UNUSUAL NUCLEIC ACID STRUCTURES IN BIOLOGY

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The objectives of this proposal are to refine and extend our structural studies on a new structural motif we have recently discovered, the "sheared GA-bracketed unpaired G stack". Repeats of the human centromere pentameric DNA sequence TGGAA form stable duplexes with themselves in which "self-complementary" [GGA]2 motifs are separated by an AT and a TA Watson-Crick pair. The (GGA]2 motif consists of sheared GA and AG pairs which bracket two unpaired but costacked (intercalated) guanosines, to form a GGGG stack. The repeated (TGGAA)n sequence occurs at chromosome centromeres and is likely to be important in the separation of chromatids at mitosis. We wish to investigate the ability of residues other than guanosine to stack in the sheared G:A-A:G sandwich, and to study the effect of changing the flanking TA and AT pairs on the structure and stability of the internal [GNA]2 motifs in repeated pentamer sequences. We will also study the ability of the four GNA sequences to adopt sheared unpaired stack [(GNA)n]2 structures when placed immediately adjacent to each other in tandem arrays, i.e. without the intervening AT and TA Watson-Crick pair's The results will be of importance concerning the mechanism of repeat expansion in the triplet-repeat diseases muscular dystrophy, muscular atrophy, and Huntington's disease - all of which appear to be (GCA)n repeats. We will also study the (GCG)n repeats found in Fragile X infant mental retardation. The structure and stability of [GGA]2 motifs, with and without additional looped out residues, will also be investigated in short RNA "internal loops", where they may be implicated in ribozyme function, retroviral recognition and retroviral dimerization. Several purine-rich DNA and RNA hairpins that we expect emerge from our surveys will also be studied by high-resolution NMR at 750 MHz.