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

这个CAREER项目的目的是用单分子方法研究逆转录病毒5'-未翻译先导RNA的折叠和结构。逆转录病毒基因组RNA体现了RNA折叠问题的两个方面。首先，尽管有许多可能的构象，但在给定的一组条件下，长病毒RNA链可以采用明确的结构。其次，大型rna，如逆转录病毒的未翻译先导区，在受到不同信号（如与宿主细胞生长相关的信号）触发时，可以及时重新排列成可选择的折叠。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研究一个分子。每个RNA分子将被拉伸和放松，就像使用光学镊子的橡皮筋一样，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替代折叠，然后检查整个先导RNA。具体来说，我们将研究TAR结构域作为可选二次折叠的一个例子，而研究DIS结构域以了解RNA-RNA相互作用中的结构重排。这些结构域的特征折叠模式稍后将用作指纹来解释整个先导RNA的折叠轨迹。此外，还与理论学家建立了合作关系，以解释实验数据并测试RNA折叠的物理模型。分子动力学模拟将用于在原子水平上检查张力下的RNA结构。统计力学模型用于弥合单分子和整体观察之间的差距。该项目更广泛的影响包括开发一种新的方法来研究大rna的替代折叠和结构重排。在后基因组世界中，扩展到最稳定结构之外的RNA折叠新模型将有助于从不断增长的序列数据库中预测非翻译RNA的多种生物学功能。这个单分子研究项目将为校准领先的折叠理论提供必要的直接观察，并将促进生物学家和物理学家、实验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分校新成立的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。该项目通过参与几个研究生课程，将RNA结构生物学和生物物理学纳入教学，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，将被开发为各种学科的本科和研究生课程的实验室演示，以及高中生的课外科学活动。

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.