2'-O-Methyl Oligoribonucleotide Analogs Used to Change the Temperature Characteristics of Immobilized Probes and to Enhance the Specificity of Hybridization

Abstract

The specificity of interactions between oligonucleotide probes immobilized in a biological microchip (biochip) and target DNA depends on a number of factors, with the main roles being played by the probe length and nucleotide sequence (GC composition primarily). The 2'-O-methyl oligoribonucleotides are known to form more stable duplexes with complementary DNA compared with 2'-deoxyribonucleotide probes. The study tested the possibility of using 2'-O-methyl oligoribonucleotides to increase the specificity of interactions between target DNA and probes immobilized in a biochip. Fluorophore-labeled target DNA was obtained via one-round asymmetric PCR with simultaneous incorporation of a fluorescent label in the PCR product. After hybridization with immobilized probes, fluorescent signals from gel pads with 2'-O-methyl oligoribonucleotide probes were, on average, two times higher than from those with 2'-deoxyribonucleotide probes, with the target DNA sequences and probe concentrations being the same. The increase in fluorescence intensity was greater in the case of perfect versus imperfect (mismatched) duplexes.