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

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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.