Utilizing RNase J1 to Degrade Ribozyme Reporter Signal in Cell-Free Systems

Abstract

Ribozymes are noncoding RNAs that are able to catalyze reactions, such as self-cleavage, and can be used to control the expression of downstream genes under specific conditions. In E.coli-based cell-free protein expression systems, native RNases cannot efficiently degrade transcripts generated following ribozyme self-cleavage. Exogenous ribonuclease RNase J1, from Bacillus subtilis, can selectively degrade RNA transcripts containing a 5’ hydroxyl group, and could potentially be used to turn off the expression of downstream fluorescent reporters. In this study, we designed cell-free reactions (CFRs) containing a Pistol self-cleaving ribozyme upstream of the fluorescent reporter deGFP. Pistol ribozyme reporter constructs were PCR amplified, cleaned, and quantified. RNase J1 was expressed in E.coli-based cell-free lysates and its expression was evaluated by an SDS-PAGE gel and Western blot. One-pot and two-pot CFR setups were used to evaluate the effect of RNase J1 on reporter fluorescence. Although RNase J1 expression was successfully detected, fluorescence measurements demonstrated high variability across experiments. Pistol reporter constructs produced fluorescence levels close to the negative control, making decreases attributable to RNase J1 difficult to distinguish, and RNase J1 did not consistently reduce reporter fluorescence under the conditions tested. Future work should optimize reporter fluorescence to improve signal above the background, repeat experiments to evaluate reproducibility, and investigate the effects of native RNases present in the cell-free lysate. If successful, this system could be adapted to ligand-responsive ribozyme reporters, allowing for the development of ribozyme-based biosensors for a variety of target molecules.