Design And Self-Assembly Of Two-dimensional Nucleic Acid Nanostructures For Evidence Of DNA Conformations

DNA and RNA,as genetic materials in nature,have their own characteristics of nano-sized double helix structures and constantly follow the base complementary pairing principle,making them an ideal nanotechnology material.The biocompatibility,programmability and addressability of nucleic acid molecules endow those nanomaterials with rich functions.In the 1980s,Seeman of New York University,inspired by the Holliday Junction(HJ)structure,used several DNA nucleotides to construct structural motifs and then,successfully self-assembled two-dimensional DNA nano-lattice structures.From then on,the era of DNA self-assembly began.Nucleic acid self-assembly is a process in which nucleic acids are used as molecular building blocks driven by thermodynamics to construct zero,one,two,and three dimensional(OD,1D,2D,and 3D)nano-and micro-structures.This "bottom up" method,can accurately assemble nucleic acid nanostructures with different forms,controllable size,and rich functions from different dimensions and plays an important role in nanomaterials,biosensors,biomedicine,logic calculation and other fields.Circular nucleic acids are ubiquitous in the biological world.The circular nucleic acids are connected end-to-end covalently,so during the assembly of nucleic acid structures,they lack free moving ends,which can increase the rigidity of circular tiles and help to obtain a more stable overall structure.In this thesis,2D nucleic acid nanostructures with different morphologies are constructed using circular nucleic acid molecules as scaffolds.Double Crossover(DX)tiles are the basic building blocks of nucleic acids for DNA nanostructures.The half-turn of a double-stranded DNA helix is often used as the length unit in DNA nanotechnology,that is why the most famous DX tiles are named as DAE(Double crossover,Antiparallel double helices,Even half-turns)and DAO(Double crossover,Antiparallel double helices,Odd half-turns).Through rational design,DAE and DAO tiles can be connected together and extended to DNA nanostructures with different morphologies.I participated in the self-assembly of 2D DNA nanostructures based on small circular DNA molecules of 64nt(abbreviated as c64nt)and 84nt(abbreviated as c84nt).Taking the 64nt molecule as an example,we synthesized a circular 64nt oligomer and named it c64nt,formed a HJ at the center of the c64nt with two helper strands,extended the two helper strands three half-turns outside from the center,and constructed two HJs at both poles,respectively.In this way,we coupled two DAO tiles into an integrated tile cDAO-c64nt.The perfect infinite 2D lattice structures of cDAO-c64nt-E and cDAO-c64nt-Owere obtained by connecting cDAO-c64nt with even and odd half-turns,respectively.Because the subcellular organelles and molecular machines in a cell are precisely assembled and are controllable in size,construction of finite DNA nanostructures are necessary.Using 32 cDAO-c64nt tiles,with the same tile core made from a c64nt molecule but different overhangs,we also completed the self-assembly of a finite lattice of 5×6 patches.Following the same strategy,we also succeded in constructing tHJ-c84nt-E and tHJ-c84nt-O nanostructures.DNA has different conformations,people believe that in nature,right handed B-DNA and A-DNA are dominant conformations,and left-handed Z-DNA is also involed in some cases.A few reports reported that aother DNA conformation of right-handed C-DNA existed in single crystals of synthetic oligomer.However,the C-DNA conformation has not yet been well accepted to occur in nature.In DNA nanotechnology,B-DNA is taken for granted to be dominant or even the only conformation.In our 2D cDAO-c64nt-E and tHJ-c84nt-E structures,we observed that the helical axes were not perpendicular to the laterally aligned tile array,but with a tilt angle.We guess this is due to the conformation evolution of DNA double helices.To prove this hypothesis,we designed and built slanted cDAO tiles and 2D assemblies with both c64nt and c84nt as scaffolds.When shifting the central HJ 1 to 2 bases from the center along the 5'?3' rotation direction of the circle,we obtained four 2D assemblies with dominant A-DNA-like conformations.While shifting the central HJ 1 to 2 bases from the center against the 5'?3' rotation direction of the circle,we obtained other four 2D assemblies,possibly with dominant C-DNA-like conformations.The evolution of the angle between the helical axis and the laterally aligned tile array of all eight structures proves the existence of A-DNA-like configurations,while the existence of C-DNA-like conformations still needs other characterizations for confirmation.This is the first time using DNA nanotechnology to provide experimental results to evidence the existence of A-and C-DNA conformations in solution.Further we extended this strategy to RNA/DNA hybrid structures,attempting to synthesize a circular 66nt RNA(c66nt)and then use it to assemble slanted RNA/DNA hybrid structures.Due to the RNA circularization challenge,we took the linear 66nt RNA strand as a scaffold,while linear DNAs as helper strands,to mimic the RNA/DNA cDAO tiles and 2D assemblies,designed nucleic acid tiles constructed by the C-type nucleic acid double helix and self-assembled to obtain the RNA/DNA hybrid structures.We have obtained slant hybrid structures that are not neatly arranged and tightly connected,which roughly proved that the C-type nucleic acid configuration can also exist naturally in the RNA/DNA hybrid structures.