Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis

Abstract

A phosphorothioate substitution interference assay was used to investigate the role of the pro-Rp oxygens of phosphate groups in the self-cleavage reaction of the genomic human hepatitis delta virus (HDV) ribozyme. Incorporation of several different phosphorothioates (NTPαS) into the HDV ribozyme inhibited the self-cleavage activity. Incorporation of uridine 5′ phosphorothioate or adenosine 5′ phosphorothioate maintained 72% of the original selfcleavage activity whereas incorporation of guanosine 5′ phosphorothioate or cytosine 5′ phosphorothioate into the precursor reduced self-cleavage activity to about 20% in each case. Using partially substituted phosphorothioate-modified transcripts, we identified the pro-Rp oxygens that are important for the ribozyme 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 positions 0, 1 and 21 are appear to be critical for the self-cleavage activity of the HDV ribozyme. © 1994 Oxford University Press.