CAREER: Defining the Chemical Basis of RNA Function by Nucleotide Analog Interference Mapping

斯特罗贝尔教授的研究重点是建立一种新的方法来确定结构上和催化上重要的RNA分子的结构-活性关系。目标分子包括一系列具有氧化不稳定的5‘-O-(1-硫代)三磷酸取代基的核苷酸类似物。单个类似物将通过体外转录被结合到RNA中，以创建一个突变RNA文库，其中单个核苷酸已被类似物随机取代。在特定生物系统中表现出活性的单个RNA突变体与那些没有表现出活性的RNA突变体是不同的。在温和的条件下，硫代磷酸基团与碘的氧化裂解，以及测序凝胶上活性和非活性类似物的比较分析，为鉴定活性化学基团提供了一种直接的手段。这一方法被称为核苷酸模拟干扰图谱(NAIM)，将用于鉴定RNA中与以下相关的化学基团：(1)5S rRNA与核糖体蛋白L25的相互作用；(2)U6 SnRNA在前mRNA剪接中的作用；(3)自催化与来自脉孢菌的核酶；以及(4)α操纵子mRNA与S4蛋白的相互作用。教育活动包括开发一门生物有机课程，强调合成有机设计和大分子功能，并让当地本科生和少数民族学生参与研究计划。通过这个职业奖项，有机动力学项目支持耶鲁大学分子生物物理和生物化学系的Scott Strobel教授的研究和教育活动。斯特罗贝尔教授的研究重点是开发一种名为核苷酸类似干扰图谱的方法，该方法将显著加快识别结构上和催化上重要的RNA分子中的结构-活性关系的过程。斯特罗贝尔教授的教育活动包括开发一门多部门课程，探索使用有机化学合成来研究和了解生物系统，并通过让当地和少数族裔本科生参与研究计划来促进对化学研究的兴趣。

Professor Strobel's research focuses on establishing a new methodology to determine structure-activity relationships in structurally and catalytically important RNA molecules. Target molecules include a series of nucleotide analogues with oxidatively labile 5'-O-(1-thio)triphosphate substituents. Individual analogues will be incorporated into RNA by in vitro transcription to create a library of mutant RNA's, where individual nucleotides have been randomly replaced by an analogue. Individual RNA mutants that exhibit activity in a particular biological system are distinguished from those that do not. Subsequent oxidative cleavage at the thiophosphate group under mild conditions with iodine, and comparative analysis of the active and nonactive analogues on sequencing gels offers a straightforward means to identify the active chemical group. This methodology, called Nucleotide Analogue Interference Mapping (NAIM), will be used to identify the chemical groups in RNA relating to: (1) the interaction of 5S rRNA with ribosomal protein L25; (2) the role of U6 snRNA in pre-mRNA splicing; (3) the autocatalytic VS ribozyme from Neurospora; and (4) the interaction of alpha operon mRNA with S4 protein. Educational activities include developing a bioorganic course emphasizing synthetic organic design and macromolecular function, and involving both local undergraduates and minority students in the research program. With this CAREER award, the Organic Dynamics Program supports the research and educational activities of Professor Scott Strobel of the Department of Molecular Biophysics and Biochemistry at Yale University. Professor Strobel's research is focused on developing a methodology, called Nucleotide Analogue Interference Mapping, that will significantly accelerate the process of identifying structure-activity relationships in structurally and catalytically important RNA molecules. Professor Strobel's educational activities include developing a multidepartmental course which will explore the use of organic chemical synthesis to investigate and understand biological systems, and promoting an interest in chemical research by involving both local and minority undergraduates in the research program.