KINETIC AND THERMODYNAMIC ANALYSIS OF RNA-BINDING BY TETHERED OLIGONUCLEOTIDE PROBES - ALTERNATIVE STRUCTURES AND CONFORMATIONAL-CHANGES

Binding of tethered oligonucleotide probes (TOPs) to the Leptomonas co llosoma spliced leader RNA (SL RNA) was characterized using equilibriu m, kinetic, and chemical structure-mapping methods. Equilibrium dissoc iation constants for TOP-SL RNA complexes are in the nanomolar range, with binding DELTAG-degrees(obs) between -8.3 and -9.6 kcal mol-1 (10 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 25-degrees-C). The highest SL RNA a ffinities are observed with TOPS 1 and 4, which contain the shortest t ethers. Formation of the complex between TOP 4 and the SL RNA is chara cterized by a second-order rate constant (k(on)) of 2.2 X 10(3) M-1 s- 1 (10 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 25-degrees-C). Dissociation o f the complex between TOP 4 and the SL RNA is characterized by a first -order rate constant (k(off)) of 4.3 x 10(-4) s-1 under similar condit ions, corresponding to an average complex lifetime of 40 min. Chemical structure-mapping experiments performed on the uncomplexed SL RNA sup port a secondary structure model proposed recently by LeCuyer and Crot hers. Chemical structure-mapping experiments performed on the SL RNA-T OP 1 complex demonstrate that TOP 1 shifts the SL RNA from the LeCuyer -Crothers secondary structure into a structure that is not well popula ted in the absence of TOP 1.