Unveiling Salt-dependent Conformation Changes and Kinetics of Interfacial MicroRNA Let-7c Molecules using SFG-VS

MicroRNA Let-7c, a tumor suppressor implicated in non-small cell lung cancer (NSCLC), exhibits ion-dependent binding efficiency towards the target mRNA, which is critical for its therapeutic efficacy. Yet the mechanistic interplay between salt ions and RNA conformation remains poorly understood. In the present study, sum frequency generation vibrational spectroscopy (SFG-VS) was used to understand how calcium ions (Ca²⁺), Tris buffer, and phosphate-buffered saline (PBS) modulate the interfacial conformation of Let-7c molecules at the lamellar cationic lipid bilayer interface. Systematic SFG-VS analyses reveal that Ca²⁺ ions dominate conformational ordering via electrostatic and coordination interactions, reducing NH/NH₂ vibrational intensities and restructuring interfacial hydration networks. Tris and PBS buffers selectively perturb nucleobase orientations, with Tris destabilizing hydrogen bonding and PBS restricting rG-NH₂ bending flexibility. Notably, Ca²⁺ induces opposing trends in rG and rU tilt angles, suggesting multivalent cation-driven reorganization of RNA higher-order structures. These findings establish a framework for ion-dependent RNA folding at membrane interfaces and highlight microenvironmental engineering strategies to optimize microRNA-based therapeutics. The experimental data and analyses result from the present study demonstrate that SFG-VS can work as a pivotal tool for resolving nucleic acid structure at biological interfaces, offering microscopic insights into epigenetic regulation and precision drug delivery for cancer therapy.