Combining UV-Vis Absorption and FRET Melting Curves to Screen the Thermodynamic Stability and Folding Pathways of Nucleic Acids In Vitro
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 conforma-tional 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.