An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either Mal-type (GUGCAC) or Lai-type (GCGCGC) kissing complexes under various ionic conditions. The force required to disrupt the kissing interaction of the two structures, the rip force, is sensitive to concentrations of KCl and MgCl2; addition of 3 mM MgCl2 to 100 mM KCl changes the rip force of Mal from 21 ± 4 to 46 ± 3 pN. From the rip force distribution, the kinetics of breaking the kissing interaction is calculated as a function of force and cation concentration. The two kissing complexes have distinct unfolding transition states, as shown by different values of ΔX‡, which is the distance from the folded structure to the unfolding transition state. The ΔX‡ of Mal is 0.6 nm smaller than that of Lai, suggesting that fewer kissing base pairs are broken at the transition state of the former, consistent with observations that the Lai-type kissing complex is more stable and requires significantly more force to unfold than the Mal type. More importantly, neither K+ nor Mg2+ significantly changes the position of the transition state along the reaction coordinate. However, increasing concentrations of cations increase the kinetic barrier. We derived a cation-specific parameter, m, to describe how the height of the kinetic barrier depends on the concentration of cations. Our results suggest that Mg2+ greatly slows down the unfolding of the kissing complex but has moderate effects on the formation kinetics of the structure.
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