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 DNA are 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 strands of DNA only in the presence of metal ions.  ESI-MS has been successfully utilized in the study of interactions between DNA and 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 AA, CC, GG, TT, CCC, ATAT, GCAT, GCGC, CATAC, ACTCG, AGTCTG, TTAGGG, GCATGC Na +, Mn 2+, Fe 2+, Co 2+, Ni 2+, Cu 2+, Zn 2+, Fe 3+, Co 3+ Binding of oligonucleotides with metal complexes Bleomycin A 2 1,10-phenanthroline Triethylenetetramine

4 Experimental  Instruments: MDS SCIEX API 2000 and Q Trap  Duplexes of the hexamers were prepared by heating 70  M ss in 70 mM aqueous NH 4 CH 3 COO to 90 0 C 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; N 2 as the collision gas; collision voltages : -1 to -100 V in the negative mode.

5 Formation of duplexes ?! SSDS

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

7 Metallation of ds hexamers [d(5’T 2 AG 3 ) 2 - 3H] 3- [Znd(5’T 2 AG 3 ) 2 -5H)] 3- [Zn 2 d(5’T 2 AG 3 ) 2 -7H)] 3- [Zn 3 d(5’T 2 AG 3 ) 2 -9H)] 3- [Nad(5’T 2 AG 3 ) 2 -4H)] 3- [Cud(5’T 2 AG 3 ) 2 -5H)] 3- [Cu 2 d(5’T 2 AG 3 ) 2 -7H)] 3- [Cu 3 d(5’T 2 AG 3 ) 2 -9H)] 3- [Cu 4 d(5’T 2 AG 3 ) 2 -11H)] : 1 5 : 1

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 M = Mn, Fe, Co, Ni - pathway (1) dominates M = Cu, Zn - pathways (1) and (2) are nearly equal [Md(5’GCATGC) 2 - 5H] 3- [Mss - 3H] - + [ss- 2H] 2- (1) [Mss - 4H] 2- + [ss- H] - (2) Pathway (3) dominates for all metals [M 2 d(5’GCATGC) 2 - 7H] 3- [Mss - 3H] - + [Mss- 4H] 2- (3) [M 2 ss - 5H] - + [ss- 2H] 2- (4) [M 3 d(5’GCATGC) 2 - 9H] 3- [M 2 ss - 5H] - + [Mss- 4H] 2- (5)

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

11 Kryatov et al. Chem. Rev., 2005, 105, Metallated Bleomycin A 2 adducts with ds hexamers

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

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

14 Special case of Zn(II) containing complex Zn 2+ has higher affinity for the phosphate groups of DNA compared to BLM!

15 [ML n ds - 5H] 3-  [ML n-1 ds - 5H] 3- + L with n = 1-3 ESI/CID of 1,10 - phenanthroline-containing complexes Co 2+ : Phen : ss 5:5:1

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