Kinetics of hybridization of mRNA of c-myc oncogene with 111In-labeled antisense oligodeoxynucleotide probes by high-pressure liquid chromatography.

Antisense oligodeoxynucleotides (ASON) were labeled with gamma-emitting 123I, 99mTc and 111In radionuclides. The hybridization kinetics of 111In-labeled ASON probes [phosphorothioate (S) and phosphodiester (O)] with intact leukemic cells (P388) and purified mRNA was studied by gel filtration technique. The 15-mer oligodeoxynucleotide (ON) sequence was synthesized, amino linked and coupled to diethylenetriaminepentaacetic acid (DTPA)-isothiocyanate, and aliquots were lyophilized to make a kit for convenient preparation. 111In radionuclide was chelated to DTPAASON derivatives and free 111In was separated by gel filtration. The probe was incubated with P388 cells and mRNA extract of P388 cells. Hybridization kinetics was studied by measuring the free and mRNA-bound probe separated by the HPLC technique. The distribution of radioactivity associated with proteins, DNA and mRNAs was directly measured with a gamma camera and further quantified with an ionization chamber. The kinetics of direct and indirect hybridization of 111In-labeled antisense probes with mRNA and intact cells was similar.