Quantifying Label-Free Protein-RNA Binding Using Microscale Thermophoresis.

Microscale thermophoresis (MST) is an innovative technique for quantitatively analyzing molecular interactions in free solution at the microliter scale, requiring only minimal sample volumes. It relies on the thermophoretic movement of molecules or molecule-ligand complexes along temperature gradients, enabling the sensitive detection of changes in size, charge, hydration shell, or conformation associated with binding events. This high sensitivity allows the determination of equilibrium dissociation constants in the nanomolar to micromolar range. In this study, MST was used to characterize interactions between fluorophore-labeled RNA and unlabeled protein directly in solution, avoiding any need for immobilization or additional labeling. The results highlight the technique's versatility and its suitability for probing complex protein-RNA systems and competitive binding processes. To support the MST data, Electrophoretic Mobility Shift Assay (EMSA) was also employed to qualitatively assess and visualize nucleoprotein complex formation. While EMSA provides direct observation of binding through mobility shifts, MST complements it by delivering rapid, quantitative affinity measurements. Together, these approaches offer a robust framework for evaluating interaction specificity and stability. Overall, MST stands out as a powerful and broadly applicable method for the quantitative characterization of biomolecular interactions, offering significant advantages for RNA-focused research and drug discovery.