NMR OF ENZYMATICALLY SYNTHESIZED UNIFORMLY (CN)-C-13-N-15-LABELED DNA OLIGONUCLEOTIDES

A procedure for the enzymatic synthesis of uniformly (CN)-C-13-N-15-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on C-13 and N-15 nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG . dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of N-15 and C-13 heteronuclear NMR experiments to isotopically labeled DNA is presented.