Stem-loop formation drives RNA folding in mechanical unzipping experiments

TL;DR

This study uses optical tweezers to measure RNA free energies and reveals that stem-loop formation significantly influences RNA folding pathways and hysteresis during mechanical unzipping experiments.

Contribution

It introduces a method to accurately derive RNA nearest-neighbor base pair energies and demonstrates the role of stem-loops in RNA folding dynamics.

Findings

Quantified RNA free energies with 0.1 kcal/mol precision.

Identified stem-loops as key kinetic traps in RNA folding.

Showed equivalence of sodium and magnesium salt effects at the NNBP level.

Abstract

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force-distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping-rezipping process, we introduce a barrier energy landscape of the stem-loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem-loops that kinetically trap the long-lived intermediates observed in the FDC. Stem-loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.