Bridged oligonucleotides as molecular probes for investigation of enzyme-substrate interaction and allele-specific analysis of DNA

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.