Analysis of diffuse K+ and Mg2+ ion binding to a two-base-pair kissing complex by single-molecule mechanical unfolding.

The folding and stability of RNA tertiary interactions depend critically on cationic conditions. It is usually difficult, however, to isolate such effects on tertiary interactions from those on the entire RNA. By manipulating conformations of single RNA molecules using optical tweezers, we distinguished individual steps of breaking and forming of a two-base-pair kissing interaction from those of secondary folding. The binding of metal ions to the small tertiary structure appeared to be saturable with an apparent Kd of 160 mM for K(+) and 1.5 mM for Mg(2+). The kissing formation was estimated to be associated with binding of ~2-3 diffuse K(+) or Mg(2+) ions. At their saturated binding, Mg(2+) provided ~3 kcal/mol more stabilizing energy to the structure than K(+). Furthermore, the cations change the unkissing forces significantly more than the kissing ones. For example, the presence of Mg(2+) ions increased the average unkissing force from 21 pN to 44 pN, surprisingly high for breaking merely two base pairs; in contrast, the mean kissing force was changed by only 4.5 pN. Interestingly, the differential salt effects on the transition forces were not caused by different changes in the height of the kinetic barriers but were instead attributed to how different molecular structures respond to the applied force. Our results showed the importance of diffuse cation binding to the stability of tertiary interaction and demonstrated the utility of mechanical unfolding in studying tertiary interactions.