Understanding in-line probing experiments by modeling cleavage of nonreactive RNA nucleotides

Ribonucleic acid (RNA) is involved in many regulatory and catalytic processes in the cell. The function of any RNA molecule is intimately related with its structure. In-line probing experiments provide valuable structural data sets for a variety of RNAs and are used to characterize conformational changes in riboswitches. However, the structural determinants that lead to differential reactivities in unpaired nucleotides have not been investigated yet. In this work, we used a combination of theoretical approaches, i.e., classical molecular dynamics simulations, multiscale quantum mechanical/molecular mechanical calculations, and enhanced sampling techniques in order to compute and interpret the differential reactivity of individual residues in several RNA motifs, including members of the most important GNRA and UNCG tetraloop families. Simulations on the multinanosecond timescale are required to converge the related free-energy landscapes. The results for uGAAAg and cUUCGg tetraloops and double helices are compared with available data from in-line probing experiments and show that the introduced technique is able to distinguish between nucleotides of the uGAAAg tetraloop based on their structural predispositions toward phosphodiester backbone cleavage. For the cUUCGg tetraloop, more advanced ab initio calculations would be required. This study is the first attempt to computationally classify chemical probing experiments and paves the way for an identification of tertiary structures based on the measured reactivity of nonreactive nucleotides.