IMPROVEMENTS FOR MEASURING H-1-H-1 COUPLING-CONSTANTS IN DNA VIA NEW STRIPE-COSY AND SUPERSTRIPE-COSY PULSE SEQUENCES COMBINED WITH A NOVELSTRATEGY OF SELECTIVE DEUTERATION

Three bond proton-proton vicinal coupling constants are of potential v alue for analyzing sugar conformations in DNA. However, self-cancellat ion in antiphase cross peaks and modulation of peak splittings by tran sverse cross relaxation can alter the apparent coupling constants such that they do not accurately reflect the sugar conformations, Transver se cross relaxation is most effective between strongly coupled geminal proton pairs. Here we report the use of stereospecific deuteration at the H2 '' position in the A5 and A6 residues in the 12 base pair DNA sequence [d(CGCGAATTCGCG)2] as a means of investigating the effect of transverse cross relaxation on P.E.COSY type cross peaks, Deuteration of the H2 '' proton is expected to reduce the transverse cross relaxat ion rate by the square of ratio of the proton to deuteron gyromagnetic ratios, i.e., by a factor of 42. Additionally, a striking eight-to ni nefold increase in the signal intensity was observed for cross peaks i nvolving the remaining H2' proton resulting from diminished dipolar re laxation, Further improvements in signal-to-noise ratio were realized by collecting P.E.COSY spectra in strips, using an experiment referred to as stripe-COSY, employing selective excitation pulses which reduce d the number of required t(1) increments by a factor of four, A final improvement was achieved by employing selective time-shared homonuclea r decoupling during the acquisition period, in an experiment referred to as superstripe-COSY, to collapse splittings due to passive coupling s. Collectively, these approaches provide P.E.COSY-type spectra with t wo to three orders of magnitude increased sensitivity per unit time an d that are relatively free from artifacts. (C) 1997 Academic Press.