Hybridization of a 99Tcm-labelled oligodeoxynucleotide to CAPL RNA.
Oligodeoxynucleotides (ODNs) labelled with an appropriate radionuclide could provide a means to identify serious diseases early on and thereby help initiate treatment at a very early phase. Regardless of important issues like in-vivo stability and membrane passage, the key issue for the oligonucleotide approach is the ability of the radiolabelled ODN to hybridize to the target mRNA. The secondary structure of mRNA does not permit all complementary ODNs to hybridize and a careful selection of the probe with consecutive testing is therefore necessary. This study was initiated to demonstrate hybridization of a 99Tcm-labelled 20-mer ODN to RNA of CAPL (S100A4), a gene reported to be overexpressed in metastatic cancers like breast carcinoma and osteosarcoma. The phosphodiester ODN GX-1 (antisense) and two control sequences (scrambled and random) were conjugated to the bifunctional chelating agent S-benzoyl-mercaptoacetyltriglycine (S-benzoyl-MAG3) and labelled with 99Tcm. The radiolabelled ODNs were purified on a C18 mini-column and characterized on a reverse-phase HPLC system. The radio-chemical purity was > 90% and the product was stable for > 6 h in aqueous medium. The hydrization properties of unlabelled, 32P-labelled and 99Tcm-labelled ODNs to transcribed RNA were studied using polyacrylamide gel electrophoresis (PAGE). Direct hybridization of GX-1 to transcribed RNA was demonstrated. A 50-fold excess of unlabelled ODN over transcribed RNA caused a near to complete consumption of RNA by RNase H activation. In 1:1 proportions of radiolabelled (32P and 99Tcm) 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'-99Tcm-labelling of ODNs does not interfere with the hybridization properties of the ODNs in solution, making 99Tcm-labelling an attractive procedure for the future development of antisense technology in imaging.
Hjelstuen
O K
OK
Department of Pharmaceutics, University of Oslo, Norway.
Maelandsmo
G M
GM
Tønnesen
H H
HH
Bremmer
P O
PO
Verbruggen
A M
AM
D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002614:Chelating Agents; D005260:Female; D005998:Glycine / Q000031:analogs & derivatives; D006801:Humans; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger / Q000235:genetics*; D011877:Radionuclide Imaging; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D013667:Technetium
D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human; D013667:Technetium; C111010:S-benzoylmercaptoacetyltriglycine; D005998:Glycine