Two-dimensional nuclear Overhauser enhancement (NOESY) spectra of labile protons were recorded in H 2 0 solutions of a protein and of a DNA duplex, using a modification of the standard NOESY experiment with all three 90" pulses replaced by jump-and-return sequences. For the protein as well as the DNA fragment the strategically important spectral regions could be recorded with good sensitivity and free of artifacts. Using this procedure, sequence-specific assignments were obtained for the imino protons, C2H of adenine, and C4NH2 of cytosine in a 23-base-pair DNA duplex which includes the 17-base-pair OR3 repressor binding site of bacteriophage A. Based on comparison with previously published results on the isolated OR3 binding site, these data were used for a study of chain termination effects on the chemical shifts of imino proton resonances of DNA duplexes.For 'H-NMR studies of nucleic acids in H 2 0 solution, where selective saturation (e.g. [l]) cannot be employed, a variety of different solvent suppression schemes by selective excitation have been applied with one-dimensional (1 D) Fourier transform experiments [2-161. Some of these techniques were recently also adapted for use with two-dimensional (2D) NMR, in particular 2D nuclear Overhauser enhancement spectroscopy (NOESY). Thereby, in all but one [17] of the modifications of the basic NOESY pulse sequence, 90"-t, -9Oo-z,,-90"-Acq. (tz) (1) only the last pulse was replaced by a semiselective excitation scheme [18-231. This leads to a 2D excitation profile which exhibits the spectral amplitude response of the semiselective pulse along wz, but is uniform along the w1 frequency axis. This could be very powerful with a phase program which assures that the combined effects of the first two pulses in the NOESY experiment always lead either to an effective 0-degree or 180-degree rotation at the frequency of the water resonance. The water magnetization would thus be parallel to the z-axis at all times except during t l , but irrespective of the radio-frequency carrier position relative to the water line such phase cycles would invariably also lead to folding of the spectrum along o1 about the water resonance [24]. On the other hand, all experimental schemes which prevent folding of the spectrum along w1 include scans which bring water magnetization into the x,y plane during the mixing time z , , so that after the semiselective observation pulse a huge water signal must be acquired. This situation would be less severe in experiments with a long mixing time, during which most of Correspondence to K. Wuthrich, Institut fur Molekularbiologie und Biophysik, ETH-Honggerberg, CH-8093 Zurich, SwitzerlandAbbreviations. NOESY, two-dimensional nuclear-Overhauser-enhancement spectroscopy; 1 D, one-dimensional; 2D, two-dimensional.the transverse magnetization could relax. Quite generally, it could also be improved with a homospoil pulse applied during the mixing time.Different approaches can be based on the use of three semiselective pulses in the NOESY experiment [17]. The ...