SYSTEMATIC SUBSTITUTION OF INDIVIDUAL BASES IN 2 IMPORTANT SINGLE-STRANDED REGIONS OF THE HDV RIBOZYME FOR EVALUATION OF THE ROLE OF SPECIFIC BASES

To elucidate the role of specific bases in the self-cleavage activity of the human hepatitis delta virus (HDV) riboxyme, systematic substitu tions of individual bases in two important single-stranded regions [be tween nucleotides 726-731 (SSrA region) and 762-766 (SSrB region)] wer e carried out by oligonucleotide-directed point mutagenesis. Among the mutants obtained, 12 mutants (G726 variants, G727A, G727C, G728C, G76 2A, G762C, C763 variants and A766C) could not tolerate the respective base-substitutions and self-cleavage activities were reduced to very l ow levels (10%), suggesting a requirement of the respective bases. In particular, G726 in the SSrA region and C763 in the SSrB region were f ound to be essential for the ribozyme activity. We could determine the preferred sequences, 5'-G-G-(G/A/U)-N-(A/U/G)-Pu-3' for SSrA and 5'-( G/U)-C-N-(A/G/U)-A-3' for SSrB regions, respectively.