Single-molecule analysis reveals low-temperature misfolding in HIV-1 TAR RNA

Temperature is one of the most fundamental physical variables governing RNA folding, yet its effects at low temperature remain incompletely understood. Recent single-molecule studies indicate that lowering temperature below a characteristic threshold (~ 20 °C) alters the folding pathways and promotes the population of non-native intermediates, including non-native conformations not predicted by standard secondary-structure models. Here, we combine temperature-controlled optical tweezers with computational free-energy landscape analysis to examine how the architecture of the HIV-1 TAR RNA hairpin modulates folding in this low-temperature regime. We show that decreasing temperature stabilizes the native hairpin but, below ~ 20 °C, promotes two distinct classes of misfolded intermediates dependent on the presence of the conserved three-nucleotide bulge. Together, the experimental data and the free-energy landscape analysis indicate that the presence of the bulge, not the loop, underlies the emergence of these low-temperature misfolded intermediates, accounting for their distinct mechanical signatures compared to those reported for RNA hairpins with a long loop.