DNA nanotechnology, commencing in 1980s, seeks to create versatile controllable structures out of DNA and explore their applications. Although it has made great progress, until now there are few theses or monographs written in Chinese systematically introducing this field. So one of the important aims of this thesis is giving a comprehensive review to DNA nanotechnology, hopefully promoting its development in China. Since it's an interdiscipline, this thesis involves knowledge of biology, chemistry, physics, mathematics, computer, nanotechnology. We encourage any researchers in relevant field work on this emerging cross-field.DNA nanotechnology can be divided into three subdivisions, which are tile-based self-assembly, DNA origami, and DNA nanodevices. For lack of space, in the first chapter of this thesis we only focused the first two. Moreover, We also indicated the inherent advantages of DNA to serve as a construction material in nanosciences, and looked into an important application in structural DNA nanotechnology, DNA nanofabrication.The main subject of this thesis is about DNA origami chip, which actually belongs to nanofabrication. DNA origami chip means a chip based on DNA origami technique. In early 2008, Ke et al. at Arizonal University successfully constructed the first, and until now the only origami chip. They prolonged the staple strands by half-probes, two of which hybridize with one target and form a stiff V-shaped structure, and detected the height difference before and after hybridization by AFM. Although their system was target label-free and had good sensitivity, the fact that they displayed a strong position-dependent hybridization effect, which meant probes at different positions in the origami chip showed significantly different hybridization efficiency, really bothered. What's more, Their chip didn't seem to have direct extended applications. So our objective is to make origami chips using other strategy, hopefully improving the limitations in Ke et al.'s chip, as well as maintaining their advantages.In this work, we generated DNA origami chips using single probe instead of V-shaped probe, and introduced biotin-STV interaction in our system. We first demonstrated that biotin-STV could serve as an effective AFM label for protruding probes on DNA origami tiles, by doing a pilot experiment. Then eight staple strands forming a―II‖pattern serve as modified points by extending 32 nucleotides at one end, which are complementary probes for a biotinylated DNA target of 32 bases. After incubation with STV, bound proteins were found clearly and specifically at the predicted position. The result demonstrated our chip worked well, so next we explored its characteristics.We first splited the eight modified positions of II pattern into two groups. One was near the edge of the chip while the other was in the middle. AFM images showed that no significant differences between the two patterns, which revealed position-dependent hybridization effect was not shown in our system. Then we discussed the impact of protruding directions of probes. we divided the II pattern into two columns. One was elongating probe from 3'end of the staple strands, which resulted proximal biotin after hybridization. The other was 5'elongation and distal biotin. The result demonstrated that proximal biotin can assist fixing the streptavidin position as well as improving imaging quality.We also did two further experiments about our chip. We examined its ability of multiplex detection, and got good specificity. We also performed a test to use a sandwich probe strategy for target detection. This new design also worked well, which had the potential of coustructing universal origami chip or label-free detection, up to whether the capture probe were designed common. In addition, we tapped the latent extensibility of our chip system. We used it to fabricate AuNP and QD, whose result was OK in despite of needing further proof. However, the states of pH-driven nanomachines, called i-motif, were not seen on our chip. Remarkably, we employed the toehold reaction for SNP detection in our chip, and AFM images could separate different strands with two consecutive different bases now. Further experiment was still in process, and a reusable origami chip for totally label-free target detection can be expected.In summary, in this work we constructed a new DNA origami chip with single strand probes on asymmetric self-assembled tile incorporating biotin-streptavidin interaction. Such a system for DNA target detection is free of index, without positional effect, and having high efficiency and specificity. We also explored two additional hybridization manner, and both worked well. These results demonstrated the potential for using DNA origami to template components with nanometer-scale precision by oligo hybridization strategy.The third chapter of this thesis looks into the prospect of DNA nanotechnology. I discuss and foresee potential breakthrough in self-assembly construction, functional nanofabrication, thermodynamic mechanism, and practicability in DNA nanotechnology field. I also mention some related projects I take part in.In the last chapter of this thesis, I give a brief introduction of another work, which I did during my doctoral years. It's an association study between two genes and bipolar disorder. Finally we found two SNPs, rs778293 in G72 and rs4680 in COMT, confered susceptibility to BP in the Chinese Han population. Both were first evidences. |