Multinuclear NMR investigation of the Interaction of DNA Liquid Crystals with Divalent Metal Ions

1. Multinuclear NMR investigation of the Interaction of DNA Liquid Crystals with Divalent Metal Ions Andrea Cattea,b, Flaminia Cesare Marincola a, Mariano Casu a, Giuseppe Saba a, Adolfo Lai a, Alexander Korobko b, Wim Jesse b, Johan van der Maarel b and Jan Reedijk b a Dipartimento di Scienze Chimiche, Cittadella Universitaria di Monserrato, University of Cagliari, Italy b Leiden Institute of Chemistry, Leiden University, The Netherlands e-mail : a.catte@mvcch3.unica.it or a.catte@chem.leidenuniv.nl • Introduction • In vitro DNA molecules form liquid crystalline phases in aqueous solution when a critical concentration, depending on the ionic strength, temperature and DNA length, is achieved [1]. The DNA liquid crystalline phases are good experimental model systems to get insights into the DNA biological activity [2]. • Considering the active biological role of divalent metal ions in the genetic processes of DNA replication, transcription and translation, we employed the31P, 23Na and 2H NMR Spectroscopy and Optical Microscopy to study the interaction of the divalent metal ions Mg2+,Mn2+,Ni2+ andCd2+with DNA liquid crystals. c b a d e • Results r=0.025 r=0.05 31P NMR 0=161.903 MHz r =[M2+]/[DNA][DNA]=275 mg/mLT =25°C • The 31P NMR spectrum of the metal free DNA aqueous solution exhibits a broad band, typical of an anisotropic system, overlapped to a downfield sharp intense signal, attributed to an isotropic phase [3]. Polarized light microscopy of the metal free DNA aqueous solution shows that the sample is mainly cholesteric (Fig. a). • Upon binding of Mg2+ and Cd2+, an increase of the intensity of the downfield signal is observed, indicating the growing up of the isotropic phase. This behaviour is confirmed by optical microscopy, showing the coexistence of small cholesteric domains with isotropic ones inboth metal-DNA systems (Fig. b for Mg-DNA and Fig. c for Cd-DNA). • In the presence of Mn2+the system is fully cholesteric (Fig. d) and a marked enlargement of the 31P NMR signal is observed. • No significant modifications are observed in the presence of Ni2+ (Fig. e). Mn2+ Ni2+ Cd2+ Mg2+ r = 0 r=0.05 r=0.025 23Na NMR 0=105.795 MHz r =[M2+]/[DNA][DNA]=275 mg/mLT =25°C 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 line shape was attributed to sample inhomogeneities [4]. The 23Na NMR spectra of the DNA liquid crystalline solutions in the presence of divalent metal ions show that the quadrupolar splitting Qincreases in the presence of all the studied ions respect to the metal free DNA sample, the more significant enhancement being observed for Ni2+ and Cd2+. The line widths of the side peaks decrease for all divalent metal ions increasing the metal ion to phosphate ratio, showing a behaviour similar to that observed in isotropic systems [5]. Mn2+ Ni2+ Cd2+ Mg2+ r = 0 r=0.05 2H NMR 0=61.394 MHz r =[M2+]/[DNA][DNA]=275 mg/mLT =25°C r=0.025 The 2H NMR spectrum of the metal free DNA sample shows the typical doublet of an oriented anisotropic sample. Upon addition of Mg2+ the quadrupolar splitting is increased, the doublet overlaps with a central signal, which confirms the presence of an isotropic phase, and the inner edges are broadened. The quadrupolar splitting remains almost unchanged after the first addition of Cd2+ions, while doubling the metal to phosphate ratio, it is increased and a central signal appears, indicating the existence of isotropic domains. The paramagnetic ions produce a marked broadening of 2H NMR signals, leading to the loss of the doublet pattern in a pronounced way for Mn2+. The deuterium NMR spectra of the Ni-DNA samples exhibit different intensities of the two spectral lines, showing a net differential line broadening (DLB), due to the dipolar interaction originating with a paramagnetic impurity [6]. The mechanism responsible for the DLB is proportional to the magnetisation of the paramagnetic impurities, as confirmed by our spectra it is increased by a factor two when the metal to phosphate ratio is doubled. Mn2+ Ni2+ Cd2+ Mg2+ r = 0 Conclusions References [1] F. Livolant and A. Leforestier, Prog. Polym. Sci., 21, 1115-1164, (1996) [2] J. Lepault, J. Dubochet, W. Baschong et al., EMBO J.,6,1507-1512, (1987) [3] T. E. Strzelecka and R. L. Rill, Macromolecules, 24,5124-5133,(1991) [4] I. Furó, B. Halle and T. C. Wong , J. Chem. Phys., 89, 5382-5397, (1988) [5] D. Vasilescu, S. Ansiss and G. Mallet, J. Biol. Phys., 19,199-209, (1994) [6] D. Petit, J. P. Korb, A. Delville, J. Grandjean and P. Laszlo, J. Magn.Reson.,96, 252-279, (1992) The comparison of the optical textures of the DNA liquid crystalline solutions without and with divalent metal ions shows the different behaviour of diamagnetic ions (Mg2+ and Cd2+) and paramagnetic ions (Mn2+and Ni2+), as also evidenced by 31P, 23Na and 2H NMR Spectroscopy. Mg2+ and Cd2+ ions interact strongly with the phosphate groups of DNA favouring the formation of an isotropic phase, while the better base binders Mn2+and Ni2+ ions do not affect the cholesteric phase formation.