The use of a convertible ribonucleoside for the site-specific modification of RNA

grantor: University of TorontoThe convertible nucleotide strategy allows for the concomitant deprotection and incorporation of a functionalized tether into an oligonucleotide. Deprotection of an RNA oligomer containing the convertible nucleoside 4-chlorophenyl uridine with cystamine disulfide allows for the introduction of a masked sulfhydryl functionality into an RNA oligonucleotide. Reduction of the masked functionality with dithiolthreitol yields a reactive sulfhydryl group that is available for further modification. Specifically, the free sulfhydryl can be derivatized with 5-(acetamido)-1,10-phenanthroline to incorporate a chemical nuclease covalently tethered to an RNA oligonucleotide. This site-specific nuclease was used to study intermolecular cleavage events of a complementary DNA molecule. In addition, the free sulfhydryl was used to create a disulfide cross-link within duplex RNA structures. The cross-link is located in the major groove of duplex RNA and reversibly constrains duplex unwinding. We tested a tRNA-like model for its ability to inhibit processing by the ribozyme RNase P. It was determined that a cross-linked tRNA model is not efficiently processed by RNase P indicating that tRNA unwinding may precede enzymatic cleavage.M.Sc