SUNY at Albany
June 19-23, 2001
Mechanism of Hybridization of Antisense Oligonucleotides with tRNA
Interaction of tRNAPhe with oligonucleotides complementary to the 3Õ half of this RNA under physiological conditions was investigated with gel-shift assay, probing of the tRNAPhe structure in the course of the hybridization with oligonucleotides and stopped-flow method. Oligonucleotides were complementary to acceptor and TyC stems (nucleotides 62-76) of tRNA. Probing experiments using RNase T1, RNase A, DMS and RNase ONE have shown that the hybridization affects the RNA structure in the D-loop, anticodon stem, TYC-hairpin and CCA-end. The probing data demonstrate invasion of oligonucleotides into the tRNA structure by displacement of the 5Õ strand of the acceptor stem. The dependencies of association rate constant versus oligonucleotide concentration determined in gel-shift experiments evidenced for two-step mechanism of oligonucleotide binding to the tRNA. At the first step, oligonucleotide binding rapidly occurs with open ACCA sequence yielding an intermediate complex. The second step is a slow unfolding of tRNA structure and extended duplex formation. Evaluation of activation energy of oligonucleotide hybridization with the RNA confirms that the interaction occurs via strand displacement mechanism.
The detailed kinetics of oligonucleotide hybridization with tRNA was studied using stopped-flow method with fluorescence quenching detection. 3Õ- or 5Õ-DABCYL labeled oligonucleotides hybridized with 3Õ-fluorescein labeled in vitro transcripts of tRNAPhe in physiological conditions. The results revealed a multi-stage invasion of oligonucleotide in tRNA structure. The data were interpreted according to minimal kinetic scheme consisting of three equilibrium stages. The first stage detected within short time interval represents the collision complex formation. The second and the third stages corresponds to intermediate and extended duplex formations as it was suggested above.
Our data evidence that the stability of RNA structure within oligonucleotide target site govern the rate of oligonucleotide binding. Factors stabilizing RNA structure affects the rate and efficacy of binding.
This work was supported by the Interdisciplinary grant and Grant in support of young scientists of Siberian Division of Russian Academy of Sciences, RFBR grants, CRDF grant REC-008.References and Footnotes
V. Petyuk, R. Serikov, V. Koval, V. Vlassov, M. Zenkova*, O. Fedorova*
Institute of Bioorganic Chemistry, Siberian Devision of Russian Academy of Sciences, Novosibirsk, 630090, Russia