¹ , Zagreb, Croatia² School of Medicine, University of Zagreb, Croatia³ Institut Ruđer Bošković, Zagreb, Croatia4 Dept. of Biological Chemistry, UCLA David Geffen School of Medicine, Los Angeles, USA
T.Vuletić ¹, S.Dolanski Babić ², S.Tomić ¹, S.Krča³, D.Ivanković³, L.Griparić4
•[email protected]; www.ifs.hr/real_science
Dielectric Spectroscopy ofDielectric Spectroscopy of Genomic DNA SolutionsGenomic DNA Solutions
Physical properties and biological functions of DNA are strongly affected by its local environment
Motivation
Experimental characterization of the counter-ion atmospheres of DNA in solution is essentialR.Das et al.,Phys.Rev.Lett.90, 188103
(2003)N.Nandi et al., Chem.Rev.100, 2013 (2000)M. Sakamoto et al., Biopolymers 18, 2769 (1979)S.Bone et al., Biochymica et Biophysica Acta 1306, 93 (1996)
DNA in solution Coulomb repulsion between PO4
- groups, DNA is stretched out to the rod-like conformation
Worm-like model: chain of N segments of length a; Contour length L = N · a
Rigid over short distance and becomes flexible over large distances
Persistence length Lp determines a boundary between the two types of behavior in 0.1 M NaCl; Lp = 50 nm : 150 bp length
200 nm
M. Daune, Molecular Biophysics (Oxford, 2003)
Kratky and Porod (1949); Kuhn...
HF mode: 10, 1- 0.8
LF mode: 100, 1- 0.8
LHF,LF= (HF.LFD)1/2
both and D from our experiments
LHF: 4 nm – 45 nm DH screening length? or DNA mesh size?
LLF: 60 nm – 750 nm Contour? or persistence length?
Two Relaxation Modes in 10 kHz – 10 MHz range
LF Mode Persistence Length
M.N. Spiteri et al., Phys.Rev.Lett.77, 5218 (1996)
DNA in pure water:0.35 ≈ 1/3 Dimensionality effect? M.Mandel, Ann.NY Acad.Sci. 303, 74 (1977)
G.S.Manning, Biophys.Chem. 9, 65 (1978)S.Bone et al., BBA 1306, 93 (1996)
/c /c ~~ L LLFLF22 in accord with theoryLLLFLF=L=L00+0.324/I+0.324/I OSF theory (below 10 mM)
T.Odijk, J.Polym.Sci.Polim.Phys.Ed., 15, 477 (1977)J.Skolnick & M.Fixman,, Macromolecules, 10, 944 (1977)
single molecule exp.
C.Baumann et al., PNAS, 94, 6185 (1997)
LFDS exp.: DNA solution + NaCl
Conductivity of Na-DNA solution in pure water due only to Na+ counter-ions
cDNA<0.1 mg/mLexp≈ (Na+)
cDNA >1 mg/mLexp ≈ ½ (Na+) cin=c(Na+)=
=cDNA ·3mol/mg
D= (kT/NAe2)·exp/cin
diffusion constant of DNA counter-ions decreases with DNA concentration
added salt ions diffusion constant is not influenced by DNA concentration
Lyophillized Na-DNA: salmon, Sigma-Aldrich (D1626, Type III); calf, Rockland (MB –102-0100)Pure water: MilliPore, Milli-Q, 0.056 S/cm
Range of DNA solutions: 0.010 – 15 mg/mLUV-spectrophotometry indicates transition from dsDNA to ssDNA below 1 mM NaCl
Samples & materials
Precision impedance analyzerAgilent 4294A: 40 Hz-100 MHzC-G, capacitance & real part of
conductance measured amplitude 20-50 mV
AgilentBNCs
Aqueous samples, conductivity range: 1.5-2000S/cm; volume: 50-200 LReproducibility 1%, Long term (2 h) 2%
Temp. range: 0° to 60°CStability: ±10 mK
Pt
cham
ber
steelcasing
Pt
Low-frequency Dielectric Spectroscopy
1
01
1
iHF∞
= (0) - ∞ relaxation process strength
0 central relaxation time
1 - symmetric broadening of the relaxation time distribution
generalized Debye function
FITS to a sum of two generalized Debye functions
• G() and C()=B()/ of DNA solutions are measured• These are subtracted for (G, C) of background (reference) NaCl solution with matching (1-100kHz) conductivity• suppression: electrode polarization effects, stray impedance effects.
=’()-i’’()Y()= G()+iB()
From complex conductance to complex dielectric function
B.Saif et al., Biopolymers 31, 1171 (1991)
()~ -iY()
DNA chain: Random sequence of segments placed in counter-ion atmosphere. With ac field applied, appear broad relaxation modes due to oscillating counter-ions at different length and time scales
Origin of dielectric dispersion in DNA solutions
S.S.Dukhin et al, Adv.Coll. Interface Sci. 13, 153 (1980)
R.W.O’Brien, J. Coll. Interface Sci 113, 81 (1986).
Modes:
1) Contour length: f0 < 1 kHz
2) LF mode: 1 kHz < f0 < 70 kHz Persistence length: distance bound by potential barriers due to variation of local conformationBehaves according to OSF theory with added salt
3) HF mode: 0.1 kHz < f0 < 15 MHz Mesh size: DNA chains form a loose mesh defining a characteristic length for relaxation– attribution is strongly supported by LHF independence of added salt I.
M. Sakamoto et al., Biopolymers 18, 2769 (1979)S.Takashima, J.Phys.Chem.70, 1372 (1966)
L
-1
Na+, Cl-
Lp
LHF
--
---
- - -
-
M.Mandel, Ann.NY Acad.Sci. 303, 74 (1977)
G.S.Manning, Biophys.Chem. 9, 65 (1978)
Na+ ions redistributed in the vicinity of DNA chain in order to screen phosphate groups – Manning condensation
Theory available only for added salt case two types of dielectric dispersion two characteristic length scales: -1 - Debye-Hückel length & contour length of molecule
HF Mode DNA mesh size
Added salt ions increase screening and strongly reduce Na+ ions active in HF relaxation
LHF is DNA concentration dependent, but added salt independent LHF can not be -1 ~ I-1/2 , Debye-Hückel length LHF can be mesh size, ie. correlation length between DNA chains in solution (such a length scale does not vary with added salt)
/c /c ~~ L LLFLF22 in accord with theory
P.G.de Gennes et al.,J.Phys.(Paris), 37, 1461 (1976)
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