Characterization of Hammerhead Ribozyme Reactions

Abstract

Hammerhead ribozymes are small catalytic RNA motifs ubiquitously present in a large variety of genomes. The reactions catalyzed by these motifs are both their self-scission and the reverse ligation reaction. Here, we describe methods for the generation of DNA templates for the subsequent in vitro transcription of hammerhead ribozymes. This is followed by a description of the preparation of suitable RNA molecules for both reaction types, and their kinetic analysis.

The ubiquitous hammerhead ribozyme

“…The difference between the resulting folding energies was then used to identify true positive HHR motifs among the primary hits. In vitro cleavage kinetics (Kalweit et al 2012) served to verify the chosen energy parameters on the example of newly identified motifs from Xenopus tropicalis (Seehafer et al 2011).…”

G17-modified hammerhead ribozymes are active in vitro and in vivo

“…Templates for in vitro transcription were generated synthetically using partially overlapping primers in recursive PCR reactions, as described earlier (Kalweit et al 2012). The primers of these PCR reactions are listed in Supplemental Table S1 and add XhoI sites at the 5 ′ and 3 ′ ends of the fragment for subsequent cloning in expression vector pDneo2a lacZ (Wiegand and Hammann 2013).…”

“…The self-cleavage reactions were analyzed as described previously (Kalweit et al 2012). Kinetic analyses were carried out for each variant at least in duplicate, and deviations in the determined kinetic parameters were below 20%.…”

“…Ligation reactions were resolved by electrophoresis on 10% or 15% polyacrylamide gels and visualized by PhosphorImaging. Analysis of ligation reactions was carried out as described recently (Kalweit et al 2012). …”