Detection of specific base sequences in solution using DNA probes labeled with d- and/or 15N-substituted spin-labels

In order to enhance the sensitivity and the accuracy of the detection by the DNA probe method in which spin-labeled oligonucleotides are used as probes, 4-amino-2,2,6,6-tetramethylpiperidine-15N-oxyl-d16 (4-amino-TEMPO-15N-d16) and 4-amino-2,2,6,6-tetramethylpiperidine-14N-oxyl-d16 (4-amino-TEMPO-14N-d16) were incorporated into the phosphate linkage of oligonucleotides by the hydrogen phosphonate method and these DNA probes were used for the detection of target DNAs in solution. The intensities of the electron paramagnetic resonance (EPR) signals of the oligonucleotides labeled with 4-amino-TEMPO-14N-d16 and 4-amino-TEMPO-15N-d16 were 3-fold and 4-fold larger than that of the oligonucleotide labeled with 4-amino-TEMPO-14N, respectively. Also, the EPR lines of these labeled oligonucleotides do not overlap each other, allowing the detection of two different regions in the same target DNA by the single EPR measurement.