The long term objectives are as follows: (1) Prepare catalytic monoclonal antibodies (Mabs) that hydrolyze phosphate esters and investigate their biochemical properties. The first successful catalytic Mab that hydrolyzed phosphate esters was prepared during the current funding cycle using a metal-hapten complex. This approach will be expanded in order to examine specificity and diversity of the isolated catalytic Mabs. (2) Develop a high yielding rapid method for synthesizing RNA and dithioate RNA on a polymer support. The approach for synthesizing RNA is built upon a completely orthogonal series of blocking groups including as a key element, a mild acid labile 2'-protecting group and a transient, easily removed 5'-silyl derivative. Using nucleoside H-phosphonothioate synthons, an approach will be developed for synthesizing dithioate RNA. The stability of this linkage toward hydrolysis by various nucleases, including RNase A and ribozymes, and its affinity for proteins such as HIV-RT will be evaluated. (3) Synthesize and evaluate the biochemical properties of new DNA analogs. The synthesis of a new derivative having boranophosphonate internucleotide derivatives will be investigated. Biochemically it will be tested for its ability to activate RNase H and form stable duplexes with complementary RNA and DNA. Another analog having nitrogen in place of the two nonbridging oxygens will also be prepared. Proposed biochemical studies include testing this derivative's ability to form stable duplexes and triplexes with complementary oligonucleotides. Additional research focusing on potential antisense applications will also be completed with dithioate DNA. These studies will include new methods for targeting DNA to cells and evaluation of a new assay for ascertaining antisense activity. (4) Develop methods for synthesizing dithiophosphoryl and boranophosphoryl peptides. Methods will be developed for incorporating these phosphorus derivatives into peptides via a solid phase synthesis strategy. Biochemical evaluation will focus on testing the activity of these phosphopeptides in blocking protein phosphorylation/dephosphorylation events in biological signaling cascades.
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