The Essential Role of Stacking Adenines in a Two-Base-Pair RNA Kissing Complex

Abstract

In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5’-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multi-equilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.

Nanomanipulation of Single RNA Molecules by Optical Tweezers

“…Alternatively, the lowest force can be set at 10 pN, higher than the kissing forces. As expected, the force-extension curve under such conditions displays only the unfolding/refolding of the hairpins (Figure 11c) 34 . Therefore, it is possible to investigate folding the kissing interaction and each of the two hairpins independently at different forces.…”

“…With many improvements, the Minitweezers display extraordinary stability as compared to the first generation optical tweezers. A number of research groups in several countries use the Minitweezers in their single-molecule research 14,15,[34][35][36][37] . The details of construction, calibration, and operation of the device, including instruction videos, are available at the "Tweezers Lab" website (http://tweezerslab.unipr.it).…”

“…Both hairpins an 9-base-pair stem, though the stem sequences are different. The mechanical unfolding path of the RNA (Figure 11a) has been characterized by force ramp methods 34 . When force is raised, the kissing complex is first broken, followed by sequential unfolding of the hairpins.…”

“…Using a combination of force ramp and force jump experiments, the thermodynamics and kinetics of individual unfolding/folding steps can be measured 28 . Direct observations of the breaking and formation of the kissing complex enables us to analyze energetic contributions of flanking bases to the minimal kissing complex 34 and to measure the salt dependence of the kissing interaction 45 . The force is first jumped from 3 pN to 22 pN to measure the lifetime of the kissing complex at 22 pN.…”

Predicting structure and stability for RNA complexes with intermolecular loop–loop base-pairing

“…Molecular dynamics simulations has the advantage of accounting for atomistic force fields. Due to the complications of conformational sampling and force field, most RNA folding simulations are focused on relatively small systems such as RNA tetraloops and small kissing complexes Kuhrova et al 2013;Stephenson et al 2013). A reliable computational sampling for flexible loop conformations with all-atom resolution requires special sampling technique (Schafer et al 2001) and can take exceedingly long computational time.…”