To elucidate the role of specific bases in the self-cleavage activity of the human hepatitis delta virus (HDV) ribozyme, systematic substitutions of individual bases in two important single-stranded regions [between nucleotides 726–731 (SSrA region) and 762–766 (SSrB region)] were carried out by oligonucleotide-directed point mutagenesis. Among the mutants obtained, 12 mutants (G726 variants, G727A, G727C, G728C, G762A, G762C, C763 variants and A766C) could not tolerate the respective base-substitutions and self-cleavage activities were reduced to very low levels (10%), suggesting a requirement of the respective bases. In particular, G726 in the SSrA region and C763 in the SSrB region were found 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.
