Femtosecond Transient Absorption Studies on the Proton-Induced Structural Transitions of Cytidine Containing Polymers Boiko Cohen Matthew H. Larson Dr. Bern Kohler 60th Ohio State University International Symposium on Molecular Spectroscopy

nucleic acid photophysics Poly(dC) Poly(rC) base monomers base multimers i-DNA hemiprotonated cytosine base stacking base pairing

hemi-protonated poly(rC) transient absorption pH 7 pH 5 pH 7

why poly(rC)? Poly(rC) pH4 Poly(dC) pH 7 d(C)18 pH 7 d(C)18 pH 8.5 CMP streak camera images from Plessow et al., J. Phys. Chem. B 2000, 104, 3695. d(C)18 pH 7 d(C)18 pH 8.5 CMP d(C)15 wavelength (nm)

temperature 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.

raman studies on poly(rC) Substantial delocalization of π electrons in the hemiprotonated base pair. This new type of cytosine ring and charge delocalization in the double stranded form may lead to a change in the base stacking motif of the double strand. Chou and Thomas, Biopolymers, 1977, 16: p. 765-78

Alternating d(CT)9 can provide some answers (Sarma et al, FEBS Lett. 1985, 205, 223 ) d(CT)9 pH 4 pH 4 typical for hemiprotonated cytosine d(C)18 pH 7 pH 8.5 d(CT)20 (Casasnovas et al, J. Mol. Biol. 1993, 233, 671 )

different coupling between the cytosine bases in d(C)18 and d(CT)9 Absorption Circular Dichroism d(C)18, pH 7 d(CT)9, pH 4 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

double stranded d(CT)9 vs d(C)18 Note the change in the relative amplitudes and the lifetime both at 280 and 570 nm 280 nm 570 nm pH a1 t1 [ps] a2 t2 a3 d(CT)9 4 0.55 2.6  0.4 0.20 205  40 0.25 d(C)18 7 4.3  0.8 0.60 320  20 pH a1 t1 [ps] a2 t2 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

The Answer Schematic representation homo oligonucleotide alternating oligonucleotide base stacking + base pairing

summary 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 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 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)

National Institutes of Health (R01-GM64563) acknowledgements Matthew H. Larson Prof. Bern Kohler Funding National Institutes of Health (R01-GM64563)

power dependence pH 7 pH 5

raman studies on poly(rC) poly(C)•poly(C+) poly(C+) poly(C) 1. Double-stranded helix 2. A-form 3. Hemiprotonated ring 4. Stacking of paired hemiprotonated bases 5. Furanose ring conformation different than in other ordered ribopolymers 1. Random chain 2. Disordered 3. Protonated ring 4. None 5. Furanose ring conformation is same as in other disordered ribopolymers 1. Single-stranded helix 2. A-form 3. Neutral ring 4. Stacking of neutral bases 5. Furanose ring conformation different than in other ordered ribopolymers Chou and Thomas, Biopolymers, 1977, 16: p. 765-78

S1 lifetimes of nucleosides J. Am. Chem. Soc. 122, 9348 (2000). J. Am. Chem. Soc. 123, 5166 (2001). J. Am. Chem. Soc. 123, 10370 (2001). S1 lifetimes of nucleosides 3.5 GW / cm2 8.3 GW / cm2 (Cyd t = 1 ps) All lifetimes are +/- 40 fs

DNA and RNA dynamics life expectancy RNA folding base stacking human life expectancy RNA folding base stacking backbone rotation origin of life age of the earth singlet state lifetimes in polymers fluorescence lifetime of Trp fluorescence lifetimes of single bases

double helix stability of d(CT)9 Circular Dichroism UV/Vis Absorption before after The typical for hemiprotonated cytosine splitting in the CD spectra is still there after 1 hour irradiation