Bridged Oligonucleotides as Molecular Probes for Investigation of Enzyme–Substrate Interaction and Allele-Specific Analysis of DNA

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I. A. Pyshnaya¹, O. A. Vinogradova¹, M. R. Kabilov¹, E. M. Ivanova^1#, and D. V. Pyshnyi^1,2*

¹Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, pr. Akademika Lavrent’eva 8, 630090 Novosibirsk, Russia; fax: (383) 333-3677; E-mail: pyshnyi@niboch.nsc.ru

²Novosibirsk State University, ul. Pirogova 2, 630090 Novosibirsk, Russia

^# Deceased.

^* To whom correspondence should be addressed.

Received July 16, 2008; Revision received September 22, 2008

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The efficiency of enzymatic conversion of DNA complexes containing non-nucleotide inserts has been studied. T4 DNA ligase and Taq DNA polymerase have been included in the study as examples of widely used DNA-dependent enzymes. A series of substrate DNA complexes have been formed using native oligonucleotides and bridged ones bearing non-nucleotide inserts based on phosphodiesters of di-, tetra-, or hexaethylene glycol, 1,5-pentanediol, 1,10-decanediol, and 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran. The perturbation in DNA located far from the site of the enzyme action had almost no influence on the substrate properties of the complex, while insertion near this site significantly deteriorated them. The use of a series of modified duplexes allows one to locate the position of the enzyme-binding site on DNA substrate with the accuracy of 1-2 nucleotides. The presence of a non-nucleotide insert in the complex has been also shown to enhance the efficiency of single mismatch discrimination upon both template-directed ligation and extension of oligonucleotides.

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KEY WORDS: bridged oligonucleotides, non-nucleotide insert, modified DNA complexes, footprinting, binding site, selectivity, T4 DNA ligase, Taq DNA polymerase

DOI: 10.1134/S0006297909090090

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