Preparation of a Multi-Part Varkud Satellite Ribozyme Variant for Kinetics Studies

Abstract

The Varkud Satellite (VS) ribozyme is the largest of the “small” nucleolytic ribozymes and is the only one for which there are no high resolution crystal structures available. The VS ribozyme comprises a catalytic domain and a substrate domain. The catalytic domain includes five helices that interact with the stem-loop substrate. The substrate is docked within a cleft that is formed by helices II and VI. This naturally brings the cleavage site in close proximity to the A730 loop in helix VI. The adenines within the A730 loop are very crucial to the cleavage reaction and any substitution causes a major decrease in the cleavage activity of the ribozyme. This study is aimed at designing and producing a variant of the Varkud Satellite ribozyme that consists of multiple parts that can be used for detailed studies of ribozyme kinetics and assembly.