CAREER: A Single Molecule Study of Alternative Folding of a Retroviral Untranslated RNA

本CAREER项目的目的是使用单分子方法研究模型逆转录病毒5 '-非翻译前导RNA的折叠和结构。 RNA折叠问题的两个方面以逆转录病毒基因组RNA为例。首先，尽管有许多可能的构象，但在给定的条件下，一条长的病毒RNA链可以采用明确定义的结构。第二，大的RNA，如逆转录病毒的非翻译前导区，可以在不同的信号（如与宿主细胞生长相关的信号）触发时及时重新排列成交替折叠。为了理解这种有趣的结构稳定性和灵活性，RNA的折叠和结构转变将一次一个分子地进行研究。每个RNA分子都将像橡皮筋一样使用光学镊子拉伸和放松，通过分子的延伸揭示结构转变。该项目的策略是分析单个结构域的交替折叠，然后检查整个前导RNA。具体而言，TAR结构域将作为替代二级折叠的一个例子进行研究，而DIS结构域将进行检查，以了解RNA-RNA相互作用中的结构重排。这些结构域的特征性折叠模式将在以后用作指纹来解释整个前导RNA的折叠轨迹。此外，与理论家建立合作，解释实验数据和测试RNA折叠的物理模型。分子动力学模拟将被用来研究RNA在原子水平上的张力下的结构。统计力学建模用于弥合单分子和批量观测之间的差距。该项目更广泛的影响包括开发一种新方法来研究大RNA的替代折叠和结构重排。在后基因组世界，RNA折叠的新模型，超出了最稳定的结构将是有用的预测非翻译RNA的生物功能，从不断增长的序列数据库。这个单分子研究项目将提供校准领先折叠理论所需的直接观察，并将促进生物学家和物理学家，实验学家和理论家之间的密切合作。该项目将在纽约州立大学奥尔巴尼新成立的RNA研究所进行，为不同背景的本科生和研究生提供RNA领域的长期研究机会。该项目将RNA结构生物学和生物物理学纳入教学，参加几个研究生课程，这些课程对来自纽约首都地区机构的所有学生开放，包括纽约州立大学，伦斯勒理工学院，纽约州卫生部和东北地区法医研究所。在这项研究中使用的技术，如拉伸DNA，将被开发为实验室示范，为本科生和研究生课程在各个学科，以及课外科学活动的高中生。

The objective of this CAREER project is to study folding and structure of a model retroviral 5'-untranslated leader RNA using single molecule methods. Two aspects of the RNA folding problem are exemplified by retroviral genomic RNAs. First, despite many possible conformations, a long viral RNA strand can adopt a well-defined structure under a given set of conditions. Second, large RNAs, such as the untranslated leader region of retroviruses, can rearrange into alternative folds in a timely fashion when triggered by different signals, such as those associated with host cell growth. To understand this intriguing structural stability and flexibility, the folding and structure transition of RNA will be studied one molecule at a time. Each RNA molecule will be stretched and relaxed like a rubber band using optical tweezers, with structure transitions revealed by extension of the molecule. The strategy of this project is to analyze alternative folding of individual domains, followed by examination of the entire leader RNA. Specifically, the TAR domain will be studied as an example of alternative secondary folding, whereas the DIS domain will be examined to understand structural rearrangements in RNA-RNA interactions. Characteristic folding patterns of these domains will be later used as fingerprints to interpret folding trajectories of the entire leader RNA. Furthermore, collaborations are established with theoreticians to interpret experimental data and to test physical models of RNA folding. Molecular dynamics simulations will be employed to examine RNA structure under tension at atomic levels. Statistical mechanical modeling is used to bridge the gap between single molecule and bulk observations. The broader impact of this project includes developing a new method to study alternative folding and structural rearrangement of large RNAs. In the post-genomic world, new models for RNA folding that extend beyond the most stable structure will be useful to predict versatile biological functions of untranslated RNAs from the ever-growing sequence database. This project with single-molecule study will provide direct observations necessary to calibrate the leading folding theories, and will foster close collaborations between biologists and physicists, experimentalists and theoreticians. The project will be conducted at the newly founded RNA Institute at SUNY Albany, providing long-term research opportunities in the RNA field for undergraduate and graduate students of diverse backgrounds. The project incorporates RNA structural biology and biophysics into teaching by participating in several graduate courses that are open to all students from the New York capital region institutions, including SUNY, Rensselaer Polytechnic Institute, New York State Department of Health, and Northeast Regional Forensics Institute. Techniques used in this research, such as stretching DNA, will be developed as lab demonstrations for undergraduate and graduate courses in various disciplines as well as ex-curricular scientific activities for high school students.