Combining UV-Vis Absorption and FRET Melting Curves to Screen the Thermodynamic Stability and Folding Pathways of...

We present a high-throughput method for characterizing nucleic acid folding pathways and thermodynamic stability by combining UV-vis absorption and ensemble FRET melting curve analyses. While UV-vis provides global information on NA stability by monitoring base stacking, FRET probes local conformational transitions via fluorophore distance changes. This dual approach allows us to resolve both global and site-specific folding trajectories. Automated data acquisition and analysis are implemented via Python-based Jupyter notebooks. Using model DNA and RNA constructs, we demonstrate ion-dependent, temperature-induced unfolding behavior and highlight the complementarity of the two techniques in probing NA folding landscapes.