Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to oligonucleotides.
The ability of T4 polynucleotide kinase (PNK) to phosphorylate non-nucleosidic moieties 5'-attached to oligodeoxynucleotides (ODNs) has been investigated. Non-nucleosidic phosphoramidite units were prepared from ethane-1,2-diol and propane-1,3-diol backbones. Some of them corresponded to pure enantiomers. They were used to obtain the corresponding 5'-end modified oligothymidylates X(pdT)10. The free primary hydroxyl of the non-nucleosidic moieties (X) of these oligomers was phosphorylated by PNK. We report the stereoselective phosphorylation of the L form of the 5'-end attached non-nucleosidic chiral fragments; the non-chiral moieties were completely phosphorylated. Dimers of glycerol analogue and thymidine 3'-phosphate were not recognized by PNK and the shortest modified ODN able to be phosphorylated was a trinucleotide X(pdT)3. A modified X(pdT)10, bearing a cyclic abasic site (X) at its 5'-end, was prepared by chemical synthesis from 1,2-dideoxyribose phosphoramidite and was phosphorylated with a 90% yield.
CEA/Départment de Recherche Fondamentale sur la matière condensée/SESAM, Grenoble, France.
M L
ML
Fontanel
H
H
Bazin
R
R
Téoule
D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D009841:Oligonucleotides; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I