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 × 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 µM(–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.