Properties of Hepatitis Delta Virus Ribozyme, Which Consists of Three RNA Oligomer Strands

Abstract

Properties of a hepatitis delta virus (HDV) RNA ribozyme system, which consists of three RNA oligomer strands (substrate 8-mer ; enzyme 16-mer plus 35-mer) and contains a hybrid sequence of genomic and antigenomic RNA cores, are reported. Effects of Mg²⁺ concentration, divalent metal ion species, pH, and temperature on the cleavage activity were examined. The substrate cleavage activity increased with increasing Mg2+ concentration (0-100 mM). Ca²⁺ and Mn²⁺ ions were the most effective divalent cations and Mg²⁺ was less effective. The cleavage activity increased with increasing pH (5-7.5). The optimum temperature for the cleavage activity was 25-40°C. The Mg²⁺ concentration, pH and temperature dependencies are different from those reported for the single-strand ribozymes (about 90-mer) although the divalent metal ion preference is very similar. Conformational change induced by Mg²⁺ ion titration was monitored by CD. The CD data and the activity-Mg²⁺ concentration data were analyzed by curve-fitting analysis using equations derived for multiple metal ion binding mechanisms. The data can be explained by a model in which three Mg²⁺ ions bind to one ribozyme unit