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Intertwisting-bend process in DNA condensationProf. Dr. Xizeng FENG

Nankai University

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Outline

Ⅰ. Experiment—Phenomenon--Model

• 1. DNA Toroids and Microstructure• 2. DNA Toroid Size and Mechanisms of

Formation

Ⅱ. Microstructure– Biofunction -- Model

• 3. Cationic-Complex Induced DNA Condensates • 4. Questions and some thinks……

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• The DNA condensates have served as models of high density packing in biological systems, in virus, and have found applications in the Non-viral gene delivery.

1. DNA Toroids and Microstructure

Ⅰ. Experiment—Phenomenon--Model

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Formation of a giant toroid from long duplex DNA

TEM image of a large DNA toroid produced by the condensation of T4 DNA by 6 mM spermidine in the presence of high salt (50 mM NaCl, 10 mM

MgCl2). Scale bar is 100 nm.

1.1 Yoshikawa Y, et al.,Langmuir,1999, 15:4085–4088.

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DNA delivery by phage as a strategy for encapsulating toroidal condensates of

arbitrary size into liposomes.

TEM image of a large toroid formed by the release of DNA from several T5 bacteriophages into a solution containing 5 mM

spermine. Empty and DNA-filled bacteriophages can be seen around the much larger DNA toroid.

1.2 Lambert O, Proc. Natl. Acad. Sci. USA, 2000 97:7248–7253.

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Cryoelectron microscopy of λ phage DNA condensates in vitreous ice: the fine structure of DNA toroids

(a) A toroid with DNA fringes visible around almost the entire circumferenceof the toroid. (b) A toroid with two small arc angles of well-defined DNA fringes that appear on opposite sides of the toroid center. Scale bar is 50 nm.

1.3 N.V. Hud, Proc. Natl. Acad. Sci. USA.,2001,98:14925–14930.

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(A) TEM images of DNA condensates formed at 22℃ by mixing DNA with an equal volume solution of 200 μM hexammine cobalt

chloride, 3.5mMMgCl2. DNA was a linearized 3-kb bacterial plasmid. DNA concentration was 10 μg/ml following mixing with

the hexammine cobalt chloride, MgCl2 solution. (B) Same solution conditions and condensation protocol as given for left image,

except carried out at 37℃. Scale bars are 200 nm.

Evidence that both kinetic and thermodynamic factors govern DNA toroid dimensions:effects of

magnesium(II) on DNA condensation by hexammine cobalt(III).

1.4 Conwell CC, Hud NV. Biochemistry,2004, 43:5380–5387.

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TEM images of particles formed by various DNA samples upon condensation with hexammine cobalt chloride

(A) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. (B) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. (C) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. (D) Condensates formed by 3kbDNA. For all samples, DNA was 15 Min base pair, and condensed by mixing with an equal volume of 200 M hexammine cobalt chloride in 5 mM Bis-Tris, 50 MEDTA (pH 7.0). Scale bar is 100 nm.

1.5 Nucleic Acids Research, 2005, 33(1):143-151.

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• Four different condensed states of DNA from a study of non-viral gene delivery carrier :

• a) Condensed negatively charged on NiCl2-treated mica,

• b) Condensed negatively charged with 0.2 mM NiCl2,

• c) Condensed positively charged,

• d) Noncondensed.

1.6 Atomic Force Microscopy (AFM) in the Investigation of Gene Delivery carrier

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Cationic Surfactant (CTAB) Induced Lambda-DNA From Lines state transition

to globes state

1.7 Science in China (Series C), 42(2)1999, P136-140

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Globe and dendrimer structures of Cetyltrimethylammonium bromide (CTAB) –-DNA condensates by Scanning Electron

micrographs (SEM)

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Globe and dendrimer structures of Cetyltrimethylammonium bromide (CTAB) –-DNA condensates by AFM

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1.8 Fluorescence dye (FI) Induced Lambda-DNA from Lines state transition

to toroidal state

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Toroidal and rod structures of Fluorescence dye–-DNA condensates by Laser Scanning Confocal Fluorescence Microscopy

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Toroidal and rod structures of Fluorescence dye–-DNA condensates by Laser Scanning Confocal Fluorescence Microscopy

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Supercoil structures of Fluorescence dye–-DNA

condensates by Atomic Force Microscopy

A

B

b)

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A

B

Cc)

A

B

C c) d)

Toroidal structures of Fluorescence dye–-DNA condensates by Atomic Force Microscopy

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Scale bar = 200nm. Images courtesy C. Roberts, University of Nottingham, UK.

1.9 Formation of toroidal DNA-polymer condensate over 35 minute time-span.

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• Imaged in Tapping Mode in 15mM salt solution. scan size: 292nm x 292nm x 5nm

• Image courtesy D. Dunlap and A. Maggi, San Raffaele, Scientific Institute, Italy.

1.10 Non-viral gene delivery with condensed DNA and a cationic polymer (polyethylenimine, PEI)

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2. DNA Toroid Size and Mechanisms of Formation

• A DNA toroid 100 nm in outside diameter with a 30 nm hole contains approximately 50 kbp of DNA.

Ⅰ. Experiment—Phenomenon--Model

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Elastic model of DNA supercoiling in the infinite-length limit

, superhelix pitch angle: • superhelix pitch;• Rex excluded volume

radius.• For plectonemic DNA, the

superhelical pitch angle is in the range 45°<<90°.

2.1 J. Chem. Phys., 1991,95(12):155

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A constant radius of curvature model for the organization of DNA in toroidal condensates

• (A) The toroid with a 900 Å outside diameter, a 300 Å diameter hole, and a 27 Å center-to-center distance between successive loops.

• (B) 10 contiguous loops depict a toroid in an early stage of development.

2.2 Proc. Natl. Acad. Sci. USA, 1995, 92: 3581-3585

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The three-state model for the dynamics of toroid formation (1)

(A) Energy state model for toroid formation.

(B) The probability (ln) that a DNA molecule in the extended state will form an n-base-pair loop depends upon the change in free energy (ΔGn) associated with the formation

The three-state model for the dynamics of toroid formation (2)

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• (C) In this simplified model for toroid initiation, two possible transitions for an existing loop, which are a return to the ground state (loop disintegration) or condensing agent binding (the completion of toroid initiation).

The three-state model for the dynamics of toroid formation (3)

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• Distributions of toroid-loop sizes are illustrated by plots of Eqs. 8 and 9.

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Controlling the size of nanoscale toroidal DNA condensates with static curvature and ionic

strength

• Scheme 1. Definition of toroid diameter and thickness.

2.3 Proc. Natl. Acad. Sci. USA, 2003, 100(16): 9296-9301

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low-salt model

• Scheme 2. Toroid nucleation and growth with equal outward and inward growth from the nucleation loop.

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Transmission electron micrographs of toroids produced by the condensation of DNA with

hexammine cobalt (III).

• (A) Atract60 (linear 3,681-bp DNA) condensed in the low-salt buffer (0.53TE: 5mMTris 0.5mMEDTA).

• (B) 3kbDNA (linear 2,961-bp plasmid) condensed in the low-salt buffer.

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• Scheme 3. Toroid nucleation and growth with preferential growth outward from the nucleation loop during the latter stage of toroid formation.

Ionic strength conditions

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• (C)3kbDNA condensed in 2.5 mM NaCl, 0.53 TE. • (D) 3kbDNA condensed in 1.75mM MgCl2 0.53 TE.

Transmission electron micrographs of toroids produced by the condensation of DNA with

hexammine cobalt (III).

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• Scheme 4.Toroid nucleation and growth with annealing to larger nucleation loop sizes during the early stage of formation, and preferential outward growth during the latter stage of toroid formation.

An annealing process occurs

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• (E) 3kbDNA condensed in 3.75 mM NaCly0.53 TE.• (F) 3kbDNA condensed in 2.5 mM MgCl2y0.53 TE.• All samples were 8.5 gyml in DNA and 100 M

hexammine cobalt chloride. (Scale bar: 100 nm.)

Transmission electron micrographs of toroids produced by the condensation of DNA with

hexammine cobalt (III).

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• Histograms of toroid diameter and thickness measurements for toroidal DNA condensates.

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• Histograms of toroid hole diameters for 3kbDNA condensed in three different salt solutions.

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Fig. 1 – Illustration of the flow field on a toroidal condensate which features additional twist. The slenderness of the torus is ξ = 1.5.

Twist-bend instability for toroidal DNA condensates

2.4 Eur. phys. Lett., 2004, 67 (3): 418–424.

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Fig. 2 – Twist order parameter τ ≡ arccos(nϕ,min) as a function of the slenderness ξ = r1/r2. The curves correspond to different ratios of elastic moduli, η = K2/K3 {∈ 0.01, 0.05, 0.1, 0.2, 0.3, 0.4},the gray arrow pointing toward increasing values. The inset illustrates the toroidal coordinate system.

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Fig. 3 – Structural phase diagram of the toroidally wound complex on a log-log scale. For small ξ = r1/r2 and η = K2/K3,the polymer is wound in a twisted way,for large ξ and η it prefers to wind straight. The dots are results from the full numerical minimization,the solid line is eq. (6),and the dashed line stems from the “improved ansatz” (see the text).

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Fig. 4 – Possible mechanism for plectonemic supercoiling [9] of the genome of giant T4 phages:(a) Initially the toroidal genome is only twisted (dark shading) at the two poles. (b) After removing the capsid,t wist propagates into the remaining DNA, but since the twist-creating confinement is removed,the remaining bundle suddenly is overtwisted. (c) This then induces a global supercoiling.

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• DNA Condensation by Multivalent Cations, Curr.Op. Struct. Biol.,1996,6:334.

• Deformation of toroidal DNA condensates under surface stress,

Eur. phys. Lett., 1996, 33 (5): 353-358.

• DNA Aggregation Induced by Polyamines and Cobalthexamine

J Biol. Chem., 1996, 271, (10): p 5656–5662.

• Toroidal Condensates of Semiflexible Polymers in Poor Solvents: Adsorption, Stretching, and Compression,

Biophysical Journal, 2001, 80: 161–168.

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• A director-field model of DNA packaging in viral capsids,

Journal of the Mechanics and Physics of Solids,

2003, 51:1815 – 1847.

• Cryoelectron microscopy of phage DNA condensates in vitreous ice: The fine structure of DNA toroids,

PNAS, 2001,98(26): 14925.

• Spontaneous condensation in DNA-polystyrene-b-poly(l-lysine) polyelectrolyte block copolymer mixtures,

Eur. Phys. J. E., 2006, 20, 1-6.

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3. Cation-Complex Induced DNA Condensates

Ⅱ. Microstructure-- Biofunction -- Model

3.1 Interactions of a novel designed polymer with DNA as a gene delivery carrier

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Synthesis of Poly(ethylene glycol) methyl ether methacrylate (poly(PEGMA)-4N)

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2

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a ba b

AFM image of DNA condensates induced by poly(PEGMA)-4N.

Image (a) is naked plasmid DNA.

Image (b) is condensed plasmid DNA

The observation of multimolecular toroidal structures

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NIH3T3 cells transfected by poly(PEGMA)-4N/DNA complexes.

(A) DNA with poly(PEGMA)-4N per well. (B) zoomed in and bright pictures of (A). (C) positive control.

Transfection of poly(PEGMA)-4N /DNA complexes

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+

+

intertwist betweenlong chains

electrostatic attraction between fidderentcharged molecules

+

too compact for longchains to inter thestructure

DNApoly(PEGMA)

quick process

tris(2-aminoethyl)amine

intertwisted strucure

DNAcompacted strucure

poly(PEGMA)compactedstrucure

slow process+

+

intertwist betweenlong chains

electrostatic attraction between fidderentcharged molecules

+

too compact for longchains to inter thestructure

DNApoly(PEGMA)

quick process

tris(2-aminoethyl)amine

intertwisted strucure

DNAcompacted strucure

poly(PEGMA)compactedstrucure

slow process

Two process during DNA condensation induced by poly(PEGMA)-4N

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The observation of spermidine-condensed DNA by atomic force microscopy and polarizing microscopy

studies

4.1 Nucleic Acids Research, 1998, Vol. 26, No. 13:3228-3234.

Spermidine

Ⅱ. Microstructure-- Biofunction -- Model

4. Questions and some thinks……

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(a) Observations reveal the coexistence of complete toroids, incomplete toroids with gaps, and U-shaped rods. (b) Close-in AFM image of a single

toroidal condensate. (c) Cross sectional profile of the toroid in (b) along the marked line. (d) A non-standard toroid.

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Acridine Orange (AO) Induced Lambda-DNA From Lines to the coil-particles-

toroidal transition

4.2 高等学校化学学报， 19（ 9） 1998 ， P1498-1500

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Toroidal structures of Acridine Orange (AO) –-DNA condensates by Scanning Electron Micrographs (SEM)

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Finger and toroidal structures of Acridine Orange (AO) –-DNA condensates by AFM