End attachment of phenol-oligonucleotide conjugates to diazotized cellulose.
The synthesis of a novel phosphoramidite reagent with a hexanediol backbone is described. This reagent has been used to incorporate a phenol moiety on an oligonucleotide (ODN) directly in the course of its automated synthesis. Multiple phenol attachments can be achieved by repetitive coupling cycles. A simple and rapid immobilization method is described where phenol-modified ODNs are covalently attached to diazotized cellulose. The binding capacity of the membrane can be modulated, depending on the ODN concentration used, to ca. 180 pmol/cm2. There is at least 80% end attachment of the ODN through the phenol group. In addition, the phenol residue can be used as a carrier for the radiolabeling with 125I. The non-nucleosidic hexanediol derivative incorporated at the 5'-end of the ODN is recognized as a substrate by the T4 polynucleotide kinase and the terminal hydroxyl group is successfully phosphorylated allowing its 32P labeling.
Fontanel
M
M
Laboratoire de Radiobiochimie, Centre d'Etudes Nucléaires de Grenoble, DRFMC/SESAM, 85X, France.
Bazin
H
H
Téoule
R
R
D001483:Base Sequence; D002482:Cellulose / Q000737:chemistry*; D003979:Diazonium Compounds / Q000737:chemistry*; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D010636:Phenols / Q000737:chemistry*; D010761:Phosphorus Radioisotopes; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism
D003979:Diazonium Compounds; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D009841:Oligonucleotides; D010636:Phenols; D010761:Phosphorus Radioisotopes; D002482:Cellulose; D011116:Polynucleotide 5'-Hydroxyl-Kinase