NMR Observation of a Novel C-Tetrad in the Structure of the SV40 Repeat Sequence GGGCGG P. K. Patel, Neel S. Bhavesh, and R. V. Hosur 1 Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India Received March 3, 2000 We report the NMR structure of the DNA sequence d-TGGGCGGT in Na  solutions at neutral pH, contain- ing a repeat sequence from SV40 viral genome. The structure is a novel quadruplex incorporating the C-tetrad formed by symmetrical pairing of four Cs via NH 2 OO 2 H-bonds in a plane. The C-tetrad has a wider cavity compared to G-tetrads and stacks well over the adjacent G4-tetrad, but poorly on the G6 tetrad. The quadruplex helix is largely underwound by 8 –10° com- pared to B-DNA except at the C5–G6 step. To our knowledge this is the first report of C-tetrad formation in DNA structures, and would be of significance from the point of view of both structural diversity and spe- cific recognition. © 2000 Academic Press Key Words: NMR structure; G-quadruplex; C-tetrad; GGGCGG repeat. Multistranded DNA structures have assumed great importance in recent years with the realization that they play important roles in DNA recombination, rep- lication, disease control, etc. on the one hand and DNA packaging inside a living cell on the other (1, 2). Among these, the quadruplex structures formed by many re- peat sequences containing stretches of Gs have exhib- ited great variety, dependence on experimental condi- tions, especially the cations (3– 6). They have created new paradigms and underscored the possibility of hith- erto not understood roles for DNA function. The vari- ability in the quadruplex structures is seen to be so large that given a repeat sequence and the experimen- tal conditions, it is hardly possible to predict the char- acteristics of the structure. Thus investigations on dif- ferent sequences under different conditions have led to the discoveries of many new structural motifs such as, G:C:G:C tetrad (7, 8), U-tetrad (9), A-tetrad (10, 11), and T-tetrad (12). We report here the observation of yet another new motif, namely, the C-tetrad, which we discovered in the sequence d-TGGGCGGT. The GGGCGG sequence contained in this DNA is biologi- cally significant for several reasons: (i) it is a repeat sequence in Simian virus (SV) 40, playing important roles in viral encapsidation (13, 14), (ii) it is a target for many anti cancer drugs (13), (iii) it is the recognition sequence of the SP1 transcription factor (15) and (iv) it is a very common sequence in CpG islands in verte- brate genomes (16). MATERIALS AND METHODS DNA samples. The oligonucleotide was synthesized on an applied Bio-systems 392 automated DNA synthesizer on 10 M scale using solid phase -cyanoethyl phosphoramidite chemistry, cleaved from support and purified by standard procedures (17, 18). The NMR sample was prepared at a monomer strand concentration range of 1–2 mM in 0.6 ml (90% H 2 O/10%D 2 O) having 10 mM sodium phos- phate, 0.2 mM EDTA, pH 7.0, and 200 mM NaCl. For experiments in D 2 O, the same sample was repeatedly lyophilized from D 2 O. NMR data acquisition and processing. NMR data were obtained on a VARIAN UNITY-plus 600 spectrometer. Temperature depen- dence one-dimensional spectra (-5–50°C) and NOESY spectra in H 2 O were recorded using jump-and-return pulse sequence (19) for H 2 O suppression. Phase sensitive NOESY (20) and TOCSY (21) spectra in D 2 O were recorded with mixing times of 80, 100, 200, and 300 ms for NOESY and 60 and 20 ms for TOCSY. A DQF-COSY was recorded for coupling constant estimation. In all the 2D experiments, the time domain data consisted of 2048 complex points in t 2 and 400 – 600 fids (free induction decay signals) in t 1 dimension. The VARIAN data were processed using VNMR, Felix-230 and Felix-97 software on an IRIS workstation. The data were apodized by shifted (60 –90°) sine bell functions prior to 2D Fourier transformations. Experimental restraints. The cross-peaks in the NOESY spectra in D 2 O were integrated and the intensities in a low mixing time NOESY were translated into interproton distances using the initial rate approximation using CH5–CH6 cross-peak intensity as the ref- erence (2.46 Å). Then, these cross-peaks in different spectra were classified as strong, medium and weak according to their relative intensities and the interproton distances were restrained with upper and lower bounds of 0.2, 0.5, and 1.0 Å from their calculated distances, respectively. The narrow bounds were mostly on strong intranucleotide cross-peaks for which the possible distance ranges are small and known. For the sequential internucleotide NOEs, loose Abbreviations used: DNA, deoxyribonucleic acid, NMR, nuclear magnetic resonance, NOESY, nuclear Overhauser enhancement spectroscopy, TOCSY, total correlation spectroscopy, DQF-COSY, double quantum filtered correlation spectroscopy, IRMA, iterative relaxation matrix analysis; MD, molecular dynamics. 1 To whom correspondence should be addressed. Fax: 091-22-215 2110. E-mail: hosur@tifr.res.in. Biochemical and Biophysical Research Communications 270, 967–971 (2000) doi:10.1006/bbrc.2000.2479, available online at http://www.idealibrary.com on 967 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.