Comparing RNA Kissing Interactions at Single-Molecule and Ensemble Levels

Abstract

Single-molecule and ensemble techniques are employed to take different perspectives to complex, heterogeneous systems. The challenge remains whether we can interpret results from the two different approaches using a unified thermodynamic framework, despite the differences in experimental procedures and conditions? In RNA mediated gene regulations, RNA kissing complexes, a type of loop-loop interaction, play a critical role in speeding RNA-RNA interactions. However, to form a kissing complex, a regulatory hairpin must compete with RNAs for various targets and non-targets. To probe such an interacting network, we use optical tweezers to determine thermodynamics of each type of kissing complexes one molecule at a time, and use mass spectrometry to take a snapshot of simultaneous equilibria of multiple kissing interactions by many molecules. Especially, we examine strength of relatively weak kissing interactions at single-molecule level and monitor their competition with formation of stronger kissing structures at ensemble level. To compare the two types of measurements, we take into account different experimental conditions, including salts, concentrations of RNAs, time vs. numerical averaging, equilibrium vs. non-equilibrium, and difference between intra- and intermolecular interactions. With these adjustments, we establish a quantitative correlation between two types of measurements, which can be used to accurately predict abundance of subpopulations, especially that of rare species, in a heterogeneous mixture. These results show that complementary information of an interacting network, generated by the two-pronged methodology, can be unified in a thermodynamic framework.