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H. Lee Weith

Area of Expertise: Synthesis, structure and function of nucleic acids; computational biochemistry

Research in computational biochemistry has become very important as molecular biologists determine nucleic acid and protein sequences with ever increasing speed. Biomolecular databases are expanding exponentially, and the ability to interpret the massive amounts of sequence information requires substantial computational methods for sequence analysis. Our research in this area has concentrated on the design of algorithms for analysis of nucleic acid secondary structure formation. We have already implemented a novel RNA structure prediction algorithm which simulates RNA folding during transcription. We have used this program to design a number of RNA sequences which can assume quite different secondary structure conformations depending on the mechanisms for RNA folding. We are studying the formation of alternative structures by these molecules during in vitro transcription reactions. The goal of these studies is to evaluate various thermodynamic and spatial parameters required for accurate computer prediction of the structure of an RNA molecule from its nucleotide sequence.

Currently our lab is developing procedures to identify and isolate hairpin loop secondary structures in DNA and RNA which possess unusual stability (high or low). The principle of the method is based on enhanced electrophoretic migration of hairpins structures relative to single strand random coils of similar chain-length and composition. Our preliminary studies have shown that it is feasible to separate hairpin loop structures on the basis of their thermodynamic stability by acrylamide gel electrophoresis in the presence of varying concentrations of denaturants. We use combinatorial chemistry methods to prepare populations of synthetic oligonucleotides containing a common helical stem and variable loop sequences. These mixed populations are subjected to acrylamide gel electrophoresis to separate various members of the population according to their stability. The oligonucleotides can be recovered from the electrophoresis gel and their sequences determined. Since a vast number of loop sequences is possible, we are initially restricting our attention to the most stable structures. Once these interesting structures are known, they will be synthesized on a larger scale for determination of thermodynamic stability by optical melting curve analysis. The new thermodynamic data for hairpins will be incorporated into structure prediction algorithms to improve their predictive capabilities. We will attempt to develop general parameters for hairpin stability based on sequence and chain-length.

Department of Biochemistry, 175 South University Street, West Lafayette, IN 47907-2063 USA, (765) 494-1600

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