Sequence-Dependent Melting and Refolding Dynamics of RNA UNCG Tetraloops Using Temperature-Jump/Drop Infrared Spectroscopy.

Abstract

Time-resolved temperature-jump/drop infrared (IR) spectroscopy has been used to measure the impact of stem base sequence on the melting and refolding dynamics of ribonucleic acid (RNA) tetraloops. A series of three 12-nucleotide RNA hairpin sequences were studied, each featuring a UACG tetraloop motif and a double-stranded stem containing four base pairs. In each case, the stem comprised three GC pairs plus a single AU base pair inserted at the closing point of the loop (RNAloop), in the middle of the stem (RNAmid), or at the stem terminus (RNAend). Results from analogous DNA tetraloop (TACG) sequences were also obtained. Inclusion of AU or AT base pairs in the stem leads to faster melting of the stem-loop structure compared to a stem sequence featuring four GC base pairs while refolding times were found to be slower, consistent with a general reduction in stem-loop stability caused by the AU/AT pair. Independent measurement of the dynamic timescales for melting and refolding of ring vibrational modes of guanine (GR) and adenine (AR) provided position-specific insight into hairpin dynamics. The GR-derived data showed that DNA sequences melted more quickly (0.5 ± 0.1 to 0.7 ± 0.1 μs at 70 °C) than analogous RNA sequences (4.3 ± 0.4 to 4.4 ± 0.3 μs at 70 °C). Position-sensitive data from the AR modes suggests that DNA hairpins begin melting from the terminal end of the stem toward the loop while RNA sequences begin melting from the loop. Refolding timescales for both RNA and DNA hairpins were found to be similar (250 ± 50 μs at 70 °C) except for RNAend and DNAloop which refolded much more slowly (746 ± 36 and 430 ± 31 μs, respectively), showing that the refolding pathway is significantly impaired by the placement of AU/AT pairs at different points in the stem. We conclude that conformational changes of analogous pairs of RNA and DNA tetraloops proceed by different mechanisms.