Gene Vectors and Nanowires Nina A. Kasyanenko Faculty of Physics - - PowerPoint PPT Presentation

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DNA Nanostructures. Gene Vectors and Nanowires

Nina A. Kasyanenko Faculty of Physics St.-Petersburg State University _______________________________________ Department of Molecular Biophysics

SLIDE 2

• J. D. Watson & F. H. C. Crick A Structure for Deoxyribose Nucleic
• Acid. Nature, 171, 737-738 (April 25, 1953).
• M. H. F. Wilkins, A.R. Stokes and H. R. Wilson. Molecular Structure of Deoxypentose Nucleic Acids Nature, 171, pages 738-740(1953)
• R. Franklin and R. Gosling. Molecular Configuration in Sodium Thymonucleate Nature, volume 171, pages 740-741, (1953)

Rosalind Franklin 1920-1958

Francis Crick 1916-2004 Maurice Wilkins 1916-2004 James Watson 1928-

1962: Nobel Prize in Physiology and Medicine “For their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material“

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Replication Reparation Transcription Translation

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DNA origami (3D-constructions). N.C. Seeman

Conformational transitions in tertiary and secondary DNA structures

High specific interactions between complementary nucleic bases DNA condensation with the formation of ordered structures DNA nanomotor (B-Z transition)

DNA-based nanotechnology -

the utilization of unique DNA properties

High charge density and exceeding chain rigidity

Nanowires

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Paul W. K. Rothemund (Departments of Computer Science and Computation & Neural Systems, California Institute of Technology, USA) Folding DNA to create nanoscale shapes and patterns Nature, Vol 440|16 March 2006| p. 297-302

DNA origami

SLIDE 6

Paul W. K. Rothemund

Nanomanipulation, 2008, 4, No. 4, 447–450 Dielectrophoretic Trapping

• f DNA Origami

Anton Kuzyk, Bernard Yurke,

• J. Jussi Toppari, Veikko Linko,

and Paivi Torma

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Reversible conformational transitions

• Melting
• DNA packaging
• DNA bending induced by

ligand binding

• B-Z transition (right-left

winding)

DNA-bleomycin complex

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• Phosphate group screening
• Change in DNA charge density (by pH

variation, ionic bonds with phosphates)

• Variation in solvent quality (by the

addition of alcohol or other poor solvent into DNA-water solution)

• Binding with ligands (alteration in DNA

hydrophility, conversion of charge density, decrease of DNA rigidity)

• Intramolecular reorganization via

simultaneous influence of counterions and conversion of solvent properties

Transformation of DNA tertiary structure can be induced by different procedures:

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Gene vectors

IMC RAS St.-Petersburg.

Nazarova O.V. Panarin E.F.

C H2 C C C C O NH C H2 n C H3 C H3 O O C H2O H H O H H O H n H O H H C H2)2 O C H3 N C H3 (

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Experimental Methods

• ATOMIC FORCE MICROSCOPY

NanoScope IV, Veeco

• DYNAMIC LIGHT SCATTERING

PhotoCor, Russia

• CIRCULAR DICHROISM
• Mark IV, Jobin Ivon
• LOW GRADIENT VISCOMETRY

Zimm-Crothers Type

• DYNAMIC BIREFRIGENCE
• SPECTROSCOPY

SF 56, Russia

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Calf Thymus DNA (Sigma)

pFL 44/EcoI (4,4 kbp)

DNA

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0,0 0,5 1,0 1,5 2,0 0,0 0,5 1,0 1,5 [n]/[ ] ([n]/[ ])0

Fe3+, 1 M NaCl Fe3+ 0,005 M NaCl La3+, 0,005 M NaCl La3+, 1 M NaCl Al3+, 0,005 M NaCl Co[(H 2O )6] 3+, 0,003 M NaCl Co[(NH 3)6] 3+, 0,003 M NaCl

C(M e3+) x 10

5, M

0,01 0,1 1 10 0,5 1,0 1,5

PDMAEM PAA PTMAEM PLL PVA

sp/ spo N/P

0,01 0,1 1 10 0,0 0,5 1,0

[n]/[ ] [n]/[ ]

N/P

DNA complexes with divalent, trivalent and multivalent ions

2 4 6 8 10 0,6 0,8 1,0

( - )/( - )0 CMex10

4,M 1 2 3 4 5 10 20 30 40 50 60 70 80 90

[ ], дл/г

0,005 M NaCl 1 M NaCl

C(FeCl3) x 10

5, M

0,0 0,2 0,4 0,6 0,8 1,0 20 40 60 80 [ ], dl/g

I(Me2+) I

• 1a
• 2a
• 3a
• 4a
• 5a
• 1b
• 2b
• 3b
• 4b
• 1c
• 2c

Ме2+ Ме3+

polycations

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0,7 N + /P N + /P 1 1,4 1

DNA complexes with polycations

DNA pFL 44

O NH CH2OH OH OH OH C O C CH2 H3C S CH2 CH2 NH C O H C H2 C C H2 C C O O CH2 CH2 N CH3 H3C CH3 p m n O NH CH2OH OH OH OH C O C CH2 H3C S CH2 CH2 NH C O H C H2 C C H2 C C O O CH2 CH2 N CH3 H3C CH3 p m n

N + /P N + /P 2

0,0 0,5 1,0 1,5 2,0 2,5 0,0 0,5 1,0 1,5 PVA15 PAA MAG-DMAEM, 55: 45 моль% sp/ sp0

N

+/P

0,01 0,1 1 10 0,0 0,5 1,0 ( 1- 2)0 ( 1- 2)

N/P

220 240 260 280 300

• 4
• 2

2 4 6

N/P=0

N/P=1,0 N/P=1,2 N/P=1,3 N/P=,4 N/P=2,5 N/P=0,7

,нм

220 240 260 280 300

• 4
• 2

2 N/P=1,2 N/P=3,3 N/P=6,6 N/P=0

нм

10

• 3

10

• 1

10 1 10 3 10 5 10 7 0,00 0,05 0,10 0,15 0,20 0,25

G 90 ,мкс

D=115 20 nm

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• N. A. Kasyanenko, D. A. Afanasieva, B. A. Dribinsky, D. V. Mukhin, O. V. Nazarova, and E.F. Panarin,

DNA interaction with synthetic polymers in solution, Structural Chemistry 18(4), 519-525, (2007).

• A. V. Slita, N. A. Kasyanenko, O. V. Nazarova, I. I. Gavrilova, E. M. Eropkina, A. K. Sirotkin, T. D.

Smirnova, O. I. Kiselev, and E. F. Panarin, DNA-polycation complexes, Effect of polycation structure

• n physico-chemical and biological properties, Journal of Biotechnology 127(4), 679-693, (2007)

Касьяненко Н.А., Захарова Н. Б., Мухин Д.А., Слита А.В., Назарова О.В., Леонтьева Е.А., Панарин Е.Ф. Комплексы ДНК с поликатионами, используемые для направленной передачи генетического материала в клетки. Биофизика, (2008,) т.53, №1, с.31-37

220 240 260 280 300

• 4
• 2

2 4 6

N/P=0

N/P=1,0 N/P=1,2 N/P=1,3 N/P=,4 N/P=2,5 N/P=0,7

,нм

220 240 260 280 300

• 4
• 2

2

N/P=1,2

N/P=3,3 N/P=6,6 N/P=0

нм

240 260 280 300

• 2

2

N/P=0

N/P=1,5 N/P=2,3

, нм

0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5

PAA (1) PAA (2) PVP PDMAEM P(MAG-DMAEM) P(MAG-DMAEM)

sp/ sp0

N

+/P

0,01 0,1 1 10 0,0 0,5 1,0 ( 1- 2)0 ( 1- 2)

N/P

10

• 3

10

• 1

10 1 10 3 10 5 10 7 0,00 0,05 0,10 0,15 0,20 0,25

G 90 ,мкс

0,0 5,0x10 91,0x10 101,5x10 10 2,0x10 102,5x10 103,0x10 10 3,5x10 104,0x10 10 0,0 5,0x10 2 1,0x10 3 1,5x10 3 2,0x10 3 2,5x10 3

1/ (sec

• 1)

q

2 (cm

• 2)

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N+/P = N+/P = 0 0,2 0,2 0,8 0,8 1 1 2 2 6 6

Block (1,2 ) and graft (3) copolymer (MAG_DMAEM) + DNA in 0.005 М NaCl СDNA=0.0001%, N/P= 2 , D = (110 ± 20) nm N+/P= 1 N+/P= 2 N+/P= 2 (1 m)

O NH CH2OH OH OH OH C O C CH2 H3C S CH2 CH2 NH C O H C H2 C C H2 C C O O CH2 CH2 N CH3 H3C CH3 p m n

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Cell lines Jurkat (I), U-937 (II), T-98G (III) with FITC-oligonucleotides (a), (c), (e) – PAA (b), (d), (f) – PDMAEM (g) – control (without transfection)

• J. Biotechnology

2007 Биофизика, 2008

T-98G cells after transfection PDMAEM+(DNA with β-Gal) after reaction with X-Gal (1:360)

A.V. Slita, N. A. Kasyanenko, O. V. Nazarova, I. I. Gavrilova,

• E. M. Eropkina, A. K. Sirotkin, T. D. Smirnova, O. I. Kiselev,
• E. F. Panarin, DNA-polycation complexes, Effect of polycation

structure on physico-chemical and biological properties, Journal of Biotechnology 127(4), 679-693, (2007)

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Conclusions (part 1)

• Gene vectors can be formed at definite relation
• f polycation/DNA concentration (N/P)
• DNA condensation is realized at N+/P>1
• DNA-polycationic gene vectors (D=120 nm) can

penetrate into cell and can provoke the protein production

• AFM and Dynamic Light Scattering belong to

rare appropriate methods for the monitoring of condensed DNA structure.

• AFM images correctly reflect DNA conformation

in complexes with polycations

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DNA Nanowires

NATURE |VOL 391 | 1998 Erez Braun, Yoav Eichen, Uri Sivan& Gdalyahu Ben-Yoseph DNA-templated assembly and electrode attachment of a conducting silver wire

• J. Mater. Chem. 14, 611-616 (2004).

Becerril, H.A.; Stoltenberg, R.M.; Monson, C.F.; Woolley, A.T. Ionic Surface Masking for Low Background in Single- and Double-Stranded DNA-Templated Silver and Copper Nanorods. Materials and Design 28 (2007)

• S. Cui et al.

Construction of silver nanowires

• n DNA template by

an electrochemical technique The TEM images of silver nanowires

• Biophys. Chem. (2009), J. Lu, et al.,

DNA-templated photo-induced silver nanowires: Fabrication and use in detection of relative humidity

Пучкова О. Стенд. (Puchkova O. Poster)

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240 260 280 300 320 0.0 0.5 1.0 1.5 DNA in AgNO3 solution

D

, nm 0.001M 0.005M 220 240 260 280 300 320

• 6
• 3

3 6

nm

DNA in 0.005 M NaCl DNA in 0,005 M AgNo3 0.005 M AgNo3

250 300 0.0 0.5 1.0 1.5

D

, nm

DNA(0.005%) DNA(0.005%)+Hydrochinon(0.002M) Hydrochinon 0.002M (DNA+Hydrochinon)-Hydrochinon

DNA metallization

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Пучкова Анастасия

. Физ. ф-т СПбГУ

DNA metallization on mica

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Plasmid DNA pFL 44 / EcoRI MM=4,4 kbp

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DNA on Silicon Surface

Mg 2+

hν = 2ev

2.5 мкм

silicon surface

р-silicon И.Л. Волков, Н.В. Базлов, А.С. Бондаренко, О.Ф. Вывенко, Н.А. Касьяненко. Светоиндуцированная нековалентная фиксация ДНК и синтеттических полиионов на поверхности монокристаллов кремния, Журнал структурной химии 50, 2009, 999-1006. И.Л. Волков, Н.В. Базлов, А.С. Бондаренко, О.Ф. Вывенко, Н.А. Касьяненко. Разработка способа нековалентной фиксации ДНК на поверхности монокристалла кремния, Вестник Санкт-Петербургского университета. Серия 4: Физика, химия, (2009), 3 45-51.

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C C H2 C C H3 O C H2 C H2 N C H3 C 2H5 C 2H5

n

Cl

SO4 2- SO4 2- SO4 2- SO4 2-

mica

n-Si

p-Si mica mica

(a)

With Na2SO4 on n-silicon (a) and p-silicon (b) with light, on mica (d, e), without Na2SO4 on mica (c). C(Na2SO4)= 5•10

• 4

M (a, b, d, e). C(pol)= 5•10

• 5

M (a, b, c, d). C(pol)= 5•10

• 6

M (d) +

SO4 2-

(b) (c) (d) (e)

Polycation on mica and silicon surface

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PAА on mica DMAEM on mica PAA on Si DNA on Si

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n-Si p-Si Without light ( 890 nm) With light ( 890нм ) Петр Соколов, Физ. ф-т СПбГУ Wiht light Without light

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Nanowires on silicon

DNA nanowires on silicon: secondary-ion microscopy, transmission and scanning electron microscopy AFM image DNA on mica Metallized DNA on mica DNA nanowires on silicon (a) (b) (c) Transmission electron microscope JEM-2100F (Jeol), Secondary-ion microzonde-microscope IMS7F Cameca, UHV Scanning Tunnel icroscope

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Project participants

Faculty of Physics, St.-Petersburg State University

аспиранты: Мухин Д.А. Дрибинский Б.А. студенты: Волков И.Л. Соколов П. А. Пучкова А.О. Лысякова Л.А.

Institute of Macromolecular Compounds RAS

к.х.н. Назарова О.В чл.-корр. РАН Панарин Е.Ф.

Research Institute of Influenza RAMS, St.-Petersburg

к.б.н. Слита А.В.

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Thank you for the attention