Structured noncoding RNA targets have been identified to play important roles in disease, and the large proportion of non-coding RNA in the genome (approximately 98%) presents many alternate drug targets for modulating disease compared to conventional protein targets. Attempts to intentionally target RNAs are largely in their infancy, with appropriate techniques for screening and characterising small molecules binding to RNA still requiring development and/or validation. Native mass spectrometry (nMS) has routinely been applied as a biophysical approach for drug discovery against protein targets due to its ability to observe biomolecules in their native folded states and maintain all noncovalent interactions. However, it’s adaption as a screening technique to the challenging target class of structured RNAs has not yet been realised.
Small, structured RNA aptamers, namely the aminoglycoside tobramycin and kanamycin B aptamers and the purine xanthine and theophylline aptamers, were utilised as example systems to study the binding of small molecules by nMS. The cognate binding ligands were initially used to develop appropriate workflows, revealing that the binding varied depending on the aptamer class. Selective binding was observed for a small panel of known binding ligands reported in the literature against the aminoglycoside aptamers, with binding responses correlating across the range of reported affinities. A screening method for a curated subset of the FDA approved drug library containing 47 antibiotics with known RNA binding mechanisms across various antibiotic classes, and 30 other drugs where RNA binding is not known was developed. This screening method allowed the identification of hit binders, the determination of binding strength for hit ranking and the analysis of structure-activity relationships for related compounds within the various classes. These methods establish the potential for nMS to be applied as an efficient biophysical screening method to identify selective and chemically diverse small molecule binders of RNA drug targets.