This competing renewal application applies a combined NMR-molecular dynamics approach to structural issues related to the formation, folding topology, stability and interactions of nucleic acid triplexes and G- quadruplexes in aqueous solution. The triplex research is directed towards a molecular understanding of oligonucleotide directed recognition of duplex DNA and addresses approaches to overcome the acidic pH requirement for formation of pyr.purpyr triplexes and the non-isomorphous positions of triples in pur.purpyr triplexes. Structural studies will also address the conformation at triplex-triplex junctions associated with alternate strand recognition and potential discontinuities at triplex-duplex junctions. The helical structures of hybrid triplexes involving DNA, RNA and PNA (peptide nucleic acid) strands will be elucidated as will triple pairing alignments and structures of triple stranded intermediates in genetic recombination. The structural studies on G-quadruplexes formed by guanine rich telomeric repeats will be extended to the single repeat G-rich Bombyx mori d(T2AG2T) and the two repeat G-rich Plasmodium d(AG3T3AG3) and Saccharomyces cerevisiae d[G3(TG)2,3TG3] telomeric sequences to elucidate the folding topologies and their potential monovalent and divalent cation dependent conformational transitions. The solution structures of the four repeat G-rich human d[AG3(T2AG3)3] and Tetrahymena d(T2G4)4 telomeric sequences determined previously in our laboratory will be monitored as a function of deletions, additions and substitutions within critical linker segments. The integrity of junctional base pairs at G-quadruplex-duplex junctions will be studied and modulations of the structure followed on insertion of junctional bulge residues. Related studies will investigate the alignment of a pair of helices projecting from a G-tetrad face and variations in their relative positioning and cavity formation following appropriate junctional bulge insertions. We shall attempt to generate and characterize G-quadruplexes from RNA and PNA sequences towards an improved understanding of the contributions of backbone conformation and charge to folding topology and stability. These structural studies will provide a molecular foundation for efforts directed towards an improved understanding of oligonucleotide directed sequence specific recognition of duplex DNA through triplex formation, as well as define the range of folding topologies adopted by guanine rich telomeric G-quadruplexes that have been implicated in chromosomal organization and association during the cell cycle.
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