Summary RNA molecules make an important contribution to viability and virulence of pathogenic bacteria and viruses, participating in the most fundamental cellular processes implicated in human disease. Many RNAs contain structured regions that are critical to function and therefore represent attractive drug targets, especially for pathogens with high mutation rates in proteins. Intervening against these RNAs with small molecules is a powerful way to treat infections. Although significant efforts are concentrated on identifying potent and specific RNA binders, most of these studies are done in vitro, leaving the actual RNA-small molecule interactions in vivo untested because a robust method to capture and identify RNA-bound small molecules in vivo does not exist. The lack of such a method could hinder optimization of candidate RNA-binding drugs and inadvertently delay the progress to preclinical and clinical studies. This proposal is focused on developing a facile, inexpensive, and robust approach to capture and identify small molecules specifically binding RNAs of interest in bacterial cells. The proposed study will use natural regulatory non-coding RNAs, riboswitches, and an in vitro-selected RNA aptamer, capable of specific binding to cellular small molecules and antibiotics, as model systems. Specific Aim 1 is devoted to the development of the biochemical approach for producing RNA species in bacterial cells, capturing cognate small molecules by RNA, extracting RNA-small molecule complexes by affinity chromatography, and identifying bound small molecules by mass spectrometry. The methodology will be benchmarked using a panel of RNAs recognizing chemically diverse small molecules. Specific Aim 2 will validate the approach on riboswitches whose cognate ligands are unclear or unknown. In this aim, we anticipate to identify a cognate ligand for an “orphan” riboswitch from the human pathogen Helicobacter pylori and characterize the riboswitch-ligand complex biochemically, biophysically, and genetically, in vitro and in vivo. This riboswitch controls genes that are essential for bacterial virulence and thus represents a potential drug target. The proposed approach is filling a methodological gap and will be essential for advancing hit-to-lead optimization of the RNA-targeting small molecules and identification of cognate small molecule binders for natural RNAs.