JOURNAL OF MAGNETIC RESONANCE 62, 378-386 (1985) Sequential Assignment of Imino- and Amino-Proton Resonances in ‘H NMR Spectra of Oligomcleotides by Two-Dimensional NMR Spectroscopy. Application to a luc Operator Fragment R. BOELENS, R. M. SCHEEK, K. DUKSTRA, AND R. KARTEIN Deparlment of Physical Chemistry, University of Groningen. Ndenborgh 16, 9747 AC Groningen, The Netherlands Received July 31, 1984; revised November 20, 1984 A sequential procedure, based on 2D NOE spectroscopy, is described for identifying the resonances of water-exchangeable imino protons in NMR spectra of oligonucleotides. The adenine H2 and amino protons are also part of the cross-relaxation network of the imino protons and their resonance frequencies are easily found in the 2D NOE spectra. The NOE contacts between the amino and H5 protons of cytidines form an independent check for the correctness of the assignments, since these CH5 protons are also part of the cross-relaxation network formed by the nonexchangeable protons, for which another sequential-assignment procedure exists (R. M. Scheek et al., J. Am. Chem. Sot. 14, 2914 (1983)). The method was applied to a 14 bp lac operator fragment. The assignments of the resonances of the water-exchangeable imino and amino protons in this DNA fragment are required for studying its complex with the luc repressor headpiece. 8 1985 Academic PIUS. Inc. INTRODUCTION A prerequisite for studies on structure and dynamics of biomoleeules by proton NMR is the assignment of resonances to specific nuclei. By two-dimensional nuclear magnetic resonance the problem of resonance assignment can be attacked in a systematic and reliable way (Z-3). In the ‘H NMR spectra of proteins most of the resonances can be identified by a combination of COSY and 2D NOE spectroscopy, which measure, respectively, coherent and incoherent transfer of magnetization between individual nuclei (4-6). In nucleic acids the same methods can be applied. Feigon et al. (7) were the first to report a number of intra- and intemucleotide NOE contacts in a 2D NMR spectrum of a double-stranded oligonucleotide. By 2D NOE spectroscopy most of the resonances of the nonexchangeable protons in small DNA fragments can be assigned by a sequential procedure, developed independently by Hare et al. (8) and in our laboratory (9, 10). Subsequently, this procedure was applied by Weiss et al. (II) to the 17 bp (base pairs) Ol operator of bacteriophage X and by us to a 14 bp lac operator fragment. In the latter case it was possible to assign the resonances of all nonexchangeable protons, except those of the HS and H5” deoxyribose protons (12). Until recently, the exchangeable imino and amino protons in these oligonucle- otides posed great problems in 2D spectroscopy and were generally studied by one- dimensional methods (13-23). Since in ‘Hz0 most of these protons are rapidly 0022-2364185 $3.00 378 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.