Oligonucleotides site-specifically spin-labeled at 5'-terminal or internucleotide linkage and their use in gene analyses.

Abstract

Spin-labeled oligonucleotides (S-probes) were synthesized and examined as DNA probes to monitor hybrid formation. TEMPO was introduced either at the internucleotide linkage of 5'-terminus (Type 1) or at the 5'-terminal hydroxyl group (Type 2) and both types of S-probes were used in this study. The presence of target DNA was detected in solution by EPR spectroscopy for both types of S-probes. Hybridization of the S-probes resulted in notable broadening of EPR line width, accompanied by a decrease in the EPR signal height ratio for I(-1)/I(0).I(-1)/I(0) of S-probes having no spacer between oligonucleotide and TEMPO decreased more markedly than that of S-probes with a spacer, indicating that TEMPO should be introduced to an oligonucleotide directly to monitor hybrid formation. When M13mp8 single-stranded DNA with or without an EcoRI recognition site was selected as a target DNA, hybrid formation was detected only for DNA containing EcoRI site in solution using spin-labeled oligonucleotides.