CAREER: Investigations of RNA Folding: A Combinatorial Approach to Thermodynamics

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9984129Bevilacqua Nucleic acids (NA) are polymers consisting of four building blocks--A, C, G, and T (or U) bases--attached in a linear fashion like beads on a string. Naturally occurring nucleic acids can be formally divided into two types, DNA and RNA, based on subtle differences of a single oxygen atom or methyl (CH3) group. Two fundamental functions essential to life are the storage of genetic information that determines an individual's identity, and the increase in the rate and specificity of chemical reactions essential to life, for example in metabolism and replication. In the cell, DNA is used to store genetic information, while proteins act as enzymes to accelerate reaction rates. Strikingly, RNA can both store genetic information, as evidenced by certain viruses such as HIV-1 which causes AIDS, and act as an enzyme, with RNA enzymes referred to as "ribozymes". These properties implicate RNA in a wide variety of biological processes and diseases, making the structures and functions of RNA of significant interest. Furthermore, RNA has been implicated as an important molecule in prebiotic chemistry and the origin of life, since RNA potentially solves the "chicken and egg problem" of which came first, enzymes or genetic information. The functional properties of RNA arise from a wide range of complex three-dimensional structures. Research described in this proposal has as its primary goal understanding how RNA folds into these structures. The sequences of RNA, or the order in which the four bases occur, can be readily obtained. For example, sequencing of the genomes of approximately 25 different organisms has been completed, and sequencing of the human genome with 3 billion DNA bases is expected to be completed in the next five years. To understand the function of these RNAs, structural models are indispensable, and computer methods to predict structures are essential for keeping pace with sequencing projects. The PI's laboratory is working to obtain rules for computer prediction using a novel methodology. Investigators are developing a combinatorial approach in which a collection of molecules containing up to a million billion different sequences are prepared and then separated in a single experiment according to thermodynamic stability, or the resistance of the structure to heat denaturation. Separations are on a gel electrophoresis apparatus containing a temperature gradient (TGGE). Exceptionally stable and unstable sequences will be isolated and identified by cloning and sequencing, and rules for structure prediction obtained by further physical characterization of selected sequences. These methods will be applied to a wide range of structural motifs in nucleic acids. The proposed research should provide a better understanding of basic principles of RNA folding.