Kinetic and thermodynamic analysis of RNA binding by tethered oligonucleotide probes: alternative structures and conformational changes

Abstract

Binding of tethered oligonucleotide probes (TOPs) to the Leptomonas collosoma spliced leader RNA (SL RNA) was characterized using equilibrium, kinetic, and chemical structure-mapping methods. Equilibrium dissociation constants for TOP-SL RNA complexes are in the nanomolar range, with binding ΔG o obs between -8.3 and -9.6 kcal mol -1 (10 mM MgCl 2 , 10 mM NaCl, 0.1 mM EDTA, 25 o C). The highest SL RNA affinities are observed with TOPS 1 and 4, which contain the shortest tethers. Formation of the complex between TOP 4 and the SL RNA is characterized by a second-order rate constant (k on ) of 2.2×10 3 M -1 s -1 (10 mM MgCl 2 , 10mM NaCl, 0.1 mM EDTA, 25 o C)