Multinuclear NMR investigation of the anisotropic system NaDNA/Ethidium Bromide

1. Multinuclear NMR investigation of the anisotropic system NaDNA/Ethidium Bromide Andrea Catte, Flaminia Cesare Marincola, Mariano Casu, Giuseppe Saba and Adolfo Lai Dipartimento di Scienze Chimiche, Cittadella Universitaria di Monserrato, University of Cagliari, Italy e-mail : a.catte@mvcch3.unica.it • Introduction • Isotropic DNA aqueous solutions undergo transitions to ordered liquid crystalline phases above a critical concentration, which depends on the ionic strength, temperature and DNA length [1]. The DNA liquid crystalline phases are good experimental models capable to give information on the DNA biological activity in the living systems [2]. • We employed the31P, 23Na and 17O NMR spectroscopies to study the effects of the intercalation of ethidium bromide (EB) on the anisotropic packaging of DNA. Ethidium Bromide (EB) Intercalation of EB • Results [DNA]= 175 mg/mL [DNA]= 252 mg/mL 31P • The 31P NMR spectrum of the drug-free DNA aqueous solution comprises a broad band overlapping a sharp downfield shifted signal, respectively attributed to anisotropic and isotropic domains [3]. • In the presence of EB, a new resonance appears at high fields, attributed to a new spatial organization of intercalated DNA rods [4]. • Populations of the DNA domains were estimated from the least-square fitting of 31P NMR spectra to a linear combination of Gaussian and Lorentzian functions. The results showed that adding EB favours a progressive growth of the new domain at the expenses of the leading anisotropic phase while the “isotropic population” remains almost unchanged. 0 = 121.421 MHz r = 0.11 r = 0.055 r = 0 Fig. 131P NMR spectra of DNA anisotropic aqueous solution at 35°C, in absence and in presence of a different amount of EB, r = [EB]/[DNA]. [DNA]= 175 mg/mL [DNA]= 252 mg/mL 23Na 0 = 105.795 MHz The 23Na NMR signal shows the typical splitting arising from the interaction of the 23Na quadrupole moment with the electric field gradient at the nucleus in an anisotropic environment. The observed asymmetry of the satellite lineshape was attributed to sample inhomogeneities [5]. Furthermore, it was observed that the addition of EB decreases the quadrupolar splitting and increases the relative intensity of the central peak, indicating the release of sodium ions from the anisotropic to the isotropic phase. r = 0.11 r = 0.055 r = 0 Fig. 223Na NMR spectra of DNA anisotropic aqueous solution at 35°C, in absence and in presence of a different amount of EB, r = [EB]/[DNA]. 17O [DNA]= 175 mg/mL [DNA]= 252 mg/mL 0 = 40.662 MHz r = 0.11 The 17O NMR spectra of the water in anisotropic DNA and DNA/EB systems show that the central peak is markedly enhanced upon intercalation, indicating a significant release of water molecules from the anisotropic domain. r = 0.055 r = 0 Fig. 317O NMR spectra of DNA anisotropic aqueous solution at 35°C, in absence and in presence of a different amount of EB, r = [EB]/[DNA]. Conclusions References [1] F. Livolant and A. Leforestier, Prog. Polym. Sci., 1996 (21) 1115 [2] J. Lepault, J. Dubochet, W. Baschong et al., EMBO J.,1987 (6) 1507 [3] T. E. Strzelecka and R. L. Rill, Macromolecules, 1991 (24) 5124 [4] G. Gottarelli, G. Spada, P. Mariani et al., Chirality, 1991 (3) 227 [5] I. Furó, B. Halle and T. C. Wong , J. Chem. Phys., 1988 (89) 5382 • Due to the complexity of the systems under investigation, a detailed analysis of the anisotropic spectra is far from being exhaustive. However, a clear indication is given that the intercalation of EB into DNA reorganizes the DNA domains in such a way that less amounts of water molecules and counterions are required.