MMS Discovers Altered Thermal Stability of RNA, Originating from Ligand-induced Changes in Base Pairing

Abstract

RNA structure and thermal stability are tightly linked through base pairing and stacking, which in turn governs functional processes such as transcription and translation. Ligand-induced conformational change resulted from these base-base interactions can significantly impact RNA stability, but conventional techniques fail to resolve detailed structural changes alongside thermal stability metrics. This limits the use of these techniques for structure-activity relationship (SAR) studies that are the backbone of drug development. Here, we demonstrate the use of Microfluidic Modulation Spectroscopy (MMS) to simultaneously measure RNA thermal stability and base-pair-specific structural transitions in aqueous solution. MMS was applied to a 29-mer RNA construct, containing a bulge and a hairpin, in the presence vs. absence of two small-molecule ligands. Comparison with AU- and GC-only duplexes enabled assignment of spectral features to specific base-pairing motifs. Thermal melts revealed distinct transitions at wavenumbers corresponding to base-pair disruption, while isothermal difference spectra highlighted ligand-induced enhancements in RNA structural stability. Ligand 1 induced larger structural and stability changes over a weaker-binding ligand, Ligand 2, highlighting the ability of MMS to rank compounds in SAR studies. These results establish MMS as a powerful tool for characterizing RNA-ligand interactions, offering analysis of structural and thermal stability effects that supports RNA-targeted drug discovery.