Selection of hammerhead ribozyme variants with low Mg2+ requirement: Importance of stem-loop II

Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg2- concentrations were in vitro selected from a library with 78 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had e sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mm Mg2- concentration in concentration, 25 degreesC, pH 75). Interestingly, this very active variant was not identified by the selection procedure, Changing loop II from UUUU to GCAA or extension of stem 11 to three or four base pairs reduced the cleavage rate by 2.0 - 2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so (or at low Mg2- concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degreesC and pH 7.5 in monophasic reactions, G ntext of several substrates with differences in the lengths of with endpoints between 75 and 90%. Such constructs promise to be advantageous for the inhibitation of gene expression in vivo