An in vivo high-throughput screening for riboswitch ligands using a reverse reporter gene system.

ABSTRACT: Riboswitches are bacterial RNA elements that regulate gene expression in response to metabolite or ion abundance and are considered as potential drug targets. In recent years a number of methods to find non-natural riboswitch ligands have been described. Here we report a high-throughput in vivo screening system that allows identifying OFF-riboswitch modulators in a 384 well bioluminescence assay format. We use a reverse reporter gene setup in Bacillus subtilis, consisting of a primary screening assay, a secondary assay as well as counter assays to detect compounds in a library of 1,280 molecules that act on the guanine-responsive xpt riboswitch from B. anthracis. With this in vivo high-throughput approach we identified several hit compounds and could validate the impact of one of them on riboswitch-mediated gene regulation, albeit this might not be due to direct binding to the riboswitch. However, our data demonstrate the capability of our screening assay for bigger high-throughput screening campaigns. Furthermore, the screening system described here can not only be generally employed to detect non-natural ligands or compounds influencing riboswitches acting as genetic OFF switches, but it can also be used to investigate natural ligands of orphan OFF-riboswitches.

SUBMITTER: Kirchner M 

PROVIDER: S-EPMC5552694 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other