Universal cold RNA phase transitions

Abstract

RNA's diversity of structures and functions impacts all life forms since primordia. We use calorimetric force spectroscopy to investigate RNA folding landscapes in previously unexplored low-temperature conditions. We find that Watson-Crick RNA hairpins, the most basic secondary structure elements, undergo a glass-like transition below [Formula: see text]C where the heat capacity abruptly changes and the RNA folds into a diversity of misfolded structures. We hypothesize that an altered RNA biochemistry, determined by sequence-independent ribose-water interactions, outweighs sequence-dependent base pairing. The ubiquitous ribose-water interactions lead to universal RNA phase transitions below T_G, such as maximum stability at [Formula: see text]C where water density is maximum, and cold denaturation at [Formula: see text]C. RNA cold biochemistry may have a profound impact on RNA function and evolution.

Keywords: RNA in the cold; RNA phase transitions; cold RNA misfolding; single-RNA force spectroscopy.

Fig. 1.

Cold RNA misfolding. (A) Unfolding (blue) and refolding (red) FDCs from H1L12A unzipping experiments (Top-Left) at temperatures 7 to 42 ^°C and 4 mM MgCl₂. The gray-dashed ellipse indicates native (N) unfolding events. Unexpected unfolding events from a misfolded (M) structure appear below $25°$C (black-dashed ellipse) that become more frequent upon lowering T from $17°$C to $7°$C. (B) Classification of N (blue dots) and M (red dots) rupture events at $T\le 25°$C and Worm-like chain (WLC) fits for each state (dashed lines). The Top panels show rupture force distributions at each T. The Inset of the Leftmost panel shows the parameters of rupture force events (see text).

Fig. 2.

Temperature-dependent ssRNA elasticity. (A) Force versus the ssRNA extension per base at different temperatures. Two methods have been used to extract the ssRNA molecular extension: the force-jump (magenta triangles up—unfolding—and down—refolding) and the two-branches method (black circles) (35, 36). Blue lines are the fits to the WLC in the high-force regime (see text). (B) Representation of the ssRNA elastic response according to the WLC model. The persistence length (l_p) measures the polymer flexibility, and the interphosphate distance (d_b) is the distance between contiguous bases. The computation of the total hairpin extension accounts for the contribution of the molecular diameter (d). (C) T dependencies of l_p (Left) and d_b (Right). Linear fits (solid lines) with error limits (dashed lines) are also shown and give slopes equal to $0.17(2)$ Å/K for l_p and $-0.04(1)$ Å/K for d_b.

Fig. 3.

Universality of cold RNA misfolding. (A) Unfolding (blue) and refolding (red) FDCs of hairpins H1L4A, H1L8A, H1L10A, H1L12U, and H2L12A at $25°$C and $7°$C. Gray-dashed ellipses indicate native (N) unfolding events. Except for H1L4A, all RNAs show unfolding events from misfolded (M) structures at $7°$C (black-dashed ellipses). Hairpins H1L12U and H2L12A (featuring a dodeca-U loop and a different stem sequence) show a second misfolded structure at low forces (zoomed Insets). Hairpin sequences are shown in each panel. (B) Bayesian classification of the unfolding events for the hairpins in panel (A) at $T=7°$C. The dashed lines are the fits to the WLC for the different states. The Top panels show the rupture force distributions.

Fig. 4.

Features of cold RNA misfolding. (A) Frequency of N, M₁, and M₂ unfolding events for the different RNA hairpins at $7°$C. (B) Unfolding FDCs of cssA RNA at $7°$C and 4 mM MgCl2 (Inset) classified into native (N) and misfolded (M₁ and M₂) states. (C) $\mathrm{\Delta }{G}_0$ values at $7°$C in 4 mM MgCl₂ (empty boxes) and 400 mM Nacl (solid boxes). Temperature axis in ^°C (Bottom label) and K (Top label). Box-and-whisker plots show the median (horizontal thick line), first and third quartiles (box), 10th and 90th percentiles (whiskers), and outliers (dots). The black dashed line is the Mfold prediction.

Fig. 5.

Cold RNA misfolding and phase transitions. (A) Illustration of a multicolored folding energy landscape (FEL) at different temperatures (SI Appendix, Fig. S11). The temperature arrow indicates the tendency to explore low-lying energy states with the FEL becoming rougher upon cooling: from high (red) to intermediate (green) and low (blue) temperatures. Transition state (TS) distances are typically shorter for M than N, denoting disordered and compact misfolded structures. The encircled schematic folds are for illustration purposes. (B) Temperature-dependent entropy (black) and enthalpy (gray) of N for H1L12A (empty symbols) and H1L4A (full symbols). The results are reported in SI Appendix, Tables S5 and S6. Fits to the entropy values in the hot (red) and cold (blue) regimes for H1L12A (solid lines) and H1L4A (dashed lines) are also shown. The transition between the two regimes occurs at $T_G\sim 293 \text{K}\sim 20°$C (dashed gray band) with a sudden change in $\mathrm{\Delta }{C}_p$. Inset Stability curves of H1L12A (empty black circles) and H1L4A (solid gray squares). Maximum stability is found at $T_S\sim 278 \text{K}\sim 5°$C (vertical black line) with melting temperatures at $T_H\sim 370 \text{K}\sim 100°$C (red lines). Extrapolations of $\mathrm{\Delta }{G}_0(T)$ in the cold regime predict cold denaturation at $T_C\sim 220 \text{K}\sim -50°$C for both hairpins (blue lines).