IDENTIFICATION OF PHOSPHATE OXYGENS THAT ARE IMPORTANT FOR SELF-CLEAVAGE ACTIVITY OF THE HDV RIBOZYME BY PHOSPHOROTHIOATE SUBSTITUTION INTERFERENCE ANALYSIS

A phosphorothioate substitution interference assay was used to investi gate the role of the pro-Rp oxygens of phosphate groups in the self-cl eavage reaction of the genomic human hepatitis delta virus (HDV) riboz yme. Incorporation of several different phosphorothioates (NTP alpha S ) into the HDV ribozyme inhibited the self-cleavage activity. Incorpor ation of uridine 5' phosphorothioate or adenosine 5' phosphorothioate maintained 72% of the original self-cleavage activity whereas incorpor ation of guanosine 5' phosphorothioate or cytosine 5' phosphorothioate into the precursor reduced self-cleavage activity to about 20% in eac h case. Using partially substituted phosphorothioate-modified transcri pts, we identified the pro-Rp oxygens that are important for the riboz yme activity, and they are located at positions 0, 1, 4, 5, 21, 24, 25 , 27, 28, 30-34, 40, 43 and 75. In particular, the pro-Rp oxygens at p ositions 0, 1 and 21 are appear to be critical for the self-cleavage a ctivity of the HDV ribozyme.