Three-Point-Star Deoxyribonucleic Acid Tiles with the Core Arm Length at Three Half-Turns for Two-Dimensional Archimedean Tilings and Beyond.

2D Archimedean tiling and complex tessellation patterns assembled from soft materials including modular DNA tiles have attracted great interest because of their specific structures and potential applications in nanofabrication, nanoelectronics, nanophotonics, biomedical sensing, drug delivery, therapeutics, etc. Traditional three- and four-point-star DNA tiles with the core arm length at two half-turns (specified as three- and four-point-star-E previously and abbreviated as 3PSE and 4PSE tiles here) have been applied to assemble intricate tessellations through tuning the size of inserted nT (n = 1-7, T is thymine) loops on helper strands at the tile center. Following our recent findings using a new type of four-point-star tiles with the core arm length at three half-turns (specified as four-point-star-O previously and abbreviated as 4PSO tiles here) to assemble DNA tubes and flat 2D arrays, we report here the cross-hybridization weaving architectures at the tile center to construct three new 3PSO tiles with circular DNA oligonucleotides of 96-nt (nucleotides) serving as the scaffolds, further the monotonous and combinatory E- and O-tilings on one type of 3PSO tiles to create 2D Archimedean tiling patterns (6.6.6) and (4.8.8), and finally, the combination of 3PSO with 4PSO as well as 2PSO tiles to tile into complex tessellation patterns. The easy realization of regular and intricate DNA tessellations with 2-4PSO tiles not only richens the fundamental DNA modules and complex DNA nanostructures in types but also broadens the potential application scopes of DNA nanostructures in nanofabrication, DNA computing, biomedicine, etc.

Structural Description of Chiral E-Tiling DNA Nanotubes with the Chiral Indices (n,m) and Handedness Defined by Microscopic Imaging.

In structural DNA nanotechnology, E-tiling DNA nanotubes are evidenced to be homogeneous in diameter and thus have great potential in biomedical applications such as cellular transport and communication, transmembrane ion/molecule channeling, and drug delivery. However, a precise structural description of chiral DNA nanotubes with chiral parameters was lacking, thus greatly hindering their application breadth and depth, until we recently raised and partly solved this problem. In this perspective, we summarize recent progress in defining the chiral indices and handedness of E-tiling DNA nanotubes by microscopic imaging, especially atomic force microscopy (AFM) imaging. Such a detailed understanding of the chiral structures of E-tiling DNA nanotubes will be very helpful in the future, on the one hand for engineering DNA nanostructures precisely, and, on the other, for realizing specific physicochemical properties and biological functions successfully.

Construction of DNA Bilayer Tiles and Arrays Using Circular DNA Molecules as Scaffolds.

Using oligonucleotides to weave 2D tiles such as double crossovers (DX) and multi-arm junction (mAJ) tiles and arrays is well-known, but weaving 3D tiles is rare. Here, we report the construction of two new bilayer tiles in high yield using small circular 84mer oligonucleotides as scaffolds. Further, we designed five E-tiling approaches to construct porous nanotubes of microns long in medium yield via solution assembly and densely covered planar microscale arrays via surface-mediated assembly.

Rapid Signal Amplification Based on Planetary Cross-Catalytic Hairpin Assembly Reactions.

Non-enzymatic nucleic acid catalytic systems based on branch migration have been developed, with applications ranging from biological sensing to molecular computation. A scalable planetary cross-catalytic (PCC) system is built up in this work by cross-cascading three planetary catalytic hairpin assembly (CHA) reactions with a central three-arm-branched CHA reaction. With the bottom-up hierarchy strategy, we designed four levels of catalytic reactions, simple CHA reactions, two-layered linear cascades, conventional one-planetary PCC reactions, and two- and three-planetary PCC reactions, and examined the reaction products and intermediates in each level via native polyacrylamide gel electrophoresis. The gel shift assay optimized the designs of hairpin strands to keep the leaking reactions at a manageable level and protect against signal attenuation during serial signal transduction in nucleic acid circuits. The reaction kinetics, measured via fluorescence, are strongly dependent on the number of planetary reactions. As a result, the three-planetary PCC system achieved an exponential amplification factor of about 3k, while the conventional one-planetary cross-catalytic system has an amplification factor of 2k (k represents the cycling number). Finally, we demonstrated the rapid detection of a cancer biomarker, microRNA141, used as the catalyst in a two-planetary PCC system. We envision that the PCC systems could be applied in biological signal transduction, biocomputing, rapid detection of single- and multi-target nucleic acid probes, etc.

Tuning the Roundabout of Four-Point-Star Tiles with the Core Arm Length of Three Half-Turns for 2D DNA Arrays.

By rationally adjusting the weaving modes of point-star tiles, the curvature inherent in the tiles can be changed, and various DNA nanostructures can be assembled, such as planar wireframe meshes, perforated wireframe tubes, and curved wireframe polyhedra. Based on the weaving and tiling architectures for traditional point-star tiles with the core arm length at two DNA half-turns, we improved the weaving modes of our newly reported four-point-star tiles with the core arm length at three half-turns to adjust their curvature and rigidity for assembling 2D arrays of DNA grids and tubes. Following our previous terms and methods to analyze the structural details of E-tiling tubes, we used the chiral indices (n,m) to describe the most abundant tube of typical assemblies; especially, we applied both one-locus and/or dual-locus biotin/streptavidin (SA) labelling strategies to define the configurations of two specific tubes, along with the absolute conformations of their component tiles. Such structural details of the DNA tubes composed of tiles with addressable concave and convex faces and packing directions should help us understand their physio-chemical and biological properties, and therefore promote their applications in drug delivery, biocatalysis, biomedicine, etc.

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.

2D DNA lattices assembled from DX-coupled tiles.

HYPOTHESIS: Formation of coupled double crossovers (DXs) in a circular 106-mer oligonucleotide (c106nt) could generate stable tiles with the tile core span of 10 half turns. The large-span tiles with complicated curvatures and mechanics could assemble 2D lattices under different environments. Hence, 2D DNA lattice structures based on tile types and sequences, tile packing modes of corrugation and non-corrugation, and assembly media of solution and the substrate-solution interface are yet to be explored. EXPERIMENTS: Two c106nt scaffold strands with different sequences were synthesized. Four types of tiles, two rectilinear and two triangular tiles, were designed and their 2D assemblies were examined by atomic force microscopy (AFM). FINDINGS: The DX-coupled tiles provided fair strength and rigidity to assemble 2D lattices. Due to the complicated curvature and mechanics of tiles, the two-tile assemblies in solution displayed a few ripe lattices of ribbons, tubes, or polycrystalline aggregates as the minor products and tile oligomers as the major products; whereas the one-tile assemblies via substrate mediation exhibited well-organized monolayer lattices covering the whole mica disk. The herringbone packing patterns were first observed in DNA nanostructures. Based on the lattice constants and the surface coverages of lattices, we estimated the lattice yields for the substrate-mediated assemblies.

Two-layer stacked multi-arm junction tiles and nanostructures assembled with small circular DNA molecules serving as scaffolds.

One-layer multi-arm junction (mAJ) motifs have been investigated extensively for many kinds of planar 2D (two-dimension) lattices, surface-curved 3D (three-dimension) polyhedra, and complex 3D wireframe and tensegrity structures. Herein, we report the weaving strategy to achieve two-layer stacked multi-arm junction tiles (abbreviated as mAJ2) of 3AJ2 and 4AJ2, and several primary tessellation nanostructures of nanocages and 2D rhombus lattices carrying beautifully embossed 4-point stars. Challenges for perfect tessellation are also raised regarding the increase of motif complexity from 2D to 3D.

Small Circular DNA Molecules as Triangular Scaffolds for the Growth of 3D Single Crystals.

DNA is a very useful molecule for the programmed self-assembly of 3D (three dimension) nanoscale structures. The organised 3D DNA assemblies and crystals enable scientists to conduct studies for many applications such as enzymatic catalysis, biological immune analysis and photoactivity. The first self-assembled 3D DNA single crystal was reported by Seeman and his colleagues, based on a rigid triangle tile with the tile side length of two turns. Till today, successful designs of 3D single crystals by means of programmed self-assembly are countable, and still remain as the most challenging task in DNA nanotechnology, due to the highly constrained conditions for rigid tiles and precise packing. We reported here the use of small circular DNA molecules instead of linear ones as the core triangle scaffold to grow 3D single crystals. Several crystallisation parameters were screened, DNA concentration, incubation time, water-vapour exchange speed, and pH of the sampling buffer. Several kinds of DNA single crystals with different morphologies were achieved in macroscale. The crystals can provide internal porosities for hosting guest molecules of Cy3 and Cy5 labelled triplex-forming oligonucleotides (TFOs). Success of small circular DNA molecules in self-assembling 3D single crystals encourages their use in DNA nanotechnology regarding the advantage of rigidity, stability, and flexibility of circular tiles.

2D DNA lattice arrays assembled from DNA dumbbell tiles using poly(A-T)-rich stems.

Poly(A-T)-rich sequences have been applied as stems of DNA dumbbell tiles for construction of single crystalline 2D DNA lattice arrays in slightly acidic solutions. These arrays show much higher stability and better organised crystalline lattice structures than those assembled from DNA dumbbell tiles with randomly sequenced stems in slightly alkaline environments. DNA nanotechnology probably provides a useful platform to study the mechanical properties of DNA duplexes with specific sequences.

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

DNA dumbbell tiles with uneven widths for 2D arrays.

Small DNA tiles built from linear oligonucleotides for the construction of DNA nanostructures often have even widths in the lateral direction against the DNA helical axis. Herein we present an example of DNA dumbbell tiles with uneven widths between the central double helical stem and the two head loops after association with helper strands. A characteristic of DNA dumbbell tiles is that the stem length controls the stereostructure of the two head motifs. A stem at 11 bp twists two head motifs of a dumbbell tile into parallel conformations, wheareas a stem at 16 bp twists two head motifs into antiparallel conformations. We successfully constructed four DNA dumbbell tiles and assembled them into two-dimensional (2D) arrays of planar nanoribbons with zebra-like patterns and fine nanotubes as well.

Construction of a Holliday Junction in Small Circular DNA Molecules for Stable Motifs and Two-Dimensional Lattices.

Design rules for DNA nanotechnology have been mostly learnt from using linear single-stranded (ss) DNA as the source material. For example, the core structure of a typical DAO (double crossover, antiparallel, odd half-turns) tile for assembling 2D lattices is constructed from only two linear ss-oligonucleotide scaffold strands, similar to two ropes making a square knot. Herein, a new type of coupled DAO (cDAO) tile and 2D lattices of small circular ss-oligonucleotides as scaffold strands and linear ss-oligonucleotides as staple strands are reported. A cDAO tile of cDAO-c64nt (c64nt: circular 64 nucleotides), shaped as a solid parallelogram, is constructed with a Holliday junction (HJ) at the center and two HJs at both poles of a c64nt; similarly, cDAO-c84nt, shaped as a crossed quadrilateral composed of two congruent triangles, is formed with a HJ at the center and four three-way junctions at the corners of a c84nt. Perfect 2D lattices were assembled from cDAO tiles: infinite nanostructures of nanoribbons, nanotubes, and nanorings, and finite nanostructures. The structural relationship between the visible lattices imaged by AFM and the corresponding invisible secondary and tertiary molecular structures of HJs, inclination angle of hydrogen bonds against the double-helix axis, and the chirality of the tile can be interpreted very well. This work could shed new light on DNA nanotechnology with unique circular tiles.

DNA nanotubes assembled from tensegrity triangle tiles with circular DNA scaffolds.

Using small circular DNA molecules of different lengths as scaffolds, we successfully synthesised DNA nanotubes consisting of Mao's DNA tensegrity triangle tiles with four-arm junctions (Holliday junctions) at all vertices. Due to the intrinsic curvature of the triangle tile and the consecutive tile alignment, the 2D arrays are organised in the form of nanotubes. Two sized triangle tiles with equilateral side lengths of 1.5 and 2.5 full helical turns are connected by the sticky ended cohesion of a duplex with a length of 2.5 helical turns respectively, and their parallel lozenge tiling lattices were demonstrated by high resolution AFM images, where the former lozenge unit cell has a lattice constant of 13.6 nm, and the latter has a larger lattice constant of 17.0 nm. Modification of the triangle tile with infinitesimal disturbance on side lengths and insertion of one thymine single stranded loop at every vertex resulted in comparably similar nanotubes.

Assembling RNA Nanoparticles.

RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

2D DNA lattices constructed from two-tile DAE-O systems possessing circular central strands.

We reported a classical two-tile system of DAE-O (doublecrossover, antiparallel, and even half-turns tiles with odd half-turns connection) to construct regular single crystalline 2D (two dimensional) DNA lattices, using pre-circularised oligonucleotides of 42-, 64-, and 84-nt (nucleotides) as the central looped strands in DAE tiles respectively. DAE tiles with 42- and 64-nt as central strands, either in circular form or in linear form, grew regular single crystalline lattices well. However DAE tiles including a circular 84-nt as the central strand grew single crystalline lattices, those including a linear 84-nt as the central strand grew polycrystalline 2D lattices. A subtle difference in the lateral rigidity of DAE tiles with regard to the duplex axis was suggested to be the cause of the morphological difference.

In-Phase Assembly of Slim DNA Lattices with Small Circular DNA Motifs via Short Connections of 11 and 16 Base Pairs.

Two kinds of stable motif were constructed: SAE (semi-crossover, antiparallel, even half-turns) tile from one small circular DNA molecule (42 or 64 nt) and two linear oligonucleotides; and DAE (double-crossover, antiparallel, even half-turns) tile from one small circular DNA molecule (42 or 64 nt) and four linear oligonucleotides. With the SAE tiles, in-phase assembly of SAE-E (SAE tiles with even half-turns as connections (-E)) with the shortest -E of 11 base pairs (bp) generated homogeneous nanotubes with an average length of over 14 µm and a diameter of 16-20 nm; with the DAE tiles, in-phase assembly of DAE-O (DAE tiles with odd half-turns as connections (-O)) with the shortest -O of 16 bp produced slim monolayer nanoyarns (25-30 nm wide), nanoscarfs (100-300 nm wide), and nanoribbons (∼100 nm wide). Interestingly, a phenomenon we term "knitting nanoyarns" into nanoscarfs was observed. Finally a curvature mechanism according to the ring rotation directions is suggested to explain the formation of nanotubes, wavy nanoyarns, nanoscarfs, and nanoribbons.

Growing Embossed Nanostructures of Polymer Brushes on Wet-Etched Silicon Templated via Block Copolymers.

Block copolymer nanolithography has attracted enormous interest in chip technologies, such as integrated silicon chips and biochips, due to its large-scale and mass production of uniform patterns. We further modified this technology to grow embossed nanodots, nanorods, and nanofingerprints of polymer brushes on silicon from their corresponding wet-etched nanostructures covered with pendent SiHx (X = 1-3) species. Atomic force microscopy (AFM) was used to image the topomorphologies, and multiple transmission-reflection infrared spectroscopy (MTR-IR) was used to monitor the surface molecular films in each step for the sequential stepwise reactions. In addition, two layers of polymethacrylic acid (PMAA) brush nanodots were observed, which were attributed to the circumferential convergence growth and the diffusion-limited growth of the polymer brushes. The pH response of PMAA nanodots in the same region was investigated by AFM from pH 3.0 to 9.0.

Small circular DNA molecules act as rigid motifs to build DNA nanotubes.

Small circular DNA molecules with designed lengths, for example 64 and 96 nucleotides (nt), after hybridization with a few 32-nt staple strands respectively, can act as rigid motifs for the construction of DNA nanotubes with excellent uniformity in ring diameter. Unlike most native DNA nanotubes, which consist of longitudinal double helices, nanotubes assembled from circular DNAs are constructed from lateral double helices. Of the five types of DNA nanotubes designed here, four are built by alternating two different rings of the same ring size, while one is composed of all the same 96-nt rings. Nanotubes constructed from the same 96-nt rings are 10-100 times shorter than those constructed from two different 96-nt rings, because there are fewer hinge joints on the rings.

Periodical assembly of repetitive RNA sequences synthesized by rolling circle transcription with short DNA staple strands to RNA-DNA hybrid nanowires.

The long single-stranded RNAs produced from Rolling Circle Transcription (RCT) were used as scaffolds to be folded with a few short DNA staples into RNA-DNA hybrid nanowires.