Small molecule screening of RNA aptamers using native mass spectrometry

The intentional targeting of RNA with small molecules is recognized as a viable pathway to new therapeutics with potential to vastly expand the druggable chemical space. The practical considerations to deliberately target RNA are however still largely under development, including optimal methods to identify small molecules for binding to RNA. Native mass spectrometry (nMS) is established as a valuable biophysical screening method for identifying small molecule hits for protein targets but has been used to a much lesser extent with oligonucleotides. Herein we applied nMS to the analysis of binding of two aminoglycoside RNA aptamers with their cognate ligands, related aminoglycoside ligands and a diverse small molecule library of FDA approved drugs. nMS confirmed cognate ligand binding and allowed semi-quantitation of binding strength and selectivity to be determined across all compounds tested. nMS library screening also identified novel binders. This work demonstrates that nMS can be applied for biophysical screening of RNA aptamers and has potential to be developed as an orthogonal screening technology within broader RNA-targeting drug discovery, however with limitations related to the specific challenges presented by the physicochemical properties of RNA.