CAREER: Understanding the Structural Basis for Ligand Recognition by RNA Aptamers

WilsonMCB 98763501。技术：这项研究探索了RNA的折叠结构及其与其他分子的相互作用。 从随机序列样本中分离的适体，包括特异性结合酶辅因子氰钴胺素和生物素以及发色团孔雀石绿绿色的RNA，将作为模型系统。 重点放在这些分子的生物化学表征和晶体和溶液样品的制备上，使其能够通过X射线衍射和NMR光谱进行分析。氰钴胺和生物素适体晶体的结构将通过X射线晶体学确定，同位素标记的孔雀石绿适体RNA通过溶液NMR光谱确定。这是为了理解稳定RNA折叠的特定原子相互作用，RNA可获得的结构折叠的多样性，RNA功能中金属和沃茨的参与，以及RNA与其他分子相互作用导致的构象变化的本质。该项目的教育部分旨在通过课堂和实验室教学相结合，利用结构生物学向学生介绍物理科学。 新的和修改后的课程，在真核生物分子生物学和结构生物学，辅之以计算机为基础的教学材料的发展，将开发建立大分子结构和功能之间的关系。 将鼓励选定数量的学生参与研究. 非技术性：核糖核酸（RNA）折叠成特定构象并由此获得功能特性（例如结合分子或催化反应的能力）的能力越来越受到重视，部分原因是通过实验，RNA从随机序列分子库中从头进化而来。 这种认识激发了关于RNA参与许多生物过程（例如核糖体指导的蛋白质合成）的猜测，并支持了将基于RNA的生物学视为现代生命前身的理论。 我们对RNA功能的结构基础的理解，对于解决这些问题至关重要，仍然很差，部分原因是很少有RNA结构在原子分辨率下被确定。体外进化的配体结合RNA将作为理解RNA结构及其与RNA功能关系的模型系统。 X射线晶体学和核磁共振波谱学将用于本研究中，以确定三种不同的RNA-配体复合物的高分辨率模型。 对这些结构的分析将揭示特定RNA序列形成独特结构的机制以及这些结构如何使其与其他分子发生特异性相互作用，并将这些研究与教学相结合，为本科生提供结构生物学的基本技能。对现有真核生物分子生物学课程的改进将强调生物过程是由大分子折叠构象所指定的分子相互作用驱动的概念。 一门新的结构生物学课程将在这一主题的基础上进行扩展，并增加一个目标，即提供应用物理科学和计算科学的跨学科培训。

WilsonMCB 98763501. Technical: This study explores the folded structures of RNAs and theirinteractions with other molecules. Aptamers isolated from random sequencepools, including RNAs that specifically bind the enzymatic cofactorscyanocobalamin and biotin and the chromophore malachite green, will serveas model systems. Focus is placed on biochemical characterization of thesemolecules and on the preparation of crystals and solution samples that willenable their analysis by x-ray diffraction and NMR spectroscopy. Thestructures of cyanocobalamin and biotin aptamer crystals will be determinedby X-ray crystallography and the isotopically-labeled malachite greenaptamer RNA by solution NMR spectroscopy. This is to understand thespecific atomic interactions that stabilize RNA folding, the variety ofstructural folds available to RNA, the involvement of metals and waters inRNA function, and the nature of conformational changes in RNA that resultfrom its interaction with other molecules. The educational component ofthis project aims to use structural biology for introducing students to thephysical sciences through a combination of both classroom and laboratoryinstruction. New and modified curricula in eukaryotic molecular biologyand structural biology, supplemented by the development of computer-basededucational materials, will be developed to establish the relationshipbetween macromolecular structure and function. Selected number ofstudents will be encouraged to engage in the research.2. Non-technical: The capacity for ribonucleic acids (RNAs) to fold intospecific conformations and thereby obtain functional properties (e.g. theability to bind molecules or to catalyze reactions) has been increasinglyappreciated, in part through experiments in which RNAs have been evolved denovo from pools of random sequence molecules. This recognition has fueledspeculation about the involvement of RNAs in many biological processes(e.g. ribosome-directed protein synthesis) and supported theories favoringan RNA-based biology as a precursor to modern life. Our understanding ofthe structural basis for RNA function, critical for addressing theseissues, remains poor in part because few RNA structures have beendetermined at atomic resolution. In vitro-evolved ligand-binding RNAs willserve as model systems for understanding RNA structure and its relationshipto RNA function. X-ray crystallography and NMR spectroscopy will be usedin this study to determine high resolution models for three differentRNA-ligand complexes. Analysis of these structures will reveal themechanisms by which specific RNA sequences adopt unique structures and howthese structures enable specific interactions with other molecules.Integrating these studies with teaching, this study provides undergraduatestudents with basic skills in structural biology. Enhancements to existingeukaryotic molecular biology curricula will emphasize the concept thatbiological processes are driven by molecular interactions specified by thefolded conformations of macromolecules. A new course in structural biologywill expand upon this theme with the additional goal of providinginterdisciplinary training in applied physical and computational sciences.