Assignment of phosphorus-31 and nonexchangeable proton resonances in a symmetrical 14 base pair lac pseudooperator DNA fragment

Abstract

The 31P chemical shifts of all 13 phosphates and the chemical shifts of nearly all of the non-exchangeable protons of a symmetrical 14 base pair lac pseudooperator DNA fragment have been assigned by regiospecific labeling with oxygen-17 and two-dimensional NMR techniques. At 22 degrees C, 8 of the 13 phosphorus resonances can distinctly be resolved while the remaining 5 resonances occur in two separate overlapping regions. The 31P chemical shifts of this particular 14 base pair oligonucleotide do not follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence the more upfield the 31P resonance occurs, as shown from other 31P assignment studies. Failure of this general rule is believed to be a result of helical distortions that occur along the oligonucleotide double helix, on the basis of the analysis of Callidine [Callidine, C.R. (1982) J. Mol. Biol. 161, 343-352]. Notable exceptions to the phosphate position relationship are 5'-Py-Pu-3' dinucleotide sequences, which resonate at a lower field strength than expected in agreement with similar results as reported by Ott and Eckstein [Ott, J., & Eckstein, F. (1985) Biochemistry 24, 253]. A reasonable correlation exists between 31P chemical shifts values of the 14-mer and the helical twist sum function of Calladine. The most unusual 31P resonance occurs most upfield in the 31P spectrum, which has been assigned to the second phosphate position (5'-GpT-3') from the 5' end. This unusual chemical shift may be the result of the predicted large helical twist angle that occurs at this position in the 14-mer sequence. Further, it is believed that the large helical twist represents a unique structural feature responsible for optimum binding contact between lac repressor protein and this 14-mer lac pseudooperator segment. Assignments of proton resonances were made from two-dimensional 1H-1H nuclear Overhauser effect (NOESY) connectivities in a sequential manner applicable to right-handed B-DNA, in conjunction with two-dimensional homonuclear and heteronuclear J-correlated spectroscopies (1H-1H COSY and 31P-1H HETCOR). Most nonexchangeable base proton and deoxyribose proton (except for some unresolved H4', H5', and H5" protons) resonances were assigned.

Two-Dimensional1H and31P NMR Spectra of a Decamer Oligodeoxyribonucleotide Duplex and a Quinoxaline ([MeCys3, MeCys7]TANDEM) Drug Duplex Complex

“…The self-complementary decamer d(CCCGATCGGG) was synthesized by a manual modification of the solid phase phosphite triester method (using 10 μmole of the starting nucleoside derivatized support) as previously described (12,(37)(38)(39). The purity of the decamer was verified by chromatography on an anion-exchange column, a silica column coated with crosslinked polyethylene imine (40).…”

“…The 17 O/ 18 O label was incorporated at the appropriate step in the DNA sequence by using 17 O/ 18 O water during the oxidation step of the DNA synthesis as previously described (12,37).…”

“…This "positional" 31 P chemical shift effect is apparently superimposed on a sequencespecific effect (12,15,58,59,63). The minor groove clash steps (pyrimidine-purine base steps) have been generally associated with a relatively downfield 31 P chemical shift.…”

“…The decamer's 31 P signals were assigned by both a 2D pure absorption phase constant time (PAC) heteronuclear correlation NMR (46,55) and 17 O/ 18 O labeling methodologies (12).…”

“…In addition, local conformational heterogeneity in the deoxyribose phosphate backbone has been most recently noted in the form of sequence-specific variations (2)(3)(4) or as the result of drug (1) or protein binding (5,6) to local regions of the DNA. While X-ray crystallography has provided much of our understanding of these DNA structural variations, increasingly, high resolution NMR has also begun to provide detailed three-dimensional structural information on duplex oligonucleotides (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) and drug duplex complexes.…”