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-TpCAnpGiB-pABzpABz, d-CAnpTpABzpCAn, d-ABzpGiBpABzpGiB, and d-GBzpCAnpT 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.
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