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

威尔逊MCB 98763501号技术：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。从随机序列的正醇中分离出的适体，包括与酶辅基-氰基钴巴拉明和生物素以及发色团孔雀绿特异结合的RNA，将作为模型系统。重点放在这些分子的生化表征以及晶体和溶液样品的制备上，以使其能够通过X射线衍射和核磁共振光谱进行分析。氰基钴胺和生物素适配子晶体的结构将通过X射线结晶学和同位素标记的孔雀石绿适配子RNA的溶液核磁共振光谱来确定。这是为了了解稳定RNA折叠的特定原子相互作用，RNA可用的结构折叠的多样性，金属和水在RNA功能中的参与，以及由于其与其他分子相互作用而导致的RNA构象变化的性质。该项目的教育部分旨在使用结构生物学，通过课堂和实验室教学相结合的方式，向学生介绍物理科学。将开发新的和修改的真核分子生物学和结构生物学课程，并辅之以开发基于计算机的演绎材料，以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：核糖核酸(RNA)折叠成特定构象从而获得功能特性(例如结合分子或催化反应的能力)的能力已得到越来越多的重视，部分原因是通过实验从随机序列分子池中从头进化出RNA。这一认识引发了人们对RNA参与许多生物过程(如核糖体导向的蛋白质合成)的猜测，并支持了支持将基于RNA的生物学作为现代生活的先驱的理论。我们对RNA功能的结构基础的理解仍然很差，这在一定程度上是因为很少有RNA结构是在原子分辨率下确定的。在体外进化中，配体结合的RNA将作为模型系统来理解RNA的结构及其与RNA功能的关系。本研究将使用X射线结晶学和核磁共振波谱来确定三种不同的RNA-配体络合物的高分辨模型。对这些结构的分析将揭示特定RNA序列采用独特结构的机制，以及这些结构如何使特定的分子与其他分子发生特定的相互作用。本研究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。对现有真核分子生物学课程的改进将强调这样一个概念，即生物过程是由大分子折叠构象指定的分子相互作用驱动的。一门新的结构生物学课程将围绕这一主题展开，另一个目标是提供应用物理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.