1. Dehydrogenation of nucleobases upon electron attachment to isolated nucleotides Steen Brøndsted Nielsen Department of Physics and Astronomy University of Aarhus June 24-27, 2004, Lyon-France

2. DNA DAMAGE

3. HOW DOES UV RADIATION CAUSE GENETIC DAMAGE ? 1)Direct absorption of light by the nucleobases followed by physical and chemical reactions and / or 2)Ionisation of water (H 2 O  H 2 O  + + e  ) followed by electron attachment to nucleobases Modification of nucleobases may lead to loss of base-pairing specificity

4. Elucidate the processes at the single-molecule level. Isolated damage to nucleotides, the basic building blocks of DNA and RNA, upon electron attachment. OUR APPROACH

5. TOOLBOX ELECTROSPRAY IONISATION MASS SPECTROMETRY: ACCELERATOR INSTRUMENT ION STORAGE RING ELISA LASERS QUANTUM CHEMICAL CALCULATIONS

6. BARE IONS ELECTROSPRAY IONISATION droplets

7. ELECTROSPRAY ION SOURCE 1 mbar 10 -3 mbar10 -5 mbar 10 -6 mbar Heated capillary ESI needle 4kV 22-pole ion trap Tube lens Skimmer Octapole Lenses Acceleration tube

8. GAS-PHASE EXPERIMENTS ON NUCLEOTIDE ANIONS PROBLEM: Attachment of an electron to a negative ion is unlikely because of the repulsion between the two negative charges. SOLUTION: High-energy collisions with sodium: electron transfer from Na to the ion. R  R   Compare: the violent reaction of sodium with water ! Nielsen et al., J. Am. Chem. Soc. 125, 9592-9593 (2003). Liu et al., Chem. Phys. Chem. 4, 1341-1344 (2003).

9. ACCELERATOR INSTRUMENT ESI source Magnet Na collision cell Electrostatic analyser 0 V  50 kV Channeltron detector +  R = 2 m B = 1.4 T T=500 K

10. COLLISION INTERACTION TIME: FEW FEMTOSECONDS Na - Nucleotide anion 10 5 m/s 1 nm The electron transfer process is nearly vertical.

11. Electron attachment to AMP anions m/q 346 Ne Na Liu et al., Chem. Phys. Chem. 4, 1341-1344 (2003).

12. High-energy collisions between dAMP anions and Na and Ne dAMP  dAMP 2  fragment ions m/q 330

13. Spectrum obtained after collisions between AMP  (H 2 O) 13 and Na AMP  (H 2 O) 13 AMP 2  AMP 2  (H 2 O) 9 AMP 

14. dG 2 dT 2 dC 2 dA 2 Electron transfer from Na to dinucleotide anions sugar phosphate nucleobase dianion -

15. IS THE NUCLEOTIDE DIANION INTACT ? H loss is observed upon electron attachment to nucleobases in vacuo. C. Desfrancois, H. Abdoul-Carime, and J. P. Schermann, J. Chem. Phys. 104, 7792 (1996). S. Gohlke, H. Abdoul-Carime, and E. Illenberger, Chem. Phys. Lett. 380, 595 (2003). G. Hanel, B. Gstir, S. Denifl, P. Scheier, M. Probst, B. Farizon, M. Farizon, E. Illenberger, and T. D. Märk, Phys. Rev. Lett. 90, 188104 (2003). H. Abdoul-Carime, S. Gohlke, and E. Illenberger, Phys. Rev. Lett. 92, 168103 (2004). H loss is observed upon electron attachment to deoxyribose in vacuo. S. Ptasińska, S. Denifl, P. Scheier, and T. D. Märk, J. Chem. Phys. 18, 8505 (2004).

16. # of D in AMP  0 1 2 3 Intact dianion  H  D AMP dissolved in CD 3 OD: Selection of deuterium- labelled ions for collision experiments Exchangeable hydrogens Liu et al., J. Chem. Phys., issued for Sept. 1 (2004).

17. WHAT IS THE ORIGIN OF THE HYDROGEN ?  POH phosphoric acid group  OH sugar  NH 2 nucleobase  CHsugar or nucleobase

18. Electron attachment to nucleotides: dehydrogenation at nitrogen sites Thymine Uracil Adenine Cytosine Guanine 1 2 3

19. Cross section for formation of dehydrogenated dianion

20. H-loss from N No D-loss from C H. Abdoul-Carime, S. Gohlke, and E. Illenberger, Phys. Rev. Lett. 92, 168103 (2004). Dissociative electron attachment to deuterated thymine

21. THE DNA DOUBLE HELIX

22. Watson-Crick base pairsBase mispairing Biological relevance?

23. WHAT IS THE LIFETIME OF THE DIANION ?

24. 1 m Micro-channel plate detector Accelerator with electrospray ion source Magnet Ion bunch Injection ELectrostatic Ion Storage Ring Aarhus (ELISA) S.P. Møller, NIM A 394, 281 (1997). J.U. Andersen, J.S. Forster, P. Hvelplund, T.J.D. Jørgensen, S.P Møller, S. Brøndsted Nielsen, U.V. Pedersen, S. Tomita, and H. Wahlgreen, Rev. Sci. Instrum. 73, 1284-1287 (2002). Channeltron Sodium

26. Electron autodetachment from the AMP dianion Liu et al., J. Chem. Phys., issued for Sept. 1 (2004).

27. 1 m Micro-channel plate detector Accelerator with electrospray ion source Magnet Ion bunch Injection Channeltron Long-lived component ? Dump of a remaining beam in the micro-channel plate detector after 34 ms of storage time. Sodium

28. Dump of beam in the detector after 34 ms of storage time AMP 2  (m/q 172.5) 13 C-AMP 2  (m/q 173)

29. CONCLUSIONS Electron transfer to nucleotide anions occurs in collisions with sodium vapour. Electron attachment leads to dehydrogenation of the nucleobase nitrogens. The dehydrogenated dianion is longlived. Other fragmentation channels than H-loss upon electron attachment ?

30. ACKNOWLEDGEMENTS University of Aarhus: Preben HvelplundJens Ulrik Andersen Shigeo Tomita Bo Liu Jimmy Rangama Leopold-Franzens Universität Innsbruck: Paul ScheierGabriel Hasan FUNDING The Danish Natural Science Research Council Aarhus Center for Atomic Physics (ACAP)

31. Collisional electron transfer to peptide dications: SELECTIVE FRAGMENTATION CHANNELS

32. FRAGMENTATION SPECTRA Substance P Amino acid sequence: RPKPQQFFGLM Na Ne [M + 2H] + [M + 2H] 2+

33. [M + 2H] + Amino acid sequence: RPKPQQFFGLM Hvelplund et al., Int. J. Mass Spectrom. 225, 83-87 (2003).