Identification of important bases in a single‐stranded region (SSrC) of the hepatitis delta (δ) virus ribozyme

Abstract

Models for the secondary structure of genomic and antigenomic self‐cleaving RNAs of human hepatitis delta (δ) 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′. Copyright © 1993, Wiley Blackwell. All rights reserved