The broad objectives of the proposed research are to understand the structure and mechanism and biological roles of self-cleaving RNA sequences (ribozymes). The ribozymes being studied are associated with the genomic and antigenomic RNAs of hepatitis delta virus (HDV). HDV is a human pathogen that infects only in association with hepatitis B virus. This combination results in more severe symptoms and higher fatalities than hepatitis B virus alone. Ribozyme activity is essential to replication of the HDV RNA genome. Greater understanding of the ribozyme structure, cleavage mechanism and role in replication could lead to a method for blocking viral replication. Beyond the specific medical aspects of HDV infection, the ribozymes from HDV are the first and, so far only, examples of self-cleaving RNAs that function naturally in human cells. Therefore, understanding features of these RNAs should facilitate the design of ribozymes to be used as therapeutics.
The specific aims are to: (1) Understand the potential of nucleobases to act as general acid-base catalysts in the antigenomic HDV ribozyme. The experiments will characterize a reaction in which an essential nucleobase hypothesized to function in proton transfer in the antigenomic HDV ribozyme reaction is replaced by an exogenous base in solution. (2) Definition of the contribution and role of metal ions in HDV ribozyme catalysis. Divalent metal ions are required for full activity but how metal ions contribute to catalysis is not known. Ribozyme variants that cleave with a variety of metal ions (mono-, di-, and trivalent) will be exploited in these studies. (3) Characterize sequences that affect folding into the ground-state structure, and define conformational changes in pre- and post-cleavage forms of the ribozyme. A model for regulating a choice between polyadenylation of an mRNA and replication of the genome by alternative structures will be tested. (4) Identify and characterized additional examples of nucleobases in catalysis. HDV codes for two related ribozymes; however, catalytic activity of the two reveals some distinct differences. Common and exclusive features of both ribozymes will be examined to obtain a more accurate and complete picture for nucleobase involvement in catalysis. Several other small ribozymes that could also use nucleobases as acid base catalysts will be studied using technologies developed in specific aim 1.
