ANALYSIS OF A RIBONUCLEASE-H DIGESTION OF N3'-]P5' PHOSPHORAMIDATE RNA DUPLEXES BY CAPILLARY GEL-ELECTROPHORESIS

Phosphodiester oligonucleotides (ODNs) and their analogs are presently being investigated as potential antisense therapeutics in the treatme nt of viral infections and various forms of cancer. Here, we would lik e to report results from an investigation of activity for a ribonuclea se H (RNase Hi) mediated RNA digestion assay in the duplexes formed by an ODN or the ODN analog, N3'-->P5' phosphoramidate (3'-phosphoramida te), and complimentary RNA strands. Capillary gel electrophoresis (CGE ) proved to be an effective method for determining RNA hydrolysis in t he presence of RNase H. RNA and an ODN or RNA and a 3'-phosphoramidate were hybridized in a Tris-HCl, MgCl2 buffer at room temperature (RT) and incubated with RNase H. Digestions were carried out at RT or at 37 degrees C. Control samples were unhybridized RNA with RNase H, RNA wi thout RNase H, and duplexes (RNA-ODN or 3'-phosphoramidate) without RN ase H. All controls were incubated in Tris-HCl, MgCl2 buffer, and samp le aliquots were analyzed at various time intervals. A homodecamer, (d T)(10), was used as an internal standard to determine the relative mig ration time of the RNA strand. The final digestion products for the du plexes and the various controls were monitored by CGE. In addition, po lyacrylamide gel electrophoresis (PAGE) was used in conjunction with S tains-All (staining) and a densitometric analysis to verify CGE result s.