DEVELOPMENT OF SENSITIVE RADIOIODINATED ANTISENSE OLIGONUCLEOTIDE PROBES BY CONJUGATION TECHNIQUE

Radioactive and nonradioactive oligonucleotide (ON) probes have been used in a variety of studies of in vitro hybridization for locations of specific genes and determination of the level of mRNA transcription in activated and proliferative cells. Nuclease-resistant phosphorothioate derivatives of oligonucleotides are also finding potential therapeutic applications in intact cells and animal models. In this study, we have developed new techniques of radioiodination by the conjugation of oligonucleotides with p-methoxyphenyl isothiocyanate (PMPITC) and optimized the conditions of the radioiodination, e.g. concentration of reactants, oxidants, pH, time, and temperature. We have used a 25-mer actin mRNA probe as a model substrate for radioiodination. The ON probe was modified by conjugation with the addition of aminohexyl (AH) group at the 5'-terminal. Both the techniques of preiodination of PMPITC followed by conjugation and radioiodination of PMPITC-conjugated AHON were evaluated; the latter was found to be more convenient. The optimum pH of radioiodination of PMPITC with I-125 was 6.5; on the other hand, the optimum pH for PMPITC-conjugated AHON was 8.5. The conditions of conjugation were optimized with I-125-labeled PMPITC. The techniques of paper chromatography and HPLC with a Spherogel column were developed for the purification and characterization of these oligonucleotide probes. Without PMPITC conjugation, the yield of direct radioiodination of the same oligonucleotide was very low (4-6%); the yield in our optimized technique is 50-60%. Radioiodination of conjugated oligonucleotides is a simple and efficient route to radiolabeled probes for hybridization studies.