Self- Assembly Of DNA From Rolling Circle Amplification

Owing to the exquisite specificity of Watson-Crick base pairing, in 1982 Seeman proposed to use DNA as a construction material for the assembly of geometrically defined objects with nanoscale features. In recent years, the controlled folding of a long single M13mp18 genomic DNA, so-called DNA origami, has been developed greatly and utilized in many areas. DNA origami, in principle, could avoid the problems of stoichiometry and purification associated with the approaches by using many short oligonucleotides for nanostructural assembly, often termed DNA tiles. It means an extraordinary breakthrough in the construction of nanometer-sized DNA objects.However, this strategy usually takes hundreds of different staple strands to fold one long scaffold strand DNA into a nanoscale shape. The scaffold strands used in DNA origami usually are long sequence-identified natural DNA strands (until recently only M13 DNA) which are inaccessible by chemical synthesis. To simplify the assembly process of DNA origami, we have endeavored to use products of rolling circle amplification (RCA) as the scaffold strand to create nanostructure, etc.RCA is an isothermal, enzymatic amplification process mediated by certain DNA polymerases such as Phi29 DNA polymerase to convert dNTPs into a long single strand DNA (ssDNA) molecule based a circularized template. DNA strands produced by this process contain hundreds of tandemly repeated copies wich are complementary to the original circularized template. This feature is particularly useful in DNA assembly.In this thesis, we described two new strategies to create nanowires and nanoplanes with RCA products respectively. The specific research works are as follows:1. Folding RCA products to create nanowire.Instead of using long natural ssDNA with definite sequence, we used RCA products as scaffolds to create DNA nanowire. According to Rothemund’s sequence design principle for origami structural engineering, we designed and created three kinds of nanowires with two different RCA products.All three target structures were observed by atomic force microscopy (AFM). And experiment results were consistent with the theoretical value. Owing to the repeating sequence of RCA products, this new strategy significantly simplify the design of DNA origami by replacing hundreds of different staple strands with a few staple strands, meanwhile, it still inherits many advantages of DNA origami, such as a short thermal annealing process and a high yields of the target structure.2. Folding RCA products create nanoplane.To further develop the usage of RCA in DNA nanotechnology, we developed a new strategy to create large scale two-dimensional DNA nanoplane with RCA products. In this strategy, we borrowed the design ideas of self-assembly of oligonucleotide, and managed to fold multiple RCA strands into one nanostructure to create a nanoplane.Owing to the repeating sequence of RCA strand, there are several equivalent folding structures for the same set, depending on the design. And based on this feature, we designed and created one kind of nanowire and two kinds of nanoplanes with same set of staple strands. All three target structures were observed by AFM. Experiment results were consistent with the theoretical value. Although this new strategy loosely bases on the design ideas of self-assembly of oligonucleotide, it still retains many characteristics of DNA origami, such as a short thermal annealing process and a high yields of the target structure.And we aslo observed that the transitions between those three nanosturctures, and demonstrated that the the transitions were based on the concentration of RCA scaffolds.