Poster abstracts

Poster number 50 submitted by Henry Nguyen

Synthesis of a Nucleotide Analogue for RNA Structural Analysis

Henry C. Nguyen (Department of Chemistry, Carnegie Mellon University)

Abstract:
New paradigms for the roles of RNA are emerging and it is clear that RNA plays a much larger role than previously identified. These discoveries are fueled through greater understandings of RNA structure and function relationships. We aim to understand better the contributions of specific structural features, such as the 2'-hydroxl group, to RNA function through atomic substitutions to RNA. We aim to test the hypothesis that RNA containing a 2'-deoxy-2'-chloro (2'-Cl) analogue is unable to be cleaved by self-cleaving ribozymes, yet still retains the inherent conformational flexibility of the natural ribo-sugar phosphate backbone of RNA. Starting with uridine (U), we synthesized in four steps the protected form of 2'-Cl-U required for solid phase RNA synthesis. The stem and loop region of the U1-RNA sequence will then be synthesized for co-crystallization with the U1A protein. The U1A protein was obtained by fusion to a (His)6-GST tag containing a TEV cleavage site for purification. It was expressed in E.coli and purified by Ni⁺² chromatography, followed by cleavage of the fusion protein using TEV. We synthesized U1-RNA containing 2'-Cl-U, and aim to co-crystallize it with the U1A protein, and solve the structure for comparison with the known structures of the natural co-complex. This will determine how well the 2'-Cl substituents mimic the natural ribo 2'-OH residues in U1-RNA. This may also help to determine what structural features of the RNA backbone conformation are important for RNA-protein recognition. Further studies will be done where the 2'-Cl-U will be incorporated into self-cleaving ribozymes to prevent cleavage. Crystallization of these non-cleaved ribozymes would give further insight to the mechanism of their cleavage.

Keywords: ribozyme, RNA analogue