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Janna Anichina Diethard K. Bohme

Electrospray Ionization Mass Spectrometry of the Interaction of Oligonucleotides with Metals, Small Molecules and Drugs. Janna Anichina Diethard K. Bohme. York University Department of Chemistry Centre for Research in Mass Spectrometry Toronto, CANADA. ASMS 2007.

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Janna Anichina Diethard K. Bohme

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  1. Electrospray Ionization Mass Spectrometry of the Interaction of Oligonucleotides with Metals, Small Molecules and Drugs Janna Anichina Diethard K. Bohme York University Department of Chemistry Centre for Research in Mass Spectrometry Toronto, CANADA ASMS 2007

  2. Why Metal Ion – Ligand – Oligonucleotides? • In vivo processes with DNAare mediated by interactions with metal ions, small molecules and proteins. • The mechanisms of action of many anti-tumor drugs include the formation of their adducts with strandsofDNA only in the presence of metal ions. • ESI-MS has been successfully utilized in the study of interactionsbetween DNAand Pt complexes important in chemotherapy (Beck et al. Mass Spec. Rev., 2001, 20, 61). • Systematic ESI-MS studies of metal ion - ligand - DNA interactions remain insufficient.

  3. Binding of oligonucleotides with metal complexes AA, CC, GG, TT, CCC, ATAT, GCAT, GCGC, CATAC, ACTCG, AGTCTG, TTAGGG, GCATGC Bleomycin A2 Na+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Fe3+, Co3+ 1,10-phenanthroline Triethylenetetramine

  4. Experimental • Instruments: MDS SCIEX API 2000 and Q Trap 2000. • Duplexes of the hexamers were prepared by heating 70 M ss in 70 mM aqueous NH4CH3COO to 900C for 10 min, neutral pH, and then cooled down slowly over a 3-hour period. • 20 M solution prepared in 20:80 (vol/vol) methanol/water was injected into the ESI sources of the mass spectrometers. • Ratios metal cation to ligand to ss were 5:varied:1, [ss]0 = 20 M. • Flow rate: 5 L/min; N2 as the collision gas; collision voltages : -1 to -100 V in the negative mode.

  5. Formation of duplexes ?! SS DS

  6. MS/MS of the duplexes of the hexamers [d(5’GCATGC)2 -3H]3- --> [d(5’GCATGC) -H] - +[d(5’GCATGC) -2H]2- [d(5’TTAGGG)2 -3H]3- --> [d(5’TTAGGG) -H] - +[d(5’TTAGGG) -2H]2- 1229 1846 922.5 Underlined species were not observed due to the limited mass range

  7. Metallation of ds hexamers [d(5’T2AG3)2 - 3H]3- 5 : 1 [Zn2d(5’T2AG3)2-7H)]3- [Znd(5’T2AG3)2-5H)]3- [Nad(5’T2AG3)2-4H)]3- [Zn3d(5’T2AG3)2-9H)]3- [Cu4d(5’T2AG3)2-11H)]3- [Cu3d(5’T2AG3)2-9H)]3- 15 : 1 [Cu2d(5’T2AG3)2-7H)]3- [Cud(5’T2AG3)2-5H)]3- [d(5’T2AG3)2 - 3H]3-

  8. Dissociation of metallated duplexes Metallated duplexes dissociate into two strands!

  9. Dissociation pathways of metallated duplexes Underlined species were not observed due to the limited mass range

  10. Tangent voltages for the dissociation of singly, doubly and triply metallated double-stranded 5’GCATGC3’ trianions. [Md(5’GCATGC)2 - 5H]3- No metal present [M2d(5’GCATGC)2 - 7H]3- [M3d(5’GCATGC)2 - 9H]3- TV ([d(5’GCATGC)2 - 3H]3-) = -(30.7 0.2) V

  11. Metallated Bleomycin A2 adducts with ds hexamers 2+ Kryatov et al. Chem. Rev., 2005,105, 2175-2226

  12. Chen, J. and Stubbe J. Cur. Op. Chem. Biol., 2004,8, 175 - 181 Arrows indicate potential hydrogen-bond donors or acceptors. Note the crescent shape of the fragment

  13. CID profiles of [MBLMd(5’GCATGC)2 - 6H]4- [MBLMd(5’GCATGC)2 - 6H]4- [d(5’GCATGC) - 2H]2- +[MBLMd(5’GCATGC) - 4H]2- BLM = (Bleomycin A2 - H+); M = Mn, Co, Ni, Cu

  14. Special case of Zn(II) containing complex Zn2+ has higher affinity for the phosphate groups of DNA compared to BLM!

  15. ESI/CID of 1,10 - phenanthroline-containing complexes Co2+ : Phen : ss 5:5:1 [MLnds - 5H]3- [MLn-1ds - 5H]3- + L with n = 1-3

  16. Relative stabilities of Phen-containing species     Coggan et al. Inorg. Chem., 1999,38, 20, 4496 -

  17. Special case of Trien as the ligand Trien forms mixed complexes with oligonucleotides only in the presence of copper (II) ! [CudsTrien]3- -(35.70.3) V [CudsPhen]3- -(29.50.2) V

  18. Conclusions • ESI/CID provides insight into Metal ion - Ligand - DNA interactions: the stoichiometry and mode of binding, the dissociation pathway and relative gas phase stabilities. Future Plans • Systematic ESI/CID studies of Metal ion - Drug - DNA interactions are needed to establish general trends in the gas-phase stability, dissociation mechanisms. • Investigation of the intrinsic reactivity of metallated biological ions toward gaseous carcinogens and other harmful compounds using the Q-trap 2000 and ESI SIFT QqQ instruments. • Solution and gas-phase experiments with double-stranded sequences containing a mismatching base pair and various intercalating species.

  19. Acknowledgements Prof. D. K. Bohme Greg Koyanagi Michael Jarvis Andrea Dasic Sara Hashemi Tuba Gozet Stefan Feil Mike Duhig Voislav Blagojevic $$ NSERC MDS SCIEX

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