Measuring RNA UNCG Tetraloop Refolding Dynamics Using Temperature-Jump/Drop Infrared Spectroscopy

[Image: see text] Determining the structural dynamics of RNA and DNA is essential to understanding their cellular function, but direct measurement of strand association or folding remains experimentally challenging. Here we illustrate a temperature-jump/drop method able to reveal refolding dynamics. Time-resolved temperature-jump/drop infrared spectroscopy is used to measure the melting and refolding dynamics of a 12-nucleotide RNA sequence comprising a UACG tetraloop and a four-base-pair double-stranded GC stem, comparing them to an equivalent DNA (TACG) sequence. Stem-loop melting occurred an order of magnitude more slowly in RNA than DNA (6.0 ± 0.1 μs versus 0.8 ± 0.1 μs at 70 °C). In contrast, the refolding dynamics of both sequences occurred on similar time scales (200 μs). While the melting and refolding dynamics of RNA and DNA hairpins both followed Arrhenius temperature dependences, refolding was characterized by an apparent negative activation energy, consistent with a mechanism involving multiple misfolded intermediates prior to zipping of the stem base pairs.