Rational design of hairpin RNA excited states reveals multi-step transitions

Abstract

RNA excited states represent a class of high-energy-level and thus low-populated conformational states of RNAs that are sequestered within the free energy landscape until being activated by cellular cues. In recent years, there has been growing interest in structural and functional studies of these transient states, but the rational design of excited states remains unexplored. Here we developed a method to design small hairpin RNAs with predefined excited states that exchange with ground states through base pair reshuffling, and verified these transient states by combining NMR relaxation dispersion technique and imino chemical shift prediction. Using van't Hoff analysis and accelerated molecular dynamics simulations, a mechanism of multi-step sequential transition has been revealed. The efforts made in this study will expand the scope of RNA rational design, and also contribute towards improved predictions of RNA secondary structure.

Fig. 1. Design and verification of T1-short and T1 RNAs.

a Left: The secondary structure of P5c. Base pairs in ES are highlighted by green bars. Right: The design principle of T1-short. Base pairs in GS are colored in red, while those in ES are highlighted by green bars. b Schematic design protocol for T1-short. c The GS structure of T1-short RNA. The residues in the variable stem are shown in red. d Three 3 × 3 internal-loop (orange) candidates. e The secondary structure of T1 RNA, which comprises a T1-short hairpin (black and red), a 3 × 3 internal-loop (orange), and a stable lower stem made of alternating G-C and C-G base pairs (gray).

Fig. 2. Verification of T1 RNA Excited State.

a Representative off-resonance ¹⁵N RD profiles (left panel) and ¹H^N CEST profiles (right panel) of residues showing significant RD signals. RD profiles of other residues are available in Supplementary Fig. 3. The error bars in ¹⁵N RD profiles represent standard deviations (SD) estimated using Monte Carlo simulation with 50 iterations. b Secondary structures of GS and ES of T1 along with their populations and forward and backward rate constants. Exchange parameters were obtained from the global fitting of RD profiles using a two-state exchange model. The green arrow on the left indicates the direction of sliding. c Correlation between predicted and experimental ¹⁵N (left panel) and ¹H^N (right panel) chemical shifts of ES for T1.

Fig. 3. T1-GAAA, T1-UUCG, T1-delAU, and T1-add1bp RNAs.

a, b Secondary structures of GS and ES for T1-GAAA (a) and T1-UUCG (b), along with their populations, and forward and backward rate constants. c, d Secondary structures of GS and ES for T1-delAU (c) and T1-add1bp (d), along with their populations, and forward and backward rate constants. Exchange parameters were obtained from the global fitting of RD profiles using a two-state exchange model. The green arrow on the left of each structure indicates the direction of sliding.

Fig. 4. T2-mirror, T2, T3, and T4 RNAs.

a, b Secondary structures of GS and ES for T2-mirror (a) and T2 (b) along with their populations, and forward and backward rate constants. T2-mirror and T2 were constructed based on T1 by swapping two strands (blue) or redesigning the sequence (red) of the upper stem, respectively. c The design principle of T3 RNA. Base pairs in ES are highlighted by green bars. d Secondary structures of GS and ES for T3, along with their populations, and forward and backward rate constants. e The design principle of T4 RNA. Base pairs in ES are highlighted by green bars. f Secondary structures of GS and ES for T4, along with their populations, and forward and backward rate constants. The green arrow beside each GS structure indicates the direction of sliding.

Fig. 5. Kinetic-thermodynamic profiles of GS-to-ES transitions.

The energy diagrams for the exchange processes between GS and ES via a transition state, in which the activation and net free energy (G), enthalpy (H), and entropy (TS) changes are shown.

Fig. 6. The representative ES-to-GS transition pathways.

a A typical downward ES-to-GS transition of T4 RNA. b A typical downward ES-to-GS transition of T1 RNA. In each state, residues with status changes relative to the previous state are highlighted in yellow. Each transition point is indicated by a capital letter with a color that matches the color of residues where the major change occurs. The number below each letter represents the time point of state transition in unit of nanoseconds (more precisely, the beginning moment of the state on the right), which can be easily located in the time courses of Supplementary Fig. 17. Please note that the time point of A has been reset to zero.

Fig. 7. Kinetic simulations of T1 RNA.

a The six-state exchange process of an upward GS-to-ES transition of T1 RNA. Shown are the structures of ground state (GS), excited state (ES), and intermediate states (I1–I4). b The free-energy diagram of a multi-step transition. The free-energy levels of the four intermediates were elevated above ES by 1.27 kcal mol⁻¹, corresponding to a 10-fold lower abundance, to ensure that these intermediates are beyond the detection of NMR RD experiments. c The free-energy diagram for the case that all base pairs are broken simultaneously during the transition. d The free-energy diagram of the equivalent two-state transition for (b).