RRM
2008 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Caged 5´-bridging phosphorothiolates to analyze ribozyme cleavage
Joy Krishna Maity, Tao Han, Ahmed Al-Harbi & Subha R Das*
Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213-3890, USA
Synthetic analogues of oligonucleotides are essential tools for probing mechanisms of enzymes that act on nucleic acids. Among the various modifications to nucleic acids, replacement of the phosphoryl oxygen atoms with sulfur has proven to be particularly useful in elucidating ribozyme mechanisms. Of the four possible phosphoryl oxygen atoms, replacement of the 5'-oxgen remains a particularly challenging modification. Access to RNA containing a 5'-S-phosphorothiolate (5'-PS) linkage requires synthesis of modified monomers and following synthesis, the 5'-PS linkage is 105 more labile than the native RNA phosphodiester. In order to preserve the integrity of the RNA containing the 5'-PS linkage the vicinal 2'-hydroxyl requires protection that persists through solid-phase synthesis and deprotection of the RNA. The photo-labile or 'caging' o-nitrobenzyl (oNBn) group fulfills the role of such an orthogonal protection in RNA synthesis. Here we describe the synthesis of three different classes of nucleoside phosphoramidites (i) containing a 5'-sulfur atom, (ii) containing a oNBn-protected 2'-hydroxyl group and (iii) containing both 5'-sulfur atom as well as (oNBn)-protected 2'-hydroxyl group. Combinations of these phosphoramidities can be used to generate 5'-S-phosphorothiolate containing RNA that are useful as hyperactivated substrates to analyze ribozyme mechanisms.
Keywords: Oligonucleotide, Phosphorothiolate, Synthesis; Ribozyme
