Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence

The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been selectively incorporated at adenosine positions in stem-loops (so called R1inv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II transcripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stopped-flow and equilibrium methods could be used to study the detailed mechanism of this RNA-RNA interaction. Formation of loop-loop kissing complexes between R1inv and R2inv hairpins, substituted with 2-AP at positions in the complementary loops, results in a 5-10-fold fluorescence emission decrease (F(max) = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average K(D) = 2.6 +/- 1.7 nM) and affinity for Rom (average K(D) = 60 +/- 24 nM) that are similar to complexes formed with equivalent unlabeled hairpins. Using stopped-flow experiments, it was found that the 2-AP probes experienced at least three different microenvironments during association of the RNA complex, thus suggesting a kinetic intermediate in the kissing pathway. In contrast, dissociation of the complex was found to fit a single exponential decay (average k(off) = 8.9 x 10(-5) s(-1)). Consistent with these observations, a two-step mechanism for RNA loop-loop complex association is proposed in which the complementary loops of R1inv and R2inv first base pair to form the loop-loop helix (average k(1) = 0.13 microM(-1)s(-1)) in the initial encounter reaction, and subsequently isomerize to the final tertiary fold in a second slower step (average k(2) = 0.09 s(-1)), where the helical stacking around the junctions is optimized.