Ultrafast deactivation of the electronic excited states of DNA bases and polynucleotides following 267 nm laser excitation explored using picosecond time-resolved infrared spectroscopy 1 Mike Towrie (m.towrie@rl.ac.uk) Central Laser Facility Rutherford Appleton Laboratory DIAMOND Diamond Tony Parker, Pavel Matousek Marina Kuimova, Joanne Dyer, Mike George, David Grills, Xuo Zhong Sun (CCLRC, RAL) (Nottingham) John Kelly, Aine Whelan (Trinity, Dublin) Transient Chemical Structures in Dense Media 14-16th March 2005,. FIAP, Paris, France
UV Excitation of DNA & Components 2 DNA is remarkably photostable UV excitation of singlet leads to ps to sub-ps internal conversion f 10-4 Low yield of potentially damaging triplets & photo-reactions Photochemical reactions UVB - Pyrimidine dimers Photoionisation Base + hνbase.+ + electron IP in solution:- 5 ev to 6 ev Ultrafast Techniques Transient absorption Fluorescence up-conversion http://www.mun.ca/biology/scarr/gr16-20,25.htm UV induced dimerisation can induce Block to transcription Cell death Mutation Review: Kohler et al Chem Rev 2004 vol 104 p1977
The Broadband Pump-Probe TRIR Spectrometer 3 64 element MCT IR array detector MIR Spectrometer (1/4m) 150 and 300 l/mm gratings 1 khz 1 mj 150fs Regen HX2 Analog Multiplexer MCT Pre-amp MCT Pre-amp 12 bit, 250 khz ADC 500 MHz PC Data acquisition >1 khz Ge Beamsplitter probe Sample Delay Generators ref. Delay Line PUMP Galvanometer (500 Hz) Pump 1 khz IR (ca. IR 200 OPA cm -1 ) ~200 cm -1 Optional 1 khz ns microlaser (700ps resolution) SENSITIVITY 10-4 A (1 s) Femtosecond time range: 0-2 ns (< 1ps resolution) Nanosecond time range: 0-100 µs (700 ps resolution)
267 nm pump IR probe in C=O stretch region 4 ps-trir spectra (1-50 ps) following 267 nm excitation NUCLEOTIDES AMP adenosine monophospate TMP thymidine monophosphate GMP guanosine monophosphate CMP cytidine monophosphate Phosphate Nucleic Acid Base TMP Sugar 5 -nucleotides (10 mm) in 50 mm phosphate D 2 O buffer.
Kinetics e.g. 5 dgmp ~ 5 0 Base 5 -dgmp 5 -dcmp lifetime 2.9 (±0.2) ps 4.7 (±0. 3) ps BLEACH RECOVERY 5 -damp 5 -TMP 4.3 (±0.2) ps 2.2 (±0.1) ps 0 20 40 time picoseconds Electronic state lifetime <1ps (see Kohler review) Kohler et al Chem Rev 2004 vol 104 p1977
What are the 2-5 ps dynamics? Martin Zanni et al 6 S 1 IR Pump-IR Probe Anharmonic downshift of 10 cm -1 ν=1 2 ν=0 1 S 0 267 nm VR Intramolecular Vibrational Relaxation, IVR Using UV transient absorption Pecourt et al Reported ~2 ps IVR in nucleosides. Cooling:- Temperature jump ~1000K Coupling of excited low frequency modes to the high frequency modes. <1 ps relaxation to ground state One possibility:- Internal conversion through a conical intersection involving π π * or dark nπ * and πσ * states? Hydrogen Bond Breaking? Would expect frequency upshift A. T. Krummel, P. Mukherjee and M. Zanni, J. Phys. Chem. B., 2003, 107, 9165. J.M. Pecourt, J. Peon, B. Kohler, J. Am. Chem. Soc 123 (2001) 10370
UV pump IR probe (poly-strands B-form) 7 (a) polydg-dc polydg-dc polydg-dc polydg-dc 0.000 O D = 0.0001 FTIR Absorbance -0.002 1593 1633 1645 1678 1687 (b) -0.004 τ =12 & 50 ps 0 50 100 150 200 250 Time / ps 4ps 0.0005 polyda-dt polyda-dt O D = 0.0003 polyda-dt polyda-dt 1700 1650 1600 1550 1500 Wavenumber / cm -1 Absorbance 0.0000-0.0005-0.0010 τ =4 &140 ps 0 100 200 300 400 500 Time / ps 1665 1632 1551 1601 1620 1645 ps-trir spectra (1-1000 ps) obtained following 300 fs, 267 nm excitation. 50mM Phosphate Buffer
Z-Form of polydgdc 8 Absorbance OD = 0.0001 1720 1680 1640 1600 4 M NaCl Wavenumber / cm -1 Absorbance 0.0000-0.0005-0.0010 τ =19 +/- 3 ps 0 50 100 150 200 250 Time / ps 1603 1616 1635 1684 1674
http://www.albany.edu/~achm110/abzdna.html http://www.albany.edu/~achm110/abztopview.html 9 Expect TRIR spectroscopy to report on changes in Base pairing & stacking Tautomerism Hydrogen bonding Hydration (not like bulk water) Solvent Accessibility Bases (Blue) Sugars (Red) Phosphates (Yellow)
Conclusions 10 Demonstrated that fs-trir can be used to follow ultrafast dynamics in DNA and its constituent nucleobases Electronic excitation leads to rapid S 1 to S 0 relaxation (in agreement with other workers) We have directly observed the rapid formation and decay of vibrationally hot (S o ν1) ground state nucleobases after relaxation of their electronic excited states. Poly nucleotide strands give complex spectra with transients living rather longer than individual bases with differing IR spectral features Future work will help define role of base pairing, stacking, conformation, solvent interactions and H-bonding. Kuimova et al Chem. Comm (2005) p1182
Acknowledgements 11 Tony Parker, Pavel Matousek Marina Kuimova, Joanne Dyer, Mike George, David Grills, Xuo Zhong Sun (CCLRC, RAL) (Nottingham) John Kelly, Aine Whelan (Trinity, Dublin)