KINETICS OF HYBRIDIZATION OF MESSENGER-RNA OF C-MYC ONCOGENE WITH IN-111-LABELED ANTISENSE OLIGODEOXYNUCLEOTIDE PROBES BY HIGH-PRESSURE LIQUID-CHROMATOGRAPHY

Antisense oligodeoxynucleotides (ASON) were labeled with gamma-emittin g I-123, 99mTc and In-111 radionuclides. The hybridization kinetics of In-111-labeled ASON probes [phosphorothioate (S) and phosphodiester ( 0)] with intact leukemic cells (P388) and purified mRNA was studied by gel filtration technique. The 15-mer oligodeoxynucleotide (ON) sequen ce was synthesized, amino linked and coupled to diethylenetriaminepent aacetic acid (DTPA)-isothiocyanate, and aliquots were lyophilized to m ake a kit for convenient preparation. In-111 radionuclide was chelated to DTPAASON derivatives and free In-111 was separated by gel filtrati on. The probe was incubated with P388 cells and mRNA extract of P388 c ells. Hybridization kinetics was studied by measuring the free and mRN A-bound probe separated by the HPLC technique. The distribution of rad ioactivity associated with proteins, DNA and mRNAs was directly measur ed with a gamma camera and further quantified with an ionization chamb er. The kinetics of direct and indirect hybridization of In-111-labele d antisense probes with mRNA and intact cells was similar.