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.

Divergence and Convergence: Complexity Emerges in Crystal Engineering from an 8-mer DNA.

Biology provides plenty of examples on achieving complicated structures out of minimal numbers of building blocks. In contrast, structural complexity of designed molecular systems is achieved by increasing the numbers of component molecules. In this study, the component DNA strand assembles into a highly complex crystal structure via an unusual path of divergence and convergence. This assembly path suggests a route to minimalists for increasing structural complexity. The original purpose of this study is to engineer DNA crystals with high resolution, which is the primary motivation and a key objective for structural DNA nanotechnology. Despite great efforts in the last 40 years, engineered DNA crystals have not yet consistently reached resolution better than 2.5 Å, limiting their potential uses. Our research has shown that small, symmetrical building blocks generally lead to high resolution crystals. Herein, by following this principle, we report an engineered DNA crystal with unprecedented high resolution (2.17 Å) assembled from one single DNA component: an 8-base-long DNA strand. This system has three unique characteristics: (1) It has a very complex architecture, (2) the same DNA strand forms two different structural motifs, both of which are incorporated into the final crystal, and (3) the component DNA molecule is only an 8-base-long DNA strand, which is, arguably, the smallest DNA motif for DNA nanostructures to date. This high resolution opens the possibility of using these DNA crystals to precisely organize guest molecules at the Å level, which could stimulate a range of new investigations.

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.

AMP Aptamer Programs DNA Tile Cohesion without Canonical Base Pairing.

Tile-based DNA self-assembly provides a versatile approach for the construction of a wide range of nanostructures for various applications such as nanomedicine and advanced materials. The inter-tile interactions are primarily programmed by base pairing, particularly Watson-Crick base pairing. To further expand the tool box for DNA nanotechnology, herein, we have designed DNA tiles that contain both ligands and aptamers. Upon ligand-aptamer binding, tiles associate into geometrically well-defined nanostructures. This strategy has been demonstrated by the assembly of a series of DNA nanostructures, which have been thoroughly characterized by gel electrophoresis and atomic force microscopy. This new inter-tile cohesion could bring new potentials to DNA self-assembly in the future. For example, the addition of free ligand could modulate the nanostructure formation. In the case of biological ligands, DNA self-assembly could be related to the presence of certain ligands.

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.

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.

Rational Design of Abasic Site-Containing DNA Triplexes To Bind Small Molecules with Low Nanomolar Dissociation Constants.

De novo design of functional biomacromolecules is of great interest to a wide range of fundamental science and technological applications, including understanding life evolution and biomacromolecular structures, developing novel catalysts, inventing medicines, and exploring high-performance materials. However, it is an extremely challenging task and its success is very limited. It requires a deep understanding of the relationships among the primary sequences, the 3D structures, and the functions of biomacromolecules. Herein, we report a rational, de novo design of a DNA aptamer that can bind melamine with high specificity and high affinity (dissociation constant Kd = 4.4 nM). The aptamer is essentially a DNA triplex, but contains an abasic site, to which the melamine binds. The aptamer-ligand recognition involves hydrogen-bonding, π-π stacking, and electrostatic interactions. This strategy has been further tested by designing aptamers to bind to guanosine. It is conceivable that such a rational strategy, with further development, would provide a general framework for designing functional DNA molecules.

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.

Solution-Phase Synthesis of DNA Amphiphiles for DNA Micellar Assembly.

Hydrophobic moieties of amphiphilic DNAs can help DNAs penetrate cell membranes, but the conjugation of hydrophobic moieties to DNAs in solution phase remains challenging. Herein we report a solution-phase synthesis method to conjugate hydrophobic molecules to DNAs. This method is simple and efficient. The resulted amphiphilic DNAs can spontaneously assemble into micelles, which may serve as nanocarriers for cellular delivery of nucleic acids and water-insoluble drugs.

Molecular Lithography on Silicon Wafers Guided by Porous, Extended Arrays of Small DNA Tiles.

Tile-based DNA self-assembly is a cost-effective fabrication method for large-scale nanopatterns. Herein, we report a protocol to directly assemble DNA 2D arrays on silicon wafers and then use the DNA nanostructures as molds to fabricate the corresponding nanostructures on the silicon wafers by hydrogen fluoride (HF) etching. Similar HF etching has been used with robust large DNA origami structures as templates. This work demonstrates that DNA nanostructures assembled from small tiles are sufficiently stable for this process. The resulting feature size (∼8.6 nm) approaches the sizes of e-beam lithography. While the reported method is parallel and inexpensive, e-beam lithography is a serial method and is expensive. We expect that this method will be very useful for preparing fine nanopatterns in large areas.

Enriching adenosine by thymine-rich DNA oligomers.

Adenosine levels are important in various physiological and pathological activities, but detecting them is difficult because of interference from a complex matrix. This study designed a series of DNA oligomers rich in thymine to enrich adenosine. Their binding affinity (Kd range: 1.25-5.0 mM) to adenosine varied based on the DNA secondary structures, with a clamped hairpin structure showing the highest binding affinity. Compared to other designs, this clamped DNA hairpin underwent the least conformational change during adenosine binding. These DNAs also suppressed the precipitation of supersaturated adenine. Taken together, these results suggest that thymine-rich DNAs could be used to enrich and separate adenosine.

Near-Quantitative Preparation of Short Single-Stranded DNA Circles.

Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA nanostructures. However, it is challenging to prepare such ssDNA circles, particularly when the DNA size becomes very small (e.g. a 20 nucleotide (nt) long ssDNA circle). Often, such short ssDNA dominantly form concatemers (either linear or circular) due to intermolecular ligation, instead of forming monomeric ssDNA circles by intramolecular ligation. Herein, a simple method to overcome this problem by designing the complementary linker molecules is reported. It is demonstrated that ssDNA, as short as 16 nts, can be enzymatically ligated (by the commonly used T4 DNA ligase) into monomeric ssDNA circles at high concentration (100 µM) with high yield (97 %). This method does not require any special sequence, thus, it is expected to be generally applicable. The experimental protocol is identical to regular DNA ligation, thus, is expected to be user friendly for general chemists and biologists.

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.

Tomography of DNA tiles influences the kinetics of surface-mediated DNA self-assembly.

This manuscript studies the impact of extruding hairpins on two-dimensional self-assembly of DNA tiles on solid surface. Hairpins are commonly used as tomographic markers in DNA nanostructures for atomic force microscopy imaging. In this study, we have discovered that hairpins play a more active role. They modulate the adsorption of the DNA tiles onto the solid surface, thus changing the tile assembly kinetics on the solid surface. Based on this discovery, we were able to promote or slow down DNA self-assembly on the surface by changing the hairpin locations on the DNA tiles. This knowledge gained will be helpful for the future design of DNA self-assembly on surface.

Mechanical deformation behaviors and structural properties of ligated DNA crystals.

DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here, we investigate the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We perform coarse-grained molecular dynamics simulations and confirm the results with nanoindentation experiments using atomic force microscopy. We observe various deformation modes, including untension, linear elasticity, duplex dissociation, and single-stranded component stretch. We find that the mechanical properties of a DNA architecture are correlated with those of its components. However, the structure shows complex behaviors which may not be predicted by components alone and the architectural design must be considered.

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.

Structure-Guided Designing Pre-Organization in Bivalent Aptamers.

Multivalent interaction is often used in molecular design and leads to engineered multivalent ligands with increased binding avidities toward target molecules. The resulting binding avidity relies critically on the rigid scaffold that joins multiple ligands as the scaffold controls the relative spatial positions and orientations toward target molecules. Currently, no general design rules exist to construct a simple and rigid DNA scaffold for properly joining multiple ligands. Herein, we report a crystal structure-guided strategy for the rational design of a rigid bivalent aptamer with precise control over spatial separation and orientation. Such a pre-organization allows the two aptamer moieties simultaneously to bind to the target protein at their native conformations. The bivalent aptamer binding has been extensively characterized, and an enhanced binding has been clearly observed. This strategy, we believe, could potentially be generally applicable to design multivalent aptamers.

Assembly of Two-Dimensional DNA Arrays Could Influence the Formation of Their Component Tiles.

Tile-based DNA self-assembly is a powerful approach for nano-constructions. In this approach, individual DNA single strands first assemble into well-defined structural tiles, which, then, further associate with each other into final nanostructures. It is a general assumption that the lower-level structures (tiles) determine the higher-level, final structures. In this study, we present concrete experimental data to show that higher-level structures could, at least in the current example, also impact on the formation of lower-level structures. This study prompts questions such as: how general is this phenomenon in programmed DNA self-assembly and can we turn it into a useful tool for fine tuning DNA self-assembly?

Boosted Productivity in Single-Tile-Based DNA Polyhedra Assembly by Simple Cation Replacement.

Cations such as divalent magnesium ion (Mg2+ ) play an essential role in DNA self-assembly. However, the strong electrostatic shielding effect of Mg2+ would be disadvantageous in some situations that require relatively weak interactions to allow a highly reversible error-correcting mechanism in the process of assembly. Herein, by substituting the conventional divalent Mg2+ with monovalent sodium ion (Na+ ), we have achieved one-pot high-yield assembly of tile-based DNA polyhedra at micromolar concentration of tiles, at least 10 times higher than the DNA concentrations reported previously. This strategy takes advantage of coexisting counterions and is expected to surmount the major obstacle to potential applications of such DNA nanostructures: large-scale production.