Optimizing Ribozyme Reporters using RNase J in E.coli based Cell-Free Systems

Abstract

Biosensors detect target molecules using biological components that generate measurable signals. RNA regulatory elements, such as ribozymes, can be engineered into biosensor designs to control the expression of downstream reporter proteins like green fluorescent protein (GFP) in response to target binding. In many synthetic ribozyme reporter systems, ligand binding either activates or inhibits ribozyme self-cleavage, regulating release of the mRNA transcript encoding a fluorescent reporter. RNase J1, a 5′-to-3′ endonuclease from Gram-positive bacteria, enables improved degradation in E. coli, unlike the native RNase E, which inefficiently degrades the transcript due to the 5'-hydroxyl group. In this study, we designed cell-free reactions incorporating ribozyme-based fluorescent reporters with either a synthetic theophylline ribozyme or the naturally occurring glmS ribozyme upstream of a deGFP reporter. We introduced RNase J1 into E. coli-based cell-free lysates and tested its effect on reporter expression. We observed that GFP expression decreased in reactions with PCR products encoding the ribozyme reporters and RNase J1, regardless of whether the ligand was present. We achieved expected performance in a two-step reaction, in which we added RNase J1 from a prior cell-free reaction to a cell-free reaction containing the ribozyme reporter PCR product. However, the fold change with and without the ligand was relatively small. Further optimization is needed to use RNase J1 to enhance ribozyme reporter activity in cell-free systems.