 RNA melting hysteresis

RNA melting hysteresis is dependence of an RNA optical melting trace on thermal history: heating and cooling can give different absorbance at the same temperature. It warns that the observed trace may not follow reversible chemical equilibrium on the measurement timescale. Two different traces do not by themselves establish two equilibrium melting temperatures.

A primary experiment illustrates the importance of history. A 21-nucleotide RNA showed a low-temperature dimer-to-hairpin transition that did not reverse on cooling, whereas its higher-temperature RNA hairpin melting transition was reversible. Measurements used a Cary 100 spectrophotometer, a 1-cm quartz cell and 268-nm light, heating at $1.0{}^{\circ }\mathrm{C}/\min$ and cooling at $0.5$ or $0.2{}^{\circ }\mathrm{C}/\min$. An adjacent solvent cell held the temperature probe.^[ref] The authors interpreted the dimer as kinetically trapped under those conditions, rather than applying a reversible equilibrium fit to that transition.

For an original simple kinetic model, let $f$ be the unfolded fraction, $f_{\mathrm{eq}}(T)$ its equilibrium value and $\tau$ an RNA melting relaxation time:After a fixed-temperature step with constant $\tau$, the deviation undergoes exponential decay:With stipulated $f(0)=0$, $f_{\mathrm{eq}}=1$ and $\tau =2\min$, the fraction reaches only 0.632 after two minutes and 0.950 after six. During a continuing temperature ramp, this lag can put heating and cooling on different branches. This single-time model is illustrative; it is not a fit to the published dimer experiment.

Useful checks compare ramp speeds and include fixed-temperature holds and repeat cycles. Verify measurement calibration and exclude evaporation or chemical degradation before attributing a discrepancy to folding kinetics. Slower scans can reduce kinetic lag, but a smooth trace alone cannot establish reversibility.