BRIDGED OLIGONUCLEOTIDES - 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 were used 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" oligonucleotides bearing non-nucleotide inserts based on phosphodiesters of di-, tetra-, and hexaethylene glycol, 1,5-pentanediol, 1,10-decanediol, and 3-hydroxy-(2-hydroxymethyl) tetrahydrofuran. The efficiency of enzymatic conversion is shown to depend significantly both on the nature of the insert and its proximity to the binding site of the enzyme. Distant disturbances in the DNA structure do not influence the substrate properties of the complex, whereas disturbances located near the binding site significantly lower the efficiency of conversion. The use of a series of modified duplexes locates the position of the binding site of the enzyme on the DNA substrate with the accuracy of 1-2 nucleotides. The presence of a non-nucleotide insert in the complex is also shown to increase the efficiency of single mismatch discrimination both during template-directed ligation and extension of oligonucleotides.