th  - 1988 - 51st ENC Conference

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CARBON-13 SPECTRAL ASSIGNMENTS OF DNA OLIGOMERS: APPLICATIONS OF PROTON-DETECTED
HETERONUCLEAR 2D-NMR: J. Ashcroft*, and D. Cowbum, The Rockefeller Univ., New York, New York, 10021-6399
Heteronuclear multi.spin coherence proton detected chemical shift correlated NMR (HMP-COSY), may be
used to obtain 2D IH-{13C} correlated spectra. Carbon resonances can then be assigned via NOESY and COSY derived
proton assignnmnts. Proton-carbon correlated spectra can also provide useful information without supplementation. In
the 1D proton spectxum of DNA oligomers, the resonances arising from the 1', 3', 4', 5' and the cytidine H5 protons all
occur within an approximam.ly 2 ppm wide region, while in the 1D carbon spectra these groups, except the 1' and 4' are
well separated. In the 1H-{tJC} correlated spectrum all groups are distinct, and.group assignments are greatly facilitated.
Proton and .cybon chemical shifts, along with single-bond coupling constants (zJcH obtaided in the HMP-COSY experi-
ment when laC decoupfing is not appfied during acquisition), can be used to assign resonances to a specific type of
nucleotide residue. For example, the adenine C2 and the cytidine C5 chemical shifts are unique, enabling identification of
these resonances. Also, the purine base CH pairs exhibit IJcrl'S at least 30 Hz. greater than pyrimidine base CH pairs.
The I-IMP-COSY experiment uses a mixing period of duration 1/2./, to ensure maximum coherence transfer
between proton and carbon. For single-bond coupling, this value ranges from 2.5 msec. to 4.0 msec., (J = 125 to 200
Hz.). If the mixing period is between 40 and 100 msec., the HMP-COSY experiment is optimized for multiple bond cou-
piing, (J = 12.5 to 5 Hz.). In such an experiment one can obtain a proton-carbon correlated spectra, which contains
proton-non-protonated carbon cross peaks. Thus, assignment of carbonyl and quaternary carbons is possible.
In principle the above two methods can be used to assign all carbon resonances in a DNA duplex. For practi-
cal reasons,-- extreme spectral crowding, cancellation of anti-phase peaks, and complications of spectral interpretation due
to strong-coupling,-- the 2', 2" and 5' carbon resonances can not be fully assigned using these techniques. The use of
proton-detected IH-{IH-13C}-RELAY experiments to obtain the 2', 2" and 5' carbon assignments is examined.
The effectiveness of different pulse sequences used to obtain IH-{13C} I-IMP-COSY spectra are compared.
Examples obtained from the study of the duplexes d(TAGCGCTA)2, d(GGTATACC)2 , d(GGAATTCC)2 , are shown.
Supported by grants from NSF, NIH, and the Keck Foundation
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