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.