Self-assembly Of DNA Nanostructures Based On Small Circular DNAs And Their Left-and Right-handed Helix Chiralities

DNA is well known as the important molecule for heredity information storage and transmission in biological systems.Because of its double helix geometry and the principle of complementary base pairing,DNA molecule is equipped with the features of biological compatibility,programmability and addressability.Hence DNA is regarded as an ideal and functional nanomaterial in the field of DNA nanotechnology.In 1982,Professor Nadrian C.Seeman,at New York university,first proposed using DNA modules to build ordered nanostructures,which means the start of DNA nanotechnology.Since then,DNA Origami,single-stranded tiles?SSTs?and other nanotechnologies have been developed.The size of nanostructures built from many kinds of tiles is continuously expanding,from nanometer scale to microns,and even to millimeter scale.DNA nanostructures include 1D nanowires and nanotubes,2D nanoribbons,and 3D nanoarrays and single crystals.The design and self-assembly of complex DNA nanostructures become easier.Nowadays DNA nanostructures are widely applied in drug delivery,biosensing,biological imaging,logic computing,and so on.In DNA nanotechnology,the linear DNAs are mostly used;however,the small circular DNAs,which have special biological and topological properties,have yet been investigated until we started using them.We assume that the application of small circular DNAs in DNA nanotechnology will obviously improve the rigidity of DNA tiles,enhance the control-ability of the geometric complexity of nanostructures,and further expand their applications.Herein,we explored the construction of 2D and 3D nanostructures based on 84 nucleotides and 64nucleotides circular DNA?c84nt DNA,c64nt DNA,c:circular?and the influencing factors for transforming the double-helix from right-handed to left-handed.Traditionally,Holliday junctions?HJs?play an important role in enhancing the rigidity of nanostructures,so we constructed HJs in c84nt and c64nt DNAs to form tiles,called 84HJ?84:c84nt,HJ:Holliday Junction?and 64HJ,respectively.In 84HJ tiles,we set aside 2×10nt from c84nt at both end sides,which will pair with helper strand,and form 84t HJ?t:triangle?tiles.84t HJ tiles can be connected together to form 2D arrays via constructing three-way junctions?TX?and double helix arms.In 64t HJ,polar HJs and extruding double helix arms can be formed at both sides for joining together to nanostructures.First,we constructed nanotubes via small circular DNA tiles.Based on 84t HJ and 84t HJ-3T tiles,we constructed Layered-crossover?LX?tiles,named 84LX_n?84:c84nt,LX:Layered-crossover,n:site number?,which were used to explore the influence of the different sites and tiles'intrinsic properties in the assembly of nanotubes.Herein,the 84t HJ-3T tile means that each of a 84t HJ tile's four corners is inserted with three bases of thymine?TTT?.In this work,we constructed four categories of layered-crossover tiles for forming nanotubes:84LX_n-S?84:c84nt DNA,LX:Layered-crossover,n:site number,S:Sticky ends?,84LX_n-3T-S?84:c84nt DNA,LX:Layered-crossover,n:site number,3T:three T bases,S:Sticky ends?,84LXD_n-A?84:c84nt DNA,LX:Layered-crossover,D:Diagonal,n:site number,A:Adjacent?and84LXD_n-I?84:c84nt DNA,LX:Layered-crossover,D:Diagonal,n:site number,I:Interval?.Next,we explored the construction of 2D and 3D nanostructures of one-layer,two-layers and three-layers based on c84nt DNA and c64nt DNA.For single-layer structures,we designed and assembled three kinds of nanostructures from 84t HJ tiles through 21bp connections,in order to form planar structures with hexagon lattices.For two-layer nanostructures,we explored the stability of bilayer tiles from 3 to 16 sites and the assembly size by the base stacking force based on 84LX_ntiles.For three-layer nanostructures,we connected two 64LX₈ tiles through 21nt intertile arms.We also tried the assembly of finite nanostructures of 2-tiles wide using two64LX₈ tiles.Finally,we proposed that the right-handed helix in 3D DNA nanostructures could transfer into the left-handed helix conformation.Therefore,we designed a series of tiles based on 64LX₈ to explore the influencing factors that make double helix conformation conversion from right-handed to left-handed by CD spectrometer.We explored the following parameters,inserting special sequences into different locations,increasing the cationic concentrations,and changing the environment temperatures.Above all,these experimental results help us learn more about the physical-chemical properties of tiles constructed by small circular DNAs,provide another strategy for construction of larger and more complex nanostructures,and lay a foundation for applying DNA nanostructures into reality.