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Because of spectral overlap, even with 2-D NMR methods, 1H and 31P signal assignments in oligonucleotides much longer than tetramers is difficult. However, by chemically introducing site-specific 17O, 13O, and S labeling in the phosphoryl groups of oligonucleotides, it is possible to unambiguously assign the 31P peaks. Thus, it is possible to assign all three phosphate 31P signals of the oligonucleotide tetramer d(ApGpCpT) by site-specific introduction of the three different oxygen isotopes into the three different phosphate diesters. Using two-dimensional 31P/1H correlated spectral methods we can also unambiguously identify the 1H NMR signals coupled to the assigned 31P signals. In the latter, only those protons which are scalar coupled to the IXP nucleus are observed in the 2-D heteronuclear spectrum. Finally by 1H/1H COSY and NOESY we can identify the other protons of the oligonucleotides. This methodology is not dependent upon any assumed B-DNA structure as is required in other recent 2-D oli...