Improvements for Measuring1H–1H Coupling Constants in DNA via New Stripe-COSY and Superstripe-COSY Pulse Sequences Combined with a Novel Strategy of Selective Deuteration

✍ Scribed by Jiping Yang; Louis Silks; Ruilian Wu; Nancy Isern; Cliff Unkefer; Michael A. Kennedy

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 146 KB
• Volume: 129
• Category: Article
• ISSN: 1090-7807
• DOI: 10.1006/jmre.1997.1261

✦ Synopsis

Three bond proton-proton vicinal coupling constants are of postripe-COSY requires significantly shorter total experimental tential value for analyzing sugar conformations in DNA. However, time than a standard COSY-35 spectrum. In the example self-cancellation in antiphase cross peaks and modulation of peak presented here, the total experimental time was reduced by splittings by transverse cross relaxation can alter the apparent a factor of 4.9. The superstripe-COSY is a stripe-COSY coupling constants such that they do not accurately reflect the experiment with additional elements for eliminating resugar conformations. Transverse cross relaxation is most effective maining unwanted passive splittings. Finally, selective deubetween strongly coupled geminal proton pairs. Here we report teration was used to further improve the spectral quality and the use of stereospecific deuteration at the H2Љ position in the A5 to effectively eliminate the deleterious effects of transverse and A6 residues in the 12 base pair DNA sequence [d(CGCGAAcross relaxation (5,6). In this example, the H2Љ proton of TTCGCG)2] as a means of investigating the effect of transverse the deoxyribose sugar was replaced with a deuteron in all cross relaxation on P.E.COSY type cross peaks. Deuteration of the H2Љ proton is expected to reduce the transverse cross relaxation adenosine nucleotides. Consequently, the magnitude of rate by the square of ratio of the proton to deuteron gyromagnetic transverse cross relaxation normally present between the ratios, i.e., by a factor of 42. Additionally, a striking eight-to geminal H2 and H2Љ protons, which is determined by the ninefold increase in the signal intensity was observed for cross square of the dipolar Hamiltonian, was reduced by the square peaks involving the remaining H2 proton resulting from diminof the ratio of the gyromagnetic ratios, i.e., (g D /g H ) 2 or ished dipolar relaxation. Further improvements in signal-to-noise about 42-fold. Altogether, in the example presented here, the ratio were realized by collecting P.E.COSY spectra in strips, using superstripe-COSY combined with selective H2Љ deuteration an experiment referred to as stripe-COSY, employing selective produced a spectrum in Ç1/5 the time required for the excitation pulses which reduced the number of required t 1 incre-COSY-35 and with about eight to nine times greater signalments by a factor of four. A final improvement was achieved by to-noise. Using a constant signal-to-noise comparison, the employing selective time-shared homonuclear decoupling during the acquisition period, in an experiment referred to as superstripesuperstripe-COSY combined with selective deuteration can COSY, to collapse splittings due to passive couplings. Collectively, potentially reduce the total experimental time by a factor of these approaches provide P.E.COSY-type spectra with two to three 200-300 compared to a standard COSY-35 experiment.

orders of magnitude increased sensitivity per unit time and that

Proton-proton coupling constants are very useful in deare relatively free from artifacts. ᭧ 1997 Academic Press termining the conformations of organic compounds and biomolecules since dihedral angles can be determined based on measured coupling constants via Karplus relationships This Communication reports new approaches for improv-(7, 8). In DNA, the coupling constants among H1 , H2, ing the measurement of proton-proton coupling constants H2Љ, H3, and H4 protons are of foremost interest for dein DNA and for diminishing the effects of transverse cross termining the sugar conformation (9). Although high-resorelaxation on P.E.COSY (1, 2) type cross peaks. The methlution correlation experiments such as phase-sensitive COSY ods are demonstrated for measuring 3 J H1 = -H2= coupling con-(10, 11) and DQF-COSY (12, 13) in principle allow the stants in a 12-base-pair DNA oligonucleotide. We introduce measurement of all coupling constants in systems of limited two modified pulse sequences: the stripe-COSY and the sucomplexity, they are less useful for complex spin systems perstripe-COSY. The stripe-COSY is essentially a phaseand for large molecules. In larger molecules, the broad lines sensitive 4) collected in narrow strips. The associated with short correlation times lead to the overlap of numerous multiplet components and to the self-cancellation of antiphase multiple components (14, 15).