevice based on conformational change between PX and its topoisomer, JX2, has been used in robust nanoscale assembly lines, as a key component in a DNA transducer, and to dictate polymer assembly. Furthermore, the PX motif has recently found a new role directly in basic biology, by possibly serving as the molecular structure for double-stranded DNA homology recognition, a prominent feature of molecular biology and essential for many crucial biological processes. This review discusses the many attributes and usages of PX-DNA-its design, characteristics, applications, and potential biological relevance-and aims to accelerate the understanding of PX-DNA motif in its many roles and manifestations.

Self-Assembly of 3D DNA Crystals Containing a Torsionally Stressed Component.

There is an increasing appreciation for structural diversity of DNA that is of interest to both DNA nanotechnology and basic biology. Here, we have explored how DNA responds to torsional stress by building on a previously reported two-turn DNA tensegrity triangle and demonstrating that we could introduce an extra nucleotide pair (np) into the original sequence without affecting assembly and crystallization. The extra np imposes a significant torsional stress, which is accommodated by global changes throughout the B-DNA duplex and the DNA lattice. The work reveals a near-atomic structure of naked DNA under a torsional stress of approximately 14%, and thus provides an example of DNA distortions that occur without a requirement for either an external energy source or the free energy available from protein or drug binding.