Kissing complexes are dimeric RNA structures stabilized by hydrogen bonding between complementary loop nucleotides. RNA segments involved in kissing complex formation have been discovered in pathogenic viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are located in highly conserved regions of the genomes, underscoring their functional importance and making them attractive pharmaceutical targets. The kissing dimerization of RNA is strongly dependent on magnesium ions (Mg 2+ ) and despite the therapeutic potential, the precise structural and energetic basis of this dependence remains poorly understood. Consequently, we studied the monomeric 55-nucleotide X55 RNA from the 3′-untranslated region of the HCV genome that adopts two distinct conformations: a two-stem-loop form (SL1-2), which forms kissing homodimers in vitro, and a three-stem-loop form (SL1-3), which forms in vitro kissing heterodimers with the 5BSL3.2 sequence in the viral open reading frame. Initial structures of both conformers were generated using the Vfold3D/ISRNA pipeline and were subjected to unbiased microsecond-scale molecular dynamics simulations in explicit solvent at Mg 2+ concentrations of 0 mM, 8 mM, and 25 mM. Root mean square fluctuation (RMSF) and entropy analyses revealed that increasing Mg 2+ concentrations progressively reduce RNA backbone flexibility and promote unfolded, linearized conformations, while the absence of Mg 2+ leads to intramolecular folding due to the system sampling misfolded substates that sequester nucleotides required for dimerization for both SL1-2 and SL1-3. These results suggest that Mg 2+ functions as a structural regulator by suppressing non-productive folding pathways and preserving dimerization-competent substates. This establishes a foundation for pathway investigations to unravel the Mg 2+ dependency of the energetic barriers and structural transitions that underlie kissing complexation in X55 as well as other conserved RNA elements.
Puthumana et al. (Sun,) studied this question.