Double electron-electron resonance (DEER): a convenient method to probe DNA conformational changes.

Site-directed spin labeling combined with pulsed EPR spectroscopy represents a promising and powerful tool to elucidate the structures of chemical and biological systems, such as DNA, RNA, proteins, and protein complexes. Recently, weak interactions in trimeric protein structures, which are usually difficult to detect by other methods, were studied by double electron–electron resonance (DEER) distance measurements. Nanometer distances for the above-mentioned biomacromolecules have been determined by DEER or double quantum coherence–EPR spectroscopy. Whereas conformational changes have previously been detected on protein structures and peptides by the DEER technique, here we present the first example of the study of DNA conformational changes by using this method. Our model system was the B–A transition, which occurs in the formation of the double-stranded regions in RNA, or which is induced by spore proteins or in the presence of organic solvents, such as ethanol and methanol. The DEER technique, because of its several advantages compared to other well-known spectroscopic methods, can quantitatively monitor B–A transitions to elucidate the role of this conformation in several biological processes. Double site-directed spin-labeled (SDSL) DNA structures were made by the selective introduction of a 2,2,6,6tetramethylpiperidine-N-oxyl (TEMPO)-type spin probe on the N2 position of the guanine base (G). This procedure involves the substitution of an amino derivative of TEMPO on 2-fluorohypoxanthine (I). As described elsewhere, this derivatization pattern does not significantly alter the overall structure of the duplex. Four DNA duplexes were synthesized, two with both spin probes inserted on the same strand [(4;19) and (4;20)] and two containing one spin probe on each DNA strand [(4;14’) and (4;18’)] (Figure 1a and b). Selected CD spectra for the duplexes without nitroxide spin probes exhibit no differences compared to those registered on the double SDSL duplexes (data not shown). Buffered aqueous solutions of the SDSL duplexes, which are known to adopt a B conformation, were characterized by DEER. Interspin distances up to 5.6 nm were measured, as summarized in Table 1. The rigidity of the DNA structure was confirmed by a narrow distance distribution obtained by the Tikhonov regularization method. The B–A transition was induced by a high concentration of trifluoroethanol (TFE; higher than 70%, v/v). For all the duplexes studied, a shorter distance was observed in TFE/aqueous buffer relative to the buffered aqueous solution. These distance changes were attributed to an A-like conformation in the presence of TFE. In particular, a remarkable distance change (Dr = 0.8 nm, Figure 2) was observed for the duplex (4;20) in [D3]TFE/D2O (80:20). By stepwise addition of TFE we could detect a gradual change of the interspin distances for duplexes (4;14’) and (4;18’). Five solutions were prepared containing from 0 to 85% (v/v) of TFE (higher concentrations induce DNA precipitation). Figure 3 shows the distance changes for duplex (4;18’). Interestingly, in TFE/H2O (65:25, v/v) both species can be observed, with two maxima centered at 4.9 and 4.3 nm, which are the distances obtained for the B andA forms, respectively. Another structure that usually adopts an A-like conformation is the (DNA:RNA) hybrid duplex; its conformation is indistinguishable from the A conformation of DNA obtained by CD analysis. On the contrary, the DEER method allowed different distances to be measured for two (4;19) duplexes (see Table 1 and the Supporting Information). The distances measured for SDSL duplexes exhibit slight deviations from theoretical B/A distance ratios because of the size and placement of the nitroxide spin probes. The distance determinations described herein are supported by molecular dynamics (MD) studies, as confirmed by the values reported in Table 1. Such studies can also shed light on the relative [*] Dr. G. Sicoli, Dr. S. Gambarelli D partement de Recherche Fondamentale sur la Mati're Condens e, Laboratoire de Chimie Inorganique et Biologique, UMR-E n83 CEA-UJF Laboratoire de R sonance Magn tique, CEA-Grenoble 17 Avenue des Martyrs, 38054 Grenoble Cedex 9 (France) Fax: (+33)4-3878-5090 E-mail: serge.gambarelli@cea.fr