Oxygen Isotope and Sulfur Labeling of Phosphoryl Groups and 2-Dimensional NMR Methodology for Assignment of 31P and 1H Signals of Oligonucleotides

Because of spectral overlap, even with 2-D NMR methods, 1 H and 31 P signal assignments in oligonucleotides much longer than tetramers is difficult. However, by chemically introducing site-specific 17 O, 13 O, and S labeling in the phosphoryl groups of oligonucleotides, it is possible to unambiguously assign the 31 P peaks. Thus, it is possible to assign all three phosphate 31 P 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 31 P/ 1 H correlated spectral methods we can also unambiguously identify the 1 H NMR signals coupled to the assigned 31 P 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 1 H/ 1 H 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 oligonucleotide assignment techniques. Assignment of signals in the actinomycin D intercalating d(ApGpCpT) tetramer complex, d(CGCAGAATTCGCG), and lac operator pseudo-fragment, d(TGTGAGCGCTCACA), are described.