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, S.Krča³, D.Ivanković³, L.Griparić 4 [email protected]; www.ifs.hr/real_science Dielectric Spectroscopy of Dielectric Spectroscopy of Genomic DNA Genomic DNA Solutions 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 essential 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) 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 Persistence 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... HF mode: 10, 1- 0.8 LF mode: 100, 1- 0.8 L HF,LF = ( HF.LF D) 1/2 both and D from our experiments L HF : 4 nm – 45 nm DH screening length? or DNA mesh size? L LF : 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 L LF LF 2 in accord with theory L L LF LF =L =L 0 +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 c DNA <0.1 mg/mL exp (Na + ) c DNA >1 mg/mL exp ≈ ½ (Na + ) c in =c(Na+)= =c DNA ·3mol/mg D= (kT/N A e 2 exp /c in 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/mL UV-spectrophotometry indicates transition from dsDNA to ssDNA below 1 mM NaCl 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 Aqueous samples, conductivity range: 1.5-2000S/cm; volume: 50-200 L Reproducibility 1%, Long term (2 h) 2% Temp. range: 0° to 60°C Stability: ±10 mK Pt chamber steel casing Pt Low-frequency Dielectric Spectroscopy 1 0 1 1 i HF = (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: f 0 < 1 kHz 2) LF mode: 1 kHz < f 0 < 70 kHz Persistence length: distance bound by potential barriers due to variation of local conformation Behaves according to OSF theory with added salt 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. M. Sakamoto et al., Biopolymers 18 , 2769 (197 S.Takashima, J.Phys.Chem.70 , 1372 (1966) L -1 Na + , Cl - L p L HF - - - - - - - - - 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 L HF is DNA concentration dependent, but added salt independent L HF can not be -1 ~ I -1/2 , Debye-Hückel length L HF 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 L LF LF 2 in accord with theory P.G.de Gennes et al.,J.Phys.(Paris), 37 , 1461 (1976)

¹ , Zagreb, Croatia

Embed Size (px)

DESCRIPTION

Samples & materials. ∞. Dielectric Spectroscopy of Genomic DNA Solutions. - PowerPoint PPT Presentation

Citation preview

Page 1: ¹                                        , Zagreb, Croatia

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)