SELECTION AND ANALYSIS OF STABLE RNA 3 WAY JUNCTIONS

描述：大RNA分子的折叠可以作为一种 一个分层的过程，其中预先形成的子结构组装成 最后是复杂的三维结构。 重要的和鲜为人知的RNA 构建单元是多螺旋连接环，其中最简单的是 三路交叉口（3 WJ）。 这种结构经常出现在大 RNA;更好地了解其折叠规则将显着改善 我们预测和模拟大的三维结构的能力， 各种RNA。 体外选择和进化方法（下文 称为SELEX）将被用来探索分子间的相互作用 其稳定并限定RNA 3 WJ的结构。 里昂提斯博士 应用SELEX生成、选择和扩增RNA分子文库 能够形成3 WJ结构，其中核苷酸位置围绕 连接点是随机的。 最紧凑和热稳定的 RNA库中的物种将通过热梯度凝胶选择 电泳并粘附扩增。 在足够的循环之后， 已经完成，这样，大部分游泳池都展示了 为了获得所需的物理性质，从库中取样RNA序列将 进行克隆、测序，并进行化学和酶促探测， 表征稳定结构的原子相互作用。 分子模拟技术将用于构建三维 代表性分子结构的原子模型， 探索数据作为指导。 建模研究将在 与RNA结构领域的领导者Eric Westhof博士合作 建模 这些分子的热稳定性将使用 UV监测热分析（“UV熔融”）和等温滴定 量热法，以提供热力学参数， 结构模型 进一步的三维实验验证 结构将通过尝试结晶感兴趣的 序列的 如果获得晶体，将进行晶体学分析。 追求。 解决方案结构的特征将使用 多维/多核NMR光谱学。

DESCRIPTION: The folding of large RNA molecules can be approached as a hierarchical process in which pre-formed substructures assemble into the final, complex 3-dimensional structure. Important and little understood RNA building units are the multi-helix junction loops, the simplest of which are the three-way junctions (3WJ). Such structures occur frequently in large RNAs; a better knowledge of their folding rules would significantly improve our ability to predict and model the three-dimensional structures of a large variety of RNAs. In vitro selection and evolution methodologies (hereafter referred to as SELEX) will be employed to explore the molecular interactions which stabilize and define the structures of RNA 3WJ. Dr. Leontis will apply SELEX to generate, select, and amplify a library of RNA molecules capable of forming 3WJ structures, in which nucleotide positions surrounding the junction site are randomized. The most compact and thermally stable species in the RNA pool will be selected by thermal gradient gel electrophoresis and amplified reiteratively. After sufficient cycles have been completed, such that a large percentage of the pool exhibits the desired physical properties, a sampling of RNA sequences from the pool will be cloned, sequenced, and chemically and enzymatically probed to characterize the atomic interactions which stabilize the structures. Molecular modeling techniques will be employed to construct 3-dimensional atomic models of the structures of representative molecules using the probing data as a guide. Modeling studies will be carried out in collaboration with Dr. Eric Westhof, a leader in the field of RNA structure modeling. The thermal stabilities of these molecules will be measured using UV-monitored thermal analysis ("UV melting") and isothermal titration calorimetry to provide thermodynamic parameters to correlate with the structural models. Further experimental verification for the 3-dimensional structures will be sought by attempting to crystallize interesting sequences. If crystals are obtained, crystallographic analysis will be pursued. Solution structures will be characterized using multi-dimensional/multi-nuclear NMR spectroscopy.