Self-assembled DNA Nanotechnology And Its Application In Genotyping

DNA molecules are not merely the secret of life but also can be used as building blocks for self-assembled DNA nanotechnology. Various static DNA nanostructures such as2D DNA lattices and3DDNA crystals based-on tile motifs, discrete DNA origami nanostructures and DNA brick architectureshave been constructed via molecular self-assembly. The promising application of DNA self-assemblyin biomedicine have attracted wild attention. My research mainly focused on constructing the large-scale and complex DNA origami structures, exploiting the programmable kinetic control of self-assembly to construct dynamic molecular structures and applying DNA nanostructures as tools to sovlescience problems in biomedicine and single-molecule biophysics.We utilized long-range polymerase chain reaction (PCR) to amply a26-kb fragment of lambda DNAand obtained long single-strand DNA by using asymmetry enzymatic digestion. The alternative scaffoldstrand can be folded by rational designed staple strands into super-sized rectangular nanostructures,which is nearly four times larger than the conventional M13DNA-based origami. This work providesa reliable method to construct micrometer-sized origami structures and it is possible to amplify muchlonger DNA fragments, which provides an artificial system that can mass produce scaffold strands inhigh yield and high purity. Construct larger and more complex DNA nanostructures, even3D DNAnanoarchitectures with functional molecules and nanomaterials, which can have specific functions innanoplasmonics and electronic and open the doors to combine both bottom-up and top-down fabrication.Hybridization chain reactions (HCR) are often used to engineer complex autonomous nanodevicesin dynamic DNA nanotechnology. We combined HCR and DNA tile self-assembly to cooperativelycontrol the molecular growth. The length of DNA nanostructures can be well controlled by tuning theratio of DNA strands. This work showed HCR can aslo provide kinetic control over the isothermalassembly of one-and two-dimensional DNA nanostructures and the molecular growth of DNA tile-hairpins can self-assembled in isothermal conditions. It provided a novel approach to realize moresophisticated temporal and spatial control of dynamic DNA self-assembly.Determining the variations of the DNA sequence in a diploid is now still a major challenge. In thiswork we show that through a combination of atomic force microscope visible DNA origami probe andallele specific primer extension assisted by gold nanoparticles, it is possible to directly identification ofseveral DNA variations on individual human genomic DNA and genotyping analysis of diseasesusceptibility. Nucleic acids were used as building blocks to construct locus probes with rationallydesigned geometry and kinetics. We designed diblock single-strand DNA, which were functionalizedboth as primers that could hybridize to single nucleotide polymorphism (SNP) loci then be extended by allele specific primer extension assisted by gold nanoparticles and as bonding sites that could berecognized by specific DNA origami probes. Contrast with optical genotyping approach, this methodcan provide higher resolution genotyping. It would be a promising application of DNA self-assemblyin genomic analysis and other fields of biomedicine.