Polyclonal Antibodies against a Structure Mimicking the Covalent Linkage Unit between Picornavirus RNA and VPg: An Immunochemical Study

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O. A. Ivanova¹, A. G. Venyaminova², M. N. Repkova², and Yu. F. Drygin^1*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-3181; E-mail: drygin@belozersky.msu.ru

²Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; fax: (383) 233-3677; E-mail: ven@niboch.nsc.ru

^* To whom correspondence should be addressed.

Received August 6, 2004; Revision received September 27, 2004

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We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH₂)]Tyr-(5´P → O)Up-O-(CH₂)₆NH₂, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs “freezing” dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.

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KEY WORDS: antibody, RNA-VPg, covalent linkage unit, affinity chromatography, immunogold, picornavirus, DNA topoisomerase

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