MODIFIED NUCLEOSIDES AS TOOLS FOR MOLECULAR BIOLOGY

本研究的长期目标是开发适合于需要引入或识别核酸序列简并的应用的修饰核苷。该项目初期的结果支持了唑类羧胺在DNA识别中能够作为天然碱基模拟物的假设。此外，唑氮取代模式对碱基配对特异性有深远的影响。我们有足够的信息来合理设计第二代唑胺核苷。具体目的包括：1)合成一系列唑类羧酰胺核苷，研究DNA聚合酶-核碱基相互作用在模板定向合成中的作用。2)构建含寡核苷酸的第二代唑核碱基进行生物物理表征。3)研究含有第一代和第二代唑核碱基的寡核苷酸作为Taq DNA聚合酶的模板，并研究唑核苷三磷酸作为PCR的替代底物。4)在定向进化技术中利用唑核碱基的两种策略探索。本提案中描述的类似物在天然核碱基结构的空间限制内呈现出广泛的电子构型多样性，从而为探索酶的识别和功能提供了独特的机会。本提案中描述的技术有可能提供从起始基因序列开始的短随机漫步的多样性空间的彻底探索。虽然这一建议主要围绕DNA聚合酶展开，但含有寡核苷酸的唑核碱基与其他核酸酶的相互作用是一个未开发的领域，有可能导致对核酸-蛋白质相互作用的基本理解，以及开发用于蛋白质工程和定向进化的新工具。

The long-range goal of this research is to develop modified nucleosides suitable for applications that require either the introduction or the recognition of nucleic acid sequence degeneracy. The results from the initial period of this project support the hypothesis that azole carboxamides are able to function as natural base mimics in DNA recognition. Furthermore, the azole nitrogen substitution pattern has a profound influence on the base pairing specificity. We have sufficient information to rationally design a second generation of azole carboxamide nucleosides. Specific aims include: 1) Synthesis of a series of azole carboxamide nucleosides to investigate the role of DNA polymerase-nucleobase interactions in template directed synthesis. 2) Construction of second generation azole nucleobase containing oligonucleotides for biophysical characterization. 3) Investigation of oligonucleotides containing first and second generation azole nucleobases as templates for Taq DNA polymerase and investigation of azole nucleoside triphosphates as alternative substrates in PCR. 4) Exploration of two strategies for using the azole nucleobases in directed evolution technology. The analogs described in this proposal present a broad diversity of electronic configurations within the steric confines of natural nucleobase structure, thereby providing a unique opportunity to explore enzyme recognition and function. The techniques described in this proposal have the potential to provide a thorough exploration of diversity space in short random walks from a starting gene sequence. Although this proposal centers primarily around DNA polymerase, the interaction of azole nucleobase containing oligonucleotides with other nucleic acid enzymes is an unexplored territory with the potential to lead to both fundamental understanding of nucleic acid-protein interactions as well as the development of new tools for protein engineering and directed evolution.