The algorithm is implemented by a computer program that examines the nucleotide sequence for stem-loop structures starting at the 5' end of the sequence. In this model the nascent molecules fold sequentially in a 5' to 3' direction as they are displaced from the template strand. (3) Refinement of computer programs to predict nucleic acid secondary structure We designed a new algorithm that simulates folding of single strand DNA or RNA molecules during their synthesis. These simple 4 member libraries will be studied by electrophoretic mobility enhancement and MALDI-TOF sequence Identification of specific residues responsible for the unusual stability will utilize mini libraries of an interesting sequence where each residue in turn is substituted by all four nucleotides. Thermodynamic parameters will be estimated by curve fitting optical melting data to a two state model. (2) Stability analysis of secondary structure elements Unusually stable structures identified above will be analyzed to determine their overall thermal stability and each position in the sequence will be analyzed to identify residues responsible for the unusual thermodynamic stability. In this case libraries will be constructed containing a common ultra stable loop structure and mixtures of mismatch and bulge loop sequences in the stem. This method will also be used to study the stem portion of hairpin loop molecules. Will be accomplished by MALDI-TOF mass spectrometry. These mixed populations will be subjected to electrophoretic analysis to isolate molecules of apparent increased stability. Combinatorial chemistry will be used to prepare synthetic oligonucleotide libraries containing a common helical stem and variable loop sequences ranging in length from 5 to 8 nucleotides. The extent of mobility enhancement is determined by the overall thermodynamic stability of the molecule the most stable structure migrates fastest followed by bands containing molecules of decreasing thermodynamic stability. The method relies on enhanced migration of compact secondary structures in electrophoresis gels relative to single strand random coils of the same chain-length and nucleotide composition. Project Methods (1) Identification of secondary structural elements with unusual stability Acrylamide gel electrophoresis will be used to identify and isolate hairpin loop secondary structures in DNA and RNA that possess unusual stability.
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