Femtosecond Transient Absorption Femtosecond Transient Absorption Studies on the Proton-Induced Studies on the Proton-Induced

Structural Transitions of Cytidine Structural Transitions of Cytidine Containing PolymersContaining Polymers

Boiko Cohen

Matthew H. Larson

Dr. Bern Kohler

60th Ohio State University International Symposium on Molecular Spectroscopy

i-DNA

nucleic acid photophysicsnucleic acid photophysics

• base monomers

• base multimers

base stackingbase pairing

hemiprotonated cytosine

Poly(dC) Poly(rC)

NH

N

O

NH2

Cytosine

hemi-protonated poly(rC)hemi-protonated poly(rC)transient absorptiontransient absorption

4

3

2

1

0

AS

/ 1

0-3

6040200

Time / ps

630 nm 600 nm 570 nm

pH 7 pH 54

3

2

1

0

AS

/ 1

0-3

5004003002001000

Time / ps

630 nm 600 nm 570 nm

pH 7

1/ ps 2 / ps A2 3 / ps A3 4 / ps A4

pH 7 1.67 0.23 53 % 8.0 1.0 47 % – – – –

pH 5 – – 8.7 2.4 14 % 310 30 43 % ∞ 43 %

5

4

3

2

1

0

A

S /

10-3

10008006004002000time / ps

Poly(rC)pH4

Poly(dC)pH 7

400

300

200

100

0

AS

/ 10

-6

4003002001000time / ps

Poly(rC), pH 4, 1 = 8.1 ± 1.3 ps, 2 = 237 ± 22 ps 20deg

600

500

400

300

200

100

0

AS /

10-6

4003002001000time / ps

Poly(dC), pH 7, 1 = 7.2 ± 0.8 ps, 2 = 226 ± 27 ps

d(C)18

pH 7

why poly(rC)?why poly(rC)?

from Plessow et al., J. Phys. Chem. B 2000, 104, 3695.

streak camera imagesstreak camera images

wavelength (nm)

d(C)18

pH 8.5CMP d(C)15

1.0

0.8

0.6

0.4

0.2

0.0

OD

320300280260wavelength / nm

poly(dC) pH7 poly(rC) pH7 poly(rC) pH5

807060504030T /

0C

0.90

0.88

0.86

0.84

0.82

0.80

0.78

0.76

OD

Scanned at 266 nm

0.54

0.52

0.50

0.48

0.46

0.44

OD

807060504030T /

0C

Scanned at 266 nm

0.47

0.46

0.45

0.44

0.43

0.42

OD

706050403020T /

0C

Scanned at 266 nm.

temperature studiestemperature studies

poly(rC) - SS

poly(dC) - DS poly(rC) - DS

Single stranded poly(rC) shows a gradual decrease in the amplitude of the transient absorbance signal, while the lifetime remains relatively constant.

At 70ºC (above the melting point of the poly(rC) double helix) the long lifetime signal disappears, allowing us to assign this lifetime to the poly(rC) double helix.

600

500

400

300

200

100

0

AS /

10

-6

20151050time / ps

Single Stranded Poly(C), Temperature Dependence

8ºC 20ºC 70ºC

600

500

400

300

200

100

AS /

10-6

300250200150100500time / ps

Double Stranded Poly(C), Temperature Dependence

8ºC 20ºC 70ºC

• Substantial delocalization of π electrons in the hemiprotonated Substantial delocalization of π electrons in the hemiprotonated

base pair. base pair.

• This new type of cytosine ring and charge delocalization in the This new type of cytosine ring and charge delocalization in the

double stranded form may lead to a change in the base stacking double stranded form may lead to a change in the base stacking

motif of the double strand. motif of the double strand.

Chou and Thomas, Biopolymers, 1977, 16: p. 765-78

raman studies on poly(rC)raman studies on poly(rC)

340320300280260240220200wavelength (nm)

Alternating d(CT)Alternating d(CT)99 can provide can provide some answerssome answers

340320300280260240220wavelength (nm)

d(CT)9

d(C)18

typical for hemiprotonated cytosine

pH 4

pH 7

pH 4

pH 8.5

d(CT)3

(Sarma et al, FEBS Lett. 1985, 205, 223 )

d(CT)20

(Casasnovas et al, J. Mol. Biol. 1993, 233, 671 )

different coupling between the different coupling between the cytosine bases in cytosine bases in d(C)d(C)1818 and d(CT) and d(CT)99

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Ab

sorb

ance

320300280260240wavelenght / nm

1.2

1.0

0.8

0.6

0.4

0.2

Ab

sorb

ance

320300280260240wavelength / nm

Absorption

Circular Dichroism

320300280260240220wavelength / nm

when the d(CT)9, pH 4

absorption spectra is shifted by 6 nm the red edge of the band overlaps with the one

for d(C)18, pH 7

d(C)18, pH 7d(CT)9, pH 4

280 nm570 nm

double strandeddouble stranded d(CT) d(CT)99 vs vs

d(C)d(C)1818

pH a1 t1

[ps]

a2 t2

[ps]

a3

d(CT)9 4 0.55 2.6 0.4 0.20 205 40 0.25

d(C)18 7 0.20 4.3 0.8 0.60 320 20 0.20

pH a1 t1

[ps]

a2 t2

[ps]

a3

d(CT)9 4 0.64 2.6 0.4 0.27 205 40 0.09

d(C)18 7 0.29 4.3 0.8 0.55 320 20 0.16

10

8

6

4

2

0

A

S /

10-3

2.01.51.00.50.0time / ns

-12

-10

-8

-6

-4

-2

0

A

/ 10

-3

1.51.00.50.0time / ns

Note the change in the relative amplitudes and the

lifetime both at 280 and 570 nm

The AnswerThe Answerhomo oligonucleotide alternating oligonucleotide

Schematic representation

base stacking

base pairing

+

summarysummary

Femtosecond transient absorption of hemiprotonated poly(rC) and poly(dC) shows an unique, long lifetime (>200 ps) component consistent with the increased fluorescence quantum yield observed by Favre (Favre, A. FEBS, 1972, 22, 280)

The temperature dependence of the transient absorption signals of single-stranded Poly(rC) shows gradual decrease in the amplitude of the lifetime associated with the base stack

The temperature dependence of the transient absorption signals of double-stranded Poly(rC) shows sharp change in the behavior, associated with the opening of the double strand

We find similar long lifetime component in the transient absorption traces of the alternating d(CT)9 oligonucleotide at pH 4 consistent with existence of stacked hemiprotonated cytosine base pairs suggested by Brown et al (Biochemistry 1985, 24, 1676)

We have shown that the long lifetime for d(C)15 seen in the streak camera studies by Plessow et al (J. Phys. Chem. B 2000, 104, 3695) is due to the formation of stacked hemiprotonated cytosine base pairs and not excimer formation in the single stranded form

acknowledgementsacknowledgements

Funding National Institutes of Health (R01-GM64563)

Prof. Bern Kohler Matthew H. Larson

2.0x10-3

1.5

1.0

0.5

0.0

AS

1.51.00.50.0power / mW

t = 0 ps t = 4 ps t = 50 ps t = 200 ps water

1.2x10-3

1.0

0.8

0.6

0.4

AS /

P

1.51.00.50.0power / mW

t = 0 ps t = 4 ps t = 50 ps t = 200 ps water

2.5x10-3

2.0

1.5

1.0

0.5

0.0

AS

1.51.00.50.0power / mW

t = 0 ps t = 10 ps t = 100 ps t = 400 ps water

1.4x10-3

1.2

1.0

0.8

0.6

0.4

0.2

AS /

P

1.51.00.50.0

power / mW

t = 0 ps t = 10 ps t = 100 ps t = 400 ps water

power dependencepower dependencepH 7 pH 5

raman studies on poly(rC)raman studies on poly(rC)

poly(C) poly(C)•poly(C+) poly(C+)

1. Single-stranded helix2. A-form3. Neutral ring4. Stacking of neutral bases5. Furanose ring conformation different than in other ordered ribopolymers

1. Double-stranded helix2. A-form3. Hemiprotonated ring4. Stacking of paired hemiprotonated bases5. Furanose ring conformation different than in other ordered ribopolymers

1. Random chain2. Disordered3. Protonated ring4. None

5. Furanose ring conformation is same as in other disordered ribopolymers

Chou and Thomas, Biopolymers, 1977, 16: p. 765-78

543210Time / ps

Thd = 540 fs4

2

0

A x

10

4

543210Time / ps

Guo = 460 fs

4

2

0

A x

10

4 Ado = 290 fs CydH+ = 720 fs

N

NH

O

O

CH3

OH

H

H

OHH H

OH

SS11 lifetimes of nucleosides lifetimes of nucleosides

All lifetimes are +/- 40 fs

3.5 GW / cm2 8.3 GW / cm2

N

N

O

NH2

OH

OH

H

OHH H

OHO

H

OH

H

OHH H

OH

NN

NN

NH2

OH

OH

H

OHH H

OH

NN

NNH

O

NH2

(Cyd = 1 ps)

J. Am. Chem. Soc. 122, 9348 (2000). J. Am. Chem. Soc. 123, 5166 (2001).J. Am. Chem. Soc. 123, 10370 (2001).

1020

1015

1010

105

100

10-5

10-10

10-15

time / seconds

DNA and RNA dynamicsDNA and RNA dynamics

age of the earth

origin of life

fluorescence lifetime of Trp

human

life expectancy

RNA folding

backbone rotation

base stacking

singlet state lifetimes in polymers

fluorescence lifetimes of single bases

double helix stability of d(CT)double helix stability of d(CT)99

150

100

50

0

-50

(md

eg M

-1cm

-1)

340320300280260240220200wavelength (nm)

0.8

0.6

0.4

0.2

Ab

sorb

ance

360340320300280260240220wavelength (nm)

0.8

0.6

0.4

0.2

Ab

sorb

ance

360340320300280260240220wavelength (nm)

UV/Vis Absorption Circular Dichroism

340320300280260240220200wavelength (nm)

before

after

The typical for hemiprotonated cytosine splitting in the CD spectra is still there after 1

hour irradiation