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Association of an RNA Kissing Complex Analyzed Using 2-Aminopurine Fluorescence



M J. Rist, John Marino


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 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 (Fmax = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average KD = 2.6 1.7 nM) and affinity for ROM (average KD = 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 Koff = 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 k1 = 0.13 M-1s-1) in the initial encounter reaction, and subsequently isomerize to the final tertiary fold in a second slower step (average k2 = 0.09 s-1), where the helical stacking around the junctions is optimized.
Nucleic Acids Research
No. 11


2-aminopurine, antisense, ColE1, fluorescence, RNA


Rist, M. and Marino, J. (2001), Association of an RNA Kissing Complex Analyzed Using 2-Aminopurine Fluorescence, Nucleic Acids Research (Accessed April 25, 2024)
Created May 31, 2001, Updated October 12, 2021