1. ¹, Zagreb, Croatia ² School of Medicine, University of Zagreb, Croatia ³ Institut Ruđer Bošković, Zagreb, Croatia 4 Dept. of Biological Chemistry, UCLA David Geffen School of Medicine, Los Angeles, USA T.Vuletić ¹, S.Dolanski Babić ², S.Tomić ¹,D.Vurnek ¹, S.Krča³, D.Ivanković³, L.Griparić 4 tvuletic@ifs.hr ; www.ifs.hr/real_science tvuletic@ifs.hr ; www.ifs.hr/real_science tvuletic@ifs.hr Dielectric Spectroscopy of Genomic DNA Solutions Lyophillized DNA: salmon testes, Sigma-Aldrich (D1626, Type III); calf thymus, Rockland (MB –102-0100) Pure water: MilliPore, Milli-Q, 0.056 S/cm Range of DNA solutions: 0.011 – 18 mg/mL quantified spectrophotometrically at 260 nm SAMPLES & MATERIALS Precision impedance analyzer Agilent 4294A: 40 Hz-100 MHz C-G, capacitance & real part of conductance measured amplitude 20-50 mV Agilent BNCs Chamber for complex conductivity of liquid samples – water solutions, conductivity range: 1.5-2000S/cm; volume: 50-200 L Reproducibility 1%, Long term (2 h) 2% Temperature control unit Temp. range: 0° to 60°C Stability: ± 10 mK Pt chamber steel casing Pt Low-frequency Dielectric Spectroscopy CONDUCTIVITY of DNA-solution vs. temperature and concentration Conductivity follows the power law with exponent smaller than 1 for both salmon-DNA and calf-DNA   percolation character of the conduction path in DNA solutions Note: measured conductivity (gray line) of DNA solution was subtracted for 1.5 S/cm - the conductivity of pure water when measured in our chamber. RESULT: Conductivity follows the power law with exponent smaller than 1 for both salmon-DNA and calf-DNA   percolation character of the conduction path in DNA solutions Note: measured conductivity (gray line) of DNA solution was subtracted for 1.5 S/cm - the conductivity of pure water when measured in our chamber.  (T) – ion mobility = e/6  R  (T)  (T) – H 2 O viscosity ~ e -  H/RT  – ionic conductivity =  N A e  ~ e  H/RT  H=-18 kJ/mol ( Source: CRC Handbook ) ionic solutions theory: Note: H, enthalpy is related to energy required for a molecule to escape from its “neighbors” RESULT: DNA solution conductivity shows a temperature dependence typical for ionic solutions Resulting (G DNA -G NaCl, C DNA -C NaCl ) Complex dielectric function ∞   relaxation process strength  = (0) -  ∞    0 – central relaxation time   symmetric broadening of the relaxation time distribution 1 -  Note: C=B/ generalized Debye function FITS to a sum of two generalized Debye functions  Worldwide motivation: Transport of electrical signals in bio-materials on a molecular scale is of fundamental interest in the life sciences  Our motivation: physical and biological functions of DNA are strongly affected by its local environment  Our aim: to reveal dynamical and conformational properties of native DNA as a function of its aqueous environment MOTIVATION  Experimental characterization of the counter-ion atmospheres around DNA in solution is essential for an understanding of DNA physical properties and biological functions  Low frequency dielectric spectroscopy (LFDS) to study genomic DNA as a function of electrolyte concentration, counter-ion and pH  LFDS: powerful tool to probe charge entities and their background structure in various bio-macromolecular structures R.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) 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 This procedure enables to eliminate the electrode polarization effects, as well as other stray impedance effects. That is, since these influences are nearly the same in DNA and reference solutions, they are reduced by the subtraction. =’()-i’’() Y()= G()+iB() From complex conductance to complex dielectric function B.Saif et al., Biopolymers 31, 1171 (1991) DNA IN SOLUTION  Coulomb repulsion between PO 4 - 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  Persistance length L p determines a boundary between the two types of behavior  in 0.1 M NaCl; L p = 50 nm : 150 bp length 200 nm M. Daune, Molecular Biophysics (Oxford, 2003) Kratky and Porod (1949) Kuhn Results: Two Relaxation Modes in 10 kHz – 10 MHz range HF mode:   10, 1-  0.8 Same features for both salmon and calf DNA LF mode:   100, 1-  0.8 calf DNA salmon DNA : c-independent strong drop at low c  0 : no change at low c levels off at low c ? Electro-kinetics of Electrical Double Layer S.S.Dukhin et al, Adv.Coll. Interface Sci. 13, 153 (1980) R.W.O’Brian, J. Coll. Interface Sci 113, 81 (1986).  Na+ ions redistributed in the vicinity of DNA chain in order to screen phosphate groups  Electrical double layer with thickness  -1 is created  Suggestion: Under applied ac field  two types of dielectric dispersion  two characteristic length scales:  -1 - Debye-Hückel length & contour length of molecule ? ? ? ? ?  L HF,LF = ( HF.LF D) 1/2,  from experiments D=k B T/6R, D(25°C) = 1.5 ·10 -9 m 2 /s L HF : 4 nm – 45 nm DH screening length? or DNA mesh size? L LF : 60 nm – 750 nm Persistence length? ( Source: CRC Handbook ) Temperature dependent relaxation  Mode characteristic lengths appear temperature independent  Since characteristic length L=(D(T)·(T)) 1/2  Thus, (T)~ 1/D(T) ~ 1/(T·e -H/RT )  Therefore,  should be FIT to e H/RT /T  Energy scales of the modes are quite similar to energy scale of ionic conductivity: H= -18 kJ/mol  H= -20±2 kJ/mol Conclusion - Origin of dielectric dispersion in DNA solutions 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 Modes: 1) Contour length; f 0 < 1 kHz M. Sakamoto et al., Biopolymers 18, 2769 (1979) S.Takashima, J.Phys.Chem.70, 1372 (1966) L  -1 Na +, Cl - LpLp L HF - - - - - - - - - 2) LF mode: 1 kHz < f 0 < 70 kHz Persistence length: distance bound by potential barriers due to variation of local conformation As expected L p ~ I -1/2 when salt is added 3) HF mode: 0.1 kHz < f 0 < 15 MHz Mesh size: DNA chains form a loose mesh defining a characteristic length for relaxation– attribution is strongly supported by L HF independence of added salt I. L HF ~ c 1/2-1/3, indicates dimensionality of the web between 2 & 3. HF Mode Characteristic Length: DNA mesh size Inherent (I NaCl =0) Na + ions only: /c ~ L HF 2 in accord with Mandel- Manning model  Added salt ions (I NaCl ≠0) do not contribute to relaxation.  On the contrary, they increase screening and strongly reduce Na + ions active in HF relaxation  L HF is DNA concentration dependent, but added salt independent   L HF can not be  -1 ~ I -1/2, Debye-Hückel length  L HF given by mesh size, ie. average distance between DNA chains in solution (this length scale does not vary with added salt, I NaCl ≠0) M.N. Spiteri et al., Phys.Rev.Lett.77, 5218 (1996) LF Mode Characteristic Length: Persistence Length  Similar effect of inherent and added Na+ ions  All ions contribute to screening  L LF ~ I -1/2 implying L LF ~  -1 as expected for persistence length  Important difference in L p of salmon and calf DNA at low concentration  Certainly  LF ~ L 2 and we found /c ~ L LF 2  Both relaxation parameters should be proportional to characteristic length, L 2 according to : M.Mandel, Ann.NY Acad.Sci. 303, 74 (1977) G.S.Manning, Biophys.Chem. 9, 65 (1978) P.G.de Gennes et al.,J.Phys.(Paris), 37, 1461 (1976) M.N. Spiteri et al., Phys.Rev.Lett.77, 5218 (1996)