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.

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.

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.

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.

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.

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.

A proximity-based programmable DNA nanoscale assembly line.

Our ability to synthesize nanometre-scale chemical species, such as nanoparticles with desired shapes and compositions, offers the exciting prospect of generating new functional materials and devices by combining them in a controlled fashion into larger structures. Self-assembly can achieve this task efficiently, but may be subject to thermodynamic and kinetic limitations: reactants, intermediates and products may collide with each other throughout the assembly time course to produce non-target species instead of target species. An alternative approach to nanoscale assembly uses information-containing molecules such as DNA to control interactions and thereby minimize unwanted cross-talk between different components. In principle, this method should allow the stepwise and programmed construction of target products by linking individually selected nanoscale components-much as an automobile is built on an assembly line. Here we demonstrate that a nanoscale assembly line can be realized by the judicious combination of three known DNA-based modules: a DNA origami tile that provides a framework and track for the assembly process, cassettes containing three independently controlled two-state DNA machines that serve as programmable cargo-donating devices and are attached in series to the tile, and a DNA walker that can move on the track from device to device and collect cargo. As the walker traverses the pathway prescribed by the origami tile track, it sequentially encounters the three DNA devices, each of which can be independently switched between an 'ON' state, allowing its cargo to be transferred to the walker, and an 'OFF' state, in which no transfer occurs. We use three different types of gold nanoparticle species as cargo and show that the experimental system does indeed allow the controlled fabrication of the eight different products that can be obtained with three two-state devices.

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.

Circular RNA oligonucleotides: enzymatic synthesis and scaffolding for nanoconstruction.

We report the efficient synthesis of monomeric circular RNAs (circRNAs) in the size range of 16-44 nt with a novel DNA dumbbell splinting plus T4 DNA ligation strategy. Such a DNA dumbbell splinting strategy was developed by one group among ours recently for near-quantitative conversion of short linear DNAs into monomeric circular ones. Furthermore, using the 44 nt circRNA as scaffold strands, we constructed hybrid RNA:DNA and pure RNA:RNA double crossover tiles and their assemblies of nucleic acid nanotubes and flat arrays.

RCA strands as scaffolds to create nanoscale shapes by a few staple strands.

Thousands of nucleotide(nt)-long single strand DNAs, generated from rolling-circle-amplification (RCA), were used as scaffolds to create DNA nanoscale wires and plates with a few short staple strands by following the origami design principle with a crossover at 1.5 turns. The core sequence of the circle template, for producing tens and hundreds of tandemly repeated copies of it by RCA, was designed according to Seeman's sequence design principle for nucleic acid structural engineering (Seeman, N. C. J. Biomol. Struct. Dyn. 1990, 8, 573). The significance for folding the RCA products into nanoscale shapes lies in the design flexibility of both staple and scaffold strand codes, simplicity of a few short staple strands to fold the periodic sequence of RCA products, and lower cost.

DNA microarray fabricated on poly(acrylic acid) brushes-coated porous silicon by in situ rolling circle amplification.

Microarrays hold considerable promise in large-scale biology on account of their analytical, massive and parallel nature. In a step toward further enabling such a capability, we describe the application of rolling circle amplification (RCA) for a sensitive and multiplex detection of nucleic acid targets on oligonucleotide-conjugated polymer brushes covalently grown from porous silicon. Both RCA and polymer brushes have been taken to increase the loading quantity of target molecules and thus improve the detection sensitivity without loss of multiplexing. Besides, polymer brushes were employed to protect porous silicon and to provide biologically simulated environments, making the attached biomolecules maintain bioactivity. This approach can reach a detection limit of 0.1 nM target analytes and three orders of magnitude dynamic range of 0.1-100 nM, with a fluorescence scanner. A two-colour DNA microarray was achieved via RCA of two kinds of circular DNA targets on one chip simultaneously. The porous silicon chip-based RCA technique is promising for the multiplex detection of deoxynucleic acids on microarrays.

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.

Different EDC/NHS activation mechanisms between PAA and PMAA brushes and the following amidation reactions.

Infrared spectroscopy was applied to investigate the well-known EDC/NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) activation details of poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) brushes grafted on porous silicon. Succinimidyl ester (NHS-ester) is generally believed to be the dominant intermediate product, conveniently used to immobilize biomolecules containing free primary amino groups via amide linkage. To our surprise, the infrared spectral details revealed that the EDC/NHS activation of PMAA generated anhydride (estimated at around 76% yield and 70% composition), but not NHS-ester (around 5% yield and 11% composition) under the well-documented reaction conditions, as the predominant intermediate product. In contrast, EDC/NHS activation of PAA still follows the general rule, i.e., the expected NHS-ester is the dominant intermediate product (around 45% yield and 57% composition), anhydride the side product (40% yield and 28% composition), under the optimum reaction conditions. The following amidation on PAA-based NHS-esters with a model amine-containing compound, L-leucine methyl ester, generated approximately 70% amides and 30% carboxylates. In contrast, amidation of PAA- or PMAA-based anhydrides with L-leucine methyl ester only produced less than 30% amides but more than 70% carboxylates. The above reaction yields and percentage compositions were estimated by fitting the carbonyl stretching region with 5 possible species, NHS-ester, anhydride, N-acylurea, unreacted acid, unhydrolyzed tert-butyl ester, and using the Beer-Lambert law. The different surface chemistry mechanisms will bring significant effects on the performance of surface chemistry-derived devices such as biochips, biosensors, and biomaterials.

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.

Gel-pad microarrays templated by patterned porous silicon for dual-mode detection of proteins.

A proof-of-concept study demonstrated the feasibility of a novel gel-pad microarray on porous silicon chips, by initiation of an atom transfer radical propagation (ATRP) polymerisation of (polyethylene glycol) methacrylate (PEGMA) with surface Si-H species, stepwise chemical conversions of the gel membrane to an NTA-Ni2+/histidine-tagged protein system, and matrix-assisted laser desorption/ionisation mass spectroscopy (MALDI MS) and fluorescence detections.

Click-chemistry-conjugated oligo-angiomax in the two-dimensional DNA lattice and its interaction with thrombin.

The recently arising antithrombin drug, angiomax, was successfully conjugated with a 5'-amino oligonucleotide through click chemistry. This oligo-angiomax conjugate was assembled into a two-dimensional DNA lattice with other oligonucleotides together. Besides the plane sheet of DNA lattices, an interesting angiomax-involved DNA tubing structure, constructed by 40 to 50 angiomax stripes which are parallel to the longitudinal axis of the tube, was also imaged. After incubation of thrombins with the angiomax-involved DNA lattice, the binding of thrombins to arrayed angiomax peptides was observed. Finally a chromogenic substrate bioassay was employed to estimate the antithrombin activities as assembled oligo-angiomax DNA lattice approximately 1.1, oligo-angiomax approximately 2.7 angiomax. The functionalized DNA lattices have the potential to be used as a powerful platform for investigation of biomolecular interactions such as drug-protein, protein-protein, DNA-RNA, and DNA-protein interactions in the nano- and subnanoscales.