USE OF CHEMICALLY-MODIFIED NUCLEOTIDES TO DETERMINE A 62-NUCLEOTIDE RNA CRYSTAL-STRUCTURE - A SURVEY OF PHOSPHOROTHIOATES, BR, PT AND HG

Two important challenges confronting RNA crystallographers are produci ng crystals and finding isomorphous heavy-atom derivatives. Non-isomor phism can be addressed by determining the phases using the multiwavele ngth anomalous dispersion (MAD) method. These phases can be greatly im proved by combining phases from MAD experiments done on different heav y-atom derivatives. Heavy-atom derivatives can be created by chemicall y modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH3)3](2+) to N7 of guanine. While phospho rothioates can provide mercury binding sites, disorder can reduce thei r value for phase determination. The location of these chemical modifi cations is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crysta l packing.