IDENTIFICATION OF IMPORTANT BASES IN A SINGLE-STRANDED REGION (SSRC) OF THE HEPATITIS-DELTA (DELTA) VIRUS RIBOZYME

Models for the secondary structure of genomic and antigenomic self-cleaving RNAs of human hepatitis delta (6) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single-stranded regions [nucleotides 726-731 (SSrA) and nucleotides 762-766 (SSrB)]. For the identification of the important residues in the remaining single-stranded region, nucleotides 708-715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single-base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in-vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild-type SSrC (HDV86). By analyzing the self-cleavage activities of various mutants, we determined the base requirements for SSrC as 5'-(U/C/G)-C-N-N-(C/A/G)-(G/A/U)-N-N-3'.