An Unintentional Discovery of a Fluorogenic DNA Probe for Ribonuclease†I.

ABSTRACT

Ribonuclease I belongs to a class of nonspecific endoribonucleases and plays many important roles in a variety of biological and cellular processes. While their ubiquitous nature and high activity contribute to the well-known problem of RNase contamination in experimentation, their abundance in bacteria can potentially be leveraged as a biosensor target. As a result, there is substantial interest in generating a specific and reliable probe for RNase detection for a variety of purposes. To that end, we report on our unintentional discovery of the RNase I probe RFA13-1 isolated through in vitro selection with the crude extracellular mixture from Clostridium difficile contaminated with Klebsiella aerogenes as a selection target. Characterization of RFA13-1 reveals that it exhibits high sensitivity to Escherichia coli RNase I with a detection limit of 1.39 pm. Furthermore, RFA13-1 also shows high specificity for RNase I produced only in select bacteria from the Enterobacteriaceae family. As a result, this probe offers a simple tool for RNase I detection with potential applications in RNase functional studies, ribonuclease contamination monitoring, and bacterial detection.