Multinuclear NMR investigation of the anisotropic system NaDNA/Ethidium Bromide
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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 : [email protected] Conclusions • 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. 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) 150 [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 The 23 Na NMR signal shows the typical splitting arising from the interaction of the 23 Na 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. -8 -6 -4 -2 0 2 4 6 8 ppm -8 -6 -4 -2 0 2 4 6 8 ppm Fig. 2 23 Na 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 r = 0.11 r = 0.055 r = 0 23 Na 0 = 105.795 MHz The 17 O 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. -30 -20 -10 0 10 20 30 ppm -60 -40 -20 0 20 40 60 ppm Fig. 3 17 O 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 r = 0.11 r = 0.055 r = 0 17 O 0 = 40.662 MHz Results The 31 P 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 31 P 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. -60 -40 -20 0 20 40 60 ppm -60 -40 -20 0 20 40 60 ppm [DNA]= 175 mg/mL [DNA]= 252 mg/mL r = 0.11 r = 0.055 r = 0 Fig. 1 31 P NMR spectra of DNA anisotropic aqueous solution at 35°C, in absence and in presence of a different amount of EB, r = [EB]/[DNA]. 31 P 0 = 121.421 MHz Introduction Intercalation of EB 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 the 31 P, 23 Na and 17 O NMR spectroscopies to study the effects of the intercalation of ethidium bromide (EB) on the anisotropic packaging of DNA. N + CH 2 N H 2 NH 2 CH 3 Br Ethidium Bromide (EB)
description
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 - PowerPoint PPT Presentation
Transcript of Multinuclear NMR investigation of the anisotropic system NaDNA/Ethidium Bromide
Multinuclear NMR investigation of the anisotropic system NaDNA/Ethidium Bromide
Andrea Catte, Flaminia Cesare Marincola, Mariano Casu, Giuseppe Saba and Adolfo LaiDipartimento di Scienze Chimiche, Cittadella Universitaria di Monserrato, University of Cagliari, Italy
e-mail : [email protected]
Conclusions
• 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.
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
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.-8-6-4-202468
ppm-8-6-4-202468
ppm
Fig. 2 23Na 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
r = 0.11
r = 0.055
r = 0
23Na
0 = 105.795 MHz
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.
-30-20-100102030ppm
-60-40-200204060ppm
Fig. 3 17O 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
r = 0.11
r = 0.055
r = 0
17O
0 = 40.662 MHz
Results
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.
-60-40-200204060ppm
-60-40-200204060ppm
[DNA]= 175 mg/mL [DNA]= 252 mg/mL
r = 0.11
r = 0.055
r = 0
Fig. 1 31P NMR spectra of DNA anisotropic aqueous solution at 35°C, in absence and in presence of a different amount of EB, r = [EB]/[DNA].
31P
0 = 121.421 MHz
Introduction
Intercalation of EB
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 the 31P, 23Na and 17O NMR spectroscopies to
study the effects of the intercalation of ethidium bromide (EB) on
the anisotropic packaging of DNA.
N+
CH2NH2
NH2
CH3
Br
Ethidium Bromide (EB)
