HYBRIDIZATION OF TC-99(M)-LABELED OLIGODEOXYNUCLEOTIDE TO CAPL RNA

Oligodeoxynucleotides (ODNs) labelled with an appropriate radionuclide could provide a means to identify serious diseases early on and there by help initiate treatment at a very early phase. Regardless of import ant issues like in-vivo stability and membrane passage, the key issue for the oligonucleotide approach is the ability of the radiolabelled O DN to hybridize to the target mRNA. The secondary structure of mRNA do es not permit all complementary ODNs to hybridize and a careful select ion of the probe with consecutive testing is therefore necessary. This study was initiated to demonstrate hybridization of a Tc-99(m)-labell ed 20-mer ODN to RNA of CAPL (S100A4), a gene reported to be overexpre ssed in metastatic cancers like breast carcinoma and osteosarcoma. The phosphodiester ODN GX-1 (antisense) and two control sequences (scramb led and random) were conjugated to the bifunctional chelating agent S- benzoyl-mercaptoacetyltriglycine (S-benzoyl-MAG3) and labelled with Tc -99(m). The radiolabelled ODNs were purified on a C18 mini-column and characterized on a reverse-phase HPLC system. The radiochemical purity was > 90% and the product was stable for > 6 h in aqueous medium. The hydrization properties of unlabelled, P-32-labelled and Tc-99(m)-labe lled ODNs to transcribed RNA were studied using polyacrylamide gel ele ctrophoresis (PAGE). Direct hybridization of GX-1 to transcribed RNA w as demonstrated. A 50-fold excess of unlabelled ODN over transcribed R NA caused a near to complete consumption of RNA by RNase H activation. In 1:1 proportions of radiolabelled (P-32 and Tc-99(m)) ODNs to RNA, only radiolabelled GX-1 was found to hybridize to RNA in a PAGE system . The radiolabelled control ODNs did not show signs of hybridization. This study demonstrates that 3'-Tc-99(m)-labelling of ODNs does not in terfere with the hybridization properties of the ODNs in solution, mak ing Tc-99(m) labelling an attractive procedure for the future developm ent of antisense technology in imaging. ((C) 1998 Lippincott Williams & Wilkins).