  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.
