【作者】 张钊；

【导师】 贺林；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 DNA纳米技术是一门新兴的交叉学科,是指利用DNA自组装构建各种一维、二维、或三维的结构,并开发其应用。DNA纳米技术从80年代中期Seeman开创至今,已经经历了长足发展。国内目前系统介绍该领域的论文或专著极少,所以本论文的目的之一就是对其研究进展做出最新最全的综述,希望能对国内的研究者有所帮助,推动该领域在国内的发展。因为是交叉学科,论文涉及到生物、化学、物理、数学、计算机、纳米技术等知识内容,相信任何相关领域的科研工作者都可以在此找到用武之地。DNA纳米技术包括tile自组装、DNA折纸术、及DNA纳米器件三个大的方向,本文受篇幅所限只涉及前两个分支。除此之外,本文还分析了DNA参与纳米技术领域的种种优势,并详述了DNA纳米技术的一项重要应用——DNA纳米排布。本课题的内容是开发一种新型的DNA折纸芯片,这其实也属于纳米排布的一种。所谓DNA折纸芯片,就是由DNA折纸术得到的图形改造为的芯片。2008年初,美国亚利桑那大学的Ke等研制出了第一张、也是此前唯一一张折纸芯片。他们把折纸图形上的订书钉链伸出V字型探针,然后与溶液中的目标RNA杂交,通过杂交前后AFM图像的亮度差实现检测。该芯片的优点是目标链不用标记,检测较灵敏,但缺点是存在位置效应(折纸图形各位置的探针杂交效率有明显差异),且可扩展性不强。我们的目的就是用新的策略制成折纸芯片,希望在尽量保持Ke等原有优点的同时,改善其不足。我们的核心思路是把V字型探针改为最常用的一字型探针,并引入biotin-STV反应来增强信号强度。具体的做法是在折纸图形特定位置的订书钉链末端伸出寡核苷酸探针,让它与末端修饰biotin的目标链一对一杂交,杂交后加入STV蛋白使得杂交位点在AFM下呈现明显亮点,从而实现检测。另外我们还把Ke等的折纸矩形换为折纸术中国地图,这是为了增加探针的可寻址性。我们先用预实验验证了biotin-STV反应的可行性,然后在正式试验中用8个探针对一个32-mer目标链检测,从AFM的图上可以看出,亮点非常特异,效率较高,与设计完全相同。实验的成功证明了方案的可行,接下来,我们就着手考察本折纸芯片的特性,特别是Ke等芯片中的不足之处。我们共分析了两种效应的影响:位置效应和biotin方向效应。我们先把上面使用过的8个探针分为两组,一组位于图形边界,一组位于中央,然后观察两组的检测效率。结果发现两组并无差异,这说明本芯片无位置效应。我们又把同样的8个探针分为另外两组,一组是订书钉链3’端延伸探针,此时杂交后biotin将朝下,另一组5’延伸,杂交后biotin朝上,也观察两组效率。结果发现3’组比5’组效率高,我们推测主要是3’的成像效果更好,于是以后都推荐用3’延伸。我们还进一步设计了两个更深入的实验。第一个是多重检测,即考察一张芯片同时对多个目标链检测的分辨能力。我们同时在地图的两个位置分别伸出一列32-mer和一列20-mer的探针,然后分别加入对应的目标链,观察图形是否在特异的位置产生亮点。结果显示特异性很好。第二个是夹心法实验,这是一种新的杂交策略,即用capture probe来固定目标链,用reporter probe来释放杂交信号。这种设计的好处要么是折纸芯片本身的通用性,要么是目标链的label-free性,取决于capture探针是否设计为通用。除此之外,我们还做了若干延伸实验。对纳米金和量子点的排布有一些好的结果,但仍需进一步验证;修饰i-motif的实验则没有很好的证据显示分子马达的工作状态。这里特别要说的是利用本折纸芯片对SNP的检测,我们又设计了一种杂交方式,引入了toehold的链竞争机制,目前已实现对两个连续碱基差异的检测。进一步的优化实验仍在进行中,本系统有望实现完全label-free、且可重用的折纸芯片。总之,本研究开发了一种新型的DNA折纸芯片,它与之前已有的折纸芯片相比,不用做索引标记、没有位置效应、具备很强的扩展潜力。本研究不仅对本折纸芯片的特性做了较系统的考察,还设计了另外两种非常有意义的杂交方式,大大推动了本折纸芯片向实用领域的发展。本文第三章对DNA纳米技术将来的几个发展方向做了展望,分别提出了自组装构图、功能化纳米排布、内在机制、实用性四个分支所可能取得突破的一些研究课题。本文最后还以附章的形式介绍了本人在研究生阶段所进行的另一项工作:关联分析。研究集中在G72与COMT两个基因与双相情感障碍(BP)疾病的关联上,最后分别在两个基因中各找到一个与BP呈阳性关联的SNP,rs778293和rs4680,暗示了两个基因对BP致病机理潜在的影响。更多还原

【Abstract】 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.更多还原