Phosphorylation of N-Protected Deoxyoligonucleotides by T₄ Polynucleotide Kinase

The substrate recognition of T₄-induced polynucleotide kinase has been investigated. It was found that the presence of base protection groups on the 5′-nucleoside moiety of an oligonucleotide did not interfere with the action of the enzyme. The deoxyoligonucleotides d-TpC^AnpG^iB-pA^BzpA^Bz, d-C^AnpTpA^BzpC^An, d-A^BzpG^iBpA^BzpG^iB, and d-G^BzpC^AnpT could be quantitatively phosphorylated at their 5′-hydroxyl terminus. No difference in the rate of phosphorylation was observed between an N-protected and unprotected deoxyoligonucleotide. The use of ³²P-labeled protected nucleotide blocks during the chemical synthesis of a polynucleotide provides a simple way to follow the rate of internucleotide bond formation and facilitates the isolation and characterization of the reaction products.