Poster abstracts

Poster number 21 submitted by Molly Evans

Chemo-genetic Analysis of the Hepatitis Delta Virus Ribozyme Active Site Network

Molly Evans (Chemistry Department, Carnegie Mellon University), Xochina El-Hilali (Chemistry Department, Carnegie Mellon University), Subha R. Das (Chemistry Department, Carnegie Mellon University)

Abstract:
The hepatitis delta virus (HDV) replication cycle requires ribozyme mediated RNA cleavage. The cleavage mechanism is noteworthy for the involvement of an active site cytosine residue, C76, that acts as a general acid. The reaction pK_a for the general acid C76 residue is significantly shifted by at least 2 pH units from the intrinsic pK_a of cytosine. A putative network of hydrogen bonding interactions may alter the intrinsic pK_a of C76 and allow it to act as a catalyst. We seek to determine the influence of the putative active site network that promotes the catalytic capability of the C76 residue. Here we replace two non-bridging oxygen atoms in an internucleotide phosphate within the network with sulfur atoms. Substitution of the oxygen atoms results in the R_P and S_P diastereomeric phosphorothioate RNAs which are separated by ion-exchange HPLC. These RNAs are each ligated to a transcribed RNA using T4 RNA ligase to provide two full-length single atom mutant ribozymes. The cleavage rate of each single atom mutant ribozyme will give insight into the influence of the pro-R_P and pro-S_P oxygen atoms in the active-site network on the catalytic cytosine residue.

Keywords: HDV ribozyme, chemo-genetic analysis