Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex

Abstract

Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.

Figure 1

Structural rearrangements associated with KC formation and the model hairpins used. (A) Possible pathways for strand displacement reactions. Strand displacement can be nucleated by formation of a KC (a), by initiation at the termini (b), or through complete unfolding of the hairpins (c). (B) Schematic diagram of the hairpin constructs used in this study: hairpins capable of rearranging into ED complexes (left), and hairpin sequences incapable of further rearrangement (right).

Figure 2

Representative ITC data. (A) (Upper) Thermograms showing formation of the HP2::HP3 kissing interaction. HP3 solution (60 μM) was titrated into 1.4 mL HP2 (3.5 μM) at 10°C in sodium-binding buffer. (Lower) Integrated data (solid squares) were fit to a single-site binding model (solid line), yielding: ΔH = −41.2 kcal mol⁻¹; −TΔS = 32.8 kcal mol⁻¹; K_A = 2.8 × 10⁶ M⁻¹; n =1.0. (B) Thermodynamic energies ΔG (upper), ΔH (middle), and −TΔS (lower) as a function of temperature.

Figure 3

Kinetics of HP2::HP3 KC formation measured by SPR. (A) Representative sensorgrams as a function of HP3 concentration (400–800 nM), with surface-immobilized HP2 RNA monitored at 15°C. Association and dissociation data were fit into a Langmuir binding model. (B) Eyring plot of HP2::HP3 association rates studied between 10°C and 40°C.

Figure 4

smFRET analysis of kissing kinetics. (A) Schematic representation of the total internal reflection setup. The donor RNA hairpin is immobilized by a biotin-streptavidin interaction on a biotin-BSA-coated quartz slide. A kissing interaction and an ED formation are shown in the presence of a 35-nM RNA hairpin with the acceptor fluorophore. (B) Typical single-molecule time trajectory. (Upper) Anticorrelated intensities of the donor (blue) and acceptor (red). (Lower) The corresponding FRET trajectory. Only the 0.0 and 0.5 FRET states are observed for the dissociated and kissing complexes. (C) Representative FRET trajectories showing several kissing interactions and their transition to the ED state. There are few molecules with a transition directly from KC to ED. (D) FRET histograms from single-molecule trajectories. (Upper) Donor-only RNA hairpin HP2 exhibits a single 0.0 FRET state. (Second from top) Preannealed noncomplementary hairpins (HP3-C1Δ,C7Δ + HP2) do not form the ED. (Third from top) Two preannealed complementary RNA hairpins (HP1 + HP2) show two distributions (0.0 and 1.0 FRET states) when preannealed before imaging. The distribution at 1.0 FRET state is due to ED formation. (Lower) Cumulative histogram built from >100 molecules. HP1 reacted with surface-immobilized HP2. This histogram shows three distributions at 0.0, 0.5, and 1.0 FRET states. The 0.0 FRET state is free HP2, the 0.5 FRET state represents the KC, and the 1.0 FRET state corresponds to the ED. ED molecules show no dynamic behavior and are trapped in that form under the experimental conditions.

Figure 5

Representative single-molecule FRET trajectories (left) and histograms (right) from selected mutants HP1-C7A::HP2 (A), HP1-C1A::HP2 (B), and HP2-A4C::HP1-U4G (C), which allowed the formation of a GC basepair by replacing the AU basepair. Vertical arrows indicate the photobleaching point for each trace.

Figure 6

Putative potential energy surface for KC and ED formation. Thermodynamic (ΔG_IJ) and activation energies (ΔG^‡_IJ) for pathways associated with KC formation and their conversion to EDs. Thermodynamic data were obtained using ITC, UV melting, and smFRET, and activation parameters were derived from kinetic data obtained by SPR and smFRET. Reaction coordinates are defined by the number of intramolecular (X) and intermolecular (Y) basepairs present in the molecular ensemble of a particular state. Stabilization energies are plotted along the z axis. Four different states are shown: unfolded strands (A), free hairpins (B), the KC (C), and the ED (D). The energy of unfolded strands (A) was used as the reference state (ΔG_A = 0).