In Vitro Selection of Varkud Satellite Ribozyme Variants that Cleave a Modified Stem-Loop Substrate

In vitro selection is an established approach to create artificial ribozymes with defined activities or to modify the properties of naturally occurring ribozymes. For the Varkud satellite ribozyme of Neurospora, an in vitro selection protocol based on its phosphodiester bond cleavage activity has not been previously reported. Here, we describe a simple protocol for cleavage-based in vitro selection that we recently used to identify variants of the Varkud satellite ribozyme able to target and cleave a non-natural stem-loop substrate derived from the HIV-1 TAR RNA. It allows quick selection of active ribozyme variants from the transcription reaction based on the size of the self-cleavage product without the need for RNA labeling. This results in a streamlined procedure that is easily adaptable to engineer ribozymes with new activities.