We then examined the interactions of Diuron, Diazinon, Alachlor and dEHP with both ODNs. The studied sequences are self-complementary and capable of assembling into duplexes upon annealing. After annealing, both single-stranded (ss) and double-stranded (ds) ODNs are present in solution. The annealed solutions of the ODNs were incubated with equimolar, 3, 5 and 10-fold excessive amounts of the contaminants. 1:1 complexes of ssODNs with Diuron were observed even at the equimolar amounts of the regents in the incubation mixtures (Figure 2).

High-Resolution Tandem Mass Spectrometry Analysis of the Interactions ofOligonucleotides with Selected River Basin Specific PollutantsJanna Anichina, Andre Schreiber, Ron Bonner, Takeo Sakuma AB SCIEX, 71 Four Valley Drive, Concord, ON, L4K 4V8 Canada

ABSTRACTIn this study we examined the interactions of two self-complementary decameric oligodeoxynucleo-tides with four river basin specific pollutants e.g., Diazinon, Diuron, Alachlor and bis(2-ethylhexyl) phthalate (Figure 1). ESI-MS/MS analysis of the incubation mixtures of Diuron with both oligo-deoxynucleotides indicated the formation of 1:1 adducts of both single-stranded oligodeoxynucleo-tides at a ratio of 1 or higher. Collected high resolution TOF MS and MS/MS data were used to confirm the elemental composition of the ions of interest as well as to gain an insight into the structure of the adducts. Similar results were observed for the incubation mixtures of Diazinon. Larger excesses of Diazinon compared to Diuron were required to observe the formation of the 1:1 adducts indicating lower affinity of Diazinon for complexation with oligodeoxynucleotides. No detectable adducts of the studied nucleic acids with Alachlor or bis(2-ethylhexyl)phthalate (dEHP) were observed under similar incubation conditions.

INTRODUCTIONSince a great number of organic compounds are annually released into the environment, the necessity to assess in a timely manner the potential risks associated with these chemicals along with the products of their environmental transformation is of high priority. In order to reduce the number of candidates for full-scale animal studies, potential toxicity can be rapidly assessed in simplified model systems. Significant advances were made in the application of ESI-MS/MS for detection, quantification and structural elucidation of non-covalent complexes between small molecule ligands and nucleic acids [1]. Also, ESI-MS/MS has proven to be a promising tool for rapid screening of the reactivity of environmental contaminants toward DNA [2]. In this study we employed high resolution tandem mass spectrometry combined with micro-flow LC separation to study interactions of the selected micro-contaminants with single- and double-stranded oligodeoxynucleotides (ODNs) of the following sequences: d(5’-GCGCGCGCGC-3’) (ODN1) and d(5’-GCGCATGCGC-3’) (ODN2). The micro-contaminants were chosen in the context of the European Union Water Framework Directive (EU WFD) based on the assessment study for 500 organic substances observed in four river basins of the Elbe, Scheldt, Danube and Llobregat [3]. Diazinon, Diuron, dEHP and Alachlor were identified among high concern river-basin pollutants with the highest priority-ranking values.

MATERIALS AND METHODSDiazinon, Diuron, Alachlor , dEHP and Malachite Green were purchased from Sigma-Aldrich, St. Louis, MO, USA. Two decameric ODNs, d(5’-GCGCATGCGC-3’) and d(5’-GCGCGCGCGC-3’) were acquired from the ACGT Corporation, Toronto, Canada.

Sample Preparation:Working solutions (20 M) of ODNs were prepared by dilution of the stock solutions with a mixed solvent of 90% water with 10% methanol. Both oligonucleotides were annealed to form double-stranded species in 100 mM ammonium acetate buffer prior to microLC-ESI-MS/MS and direct infusion analyses. Annealing was performed by heating the solutions of ODNs to eighty degrees Celsius for 10 minutes and slowly cooling them down to room temperature to ensure the formation of the duplexes.

HPLC Conditions:An Eksigent expressHT™ ultra HPLC system was utilized for chromatographic separation. A 3-µm (0.2 x 50 mm) InertSustain C18 chromatographic column (GL Sciences, Tokyo, Japan) was maintained at the temperature of 250C. De-ionized water with 5 mM ammonium acetate, pH adjusted to 8.16 with a diluted ammonium hydroxide solution, and HPLC grade methanol + 5 mM ammonium acetate were used at a flow rate of 5 µL/min. The gradient profile was as follows:

Time (min) %A %B 0 100 0 2 100 0 15 0 100 20 0 100

3-min pre-run flushing at 100% starting condition was used to equilibrate the column.

Mass Spectrometric Measurements:A TripleTOF® 5600 with Electrospray Ionization (ESI) probe was used in the negative ion mode.TOF MS scan was performed in the 100 to 1250 mass/charge range. Tandem mass spectrometric measurements (MS/MS) were performed in the product ion scan. MS/MS data were acquired at various fixed values of the laboratory frame collision energy.

Data Interpretation:AB SCIEX PeakView® software was used for the data interrogation. The FormulaFinder function of the software helped with independent assessment of the elemental composition of the peaks of interest. A prototype oligonucleotide fragmentation interpretation tool (OligoViewer 0.9) was used to gain insights into the CID pathways of the components of the reaction mixture.

Figure 5. MS/MS spectrum of the 1:1 complex of ssODN1 with Diuron (nominal m/z of 651.2) in the charge state of 5-. CE was averaged for the region of -30V to -10V.

CONCLUSIONS and FUTURE STUDIESThis study has demonstrated an elegant application of high resolution tandem mass spectrometry combined with the micro-flow liquid chromatography for rapid screening of the reactivity of high priority environmental contaminants toward DNA models. Our future studies will be directed toward quantitative investigation of the solution complexation of ODNs with micro-contaminants using high resolution microLC/MS.

REFERENCES1.V. Gabelica. In “Mass Spectrometry of Nucleosides and Nucleic Acids” Ed. J. H. Banoub and P.A. Limbach. CRC Press, 2010, pp. 283-3032.J. Anichina, Y. Zhao, S.E. Hrudey, A. Schreiber and X.-F. Li. Anal. Chem. 83, 8145-8151, 20113.P. C. von der Ohe, V. Dulio, J. Slobodnik, E. De Deckere, R. Kuhne, R.-U. Ebert, A. Ginebreda, W. De Cooman, G. Schuurmann, W. Brack. Sci. Total Environ. 409, 2064-2077, 20114.L. Orsatti, S. Di Marco, C. Volpari, A. Vannini, P. Neddermann, F. Bonelli. Anal. BioChem. 309, 11-18, 2002

ACKNOWLEDGEMENTS Mr. Ken’ichi Suzuki of GL Sciences (Tokyo) for production and provision of prototype 0.2 and 0.3 mm i.d. columns for this study. Steve Hobbs, Vlad Savchenko, Fouad Khalaf and Matthiew Furzecotte of AB SCIEX for technical assistance.

TRADEMARKS/LICENSINGFor Research Use Only. Not for use in diagnostic procedures.The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

© 2012 AB SCIEX.

The structural elucidation of the detected adducts was conducted in tandem mass spectrometric experiments. A prototype oligonucleotide fragmentation interpretation tool was utilized to match experimental CID products with the theoretically predicted product ions resulting from the dissociation of the phosphodiester backbones of the ODNs (Figure 4).Tandem mass spectrometric measurements of the 1:1 adducts of Diuron with both single-stranded ODNs demonstrated that their dissociation proceeds via the loss of a neutral ligand molecule at a relatively low value of the laboratory frame collision energy. When highly negatively charged Diuron:ODN adducts (charge states of 5-, 6- or 7-) were exposed to collision-induced dissociation (CID), we also observed a competing charge-separation channel to produce a deprotonated Diuron moiety and an oligonucleotide ion in (n-1)- charge state with n = 5,6,7 respectively (Figure 5). At the elevated values of the laboratory frame collision energy we observed further fragmentation of the primary dissociation products.

+

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At a 10-fold excess of the Diuron ligand we have also observed the formation of the 1:1 complexes of the dsODNs (Figure 6A). The CID pathways of the non-covalent assemblies of the duplexes with Diuron molecule were found to proceed via three competing fragmentation channels e.g. strands and charge separation when ligand remained attached to one of the strands; charge-separation with the elimination of a deprotonated ligand and loss of a neutral Diuron moiety to produce a bare duplex ion (Figure 6B). Our observation of the losses of neutral or deprotonated entities of Diuron upon CID of the observed ODN adducts suggests that the binding within the complexes is non-covalent. This hypothesis is consistent with the trends in CID of non-covalent complexes of groove binders and intercalators. [1]

Figure 7. (A) - XIC of the triply-charged ssODN2 from negative TOF MS of the 50 µM solution of ODN2. (B) - XIC of (Diuron-H)- from 20 µM solution of Diuron. (C) - XICs of ODN2 and Diuron from the incubation mixture (100 µM ODN2 and 300 µM Diuron). (D) - XICs of the ODN1 and Diuron, from the incubation mixture (100 µM ODN1 and 1mM Diuron).

A

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The ratio of the ODN to the ligand can be determined by dividing the measured molar concentration of the ligand by that of the injected ODN.

Therefore, we have employed micro-LC separation to the incubation mixtures of the ODNs and micro-contaminants. Our preliminary measurements demonstrate that Diuron and the ODNs elute at different times (Figure 7) and Diuron does not “stick” to the chromatographic column

The results of the experiments performed with the studied ODNs and Diazinon were similar to those observed for Diuron-containing species. However, the formation of the 1:1 adducts was observed at a higher excess of Diazinon (50-fold or higher). CID measurements of the Diazinon adducts demonstrated an earlier (compared to Diuron) CID onset which indicates a weaker binding with the ODNs. No detectable adducts of Alachlor or dEHP with either ODN were observed even at a 1000-fold excesses of the compounds.

Figure 3. (A) - TOF MS spectrum of the 20 µM solution of ODN1 and 3-fold excess of Malachite Green (MG). (B) - Product Ion spectrum of 1:1 complex of MG with ODN1 acquired at the CE of -30V.

Figure 1. Chemical structures of the micro-contaminants.

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Figure 2. TOF MS spectrum of the equimolar solution of ODN1 and Diuron. Inserted peaks comprise theoretical isotopic distribution.

[ssODN1]3-

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A B

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Figure 6. (A) - ESI TOF MS spectrum of 1:10 mixture of ODN1 and Diuron. m/z = 1211 corresponds to the ds5-. (B) - Schematic representation of the dissociation of 1:1 complex of the duplex with Diuron.

[dsODN1]5-[dsODN1:Diuron]5-

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RESULTSIn order to assess the feasibility of the chosen approach to study non-covalent interactions of nucleic acids with small ligands, we have incubated the ODNs with various excesses of Malachite Green (MG). Malachite Green is a triarylmethane dye which is known to strongly intercalate into DNA.Figure 3A demonstrates the formation of 1:1; 1:2 and 1:3 complexes of MG with the studied ODNs. The dissociation pathways of the MG complexes were found to occur either via the loss of a neutral ligand (Figure 3B) for lower charge states of the complexes or via the loss of a neutral ligand and charge separation (for higher charge states). Our measurements with MG-ligand are consistent with the literature data with respect to the dissociation of the non-covalent complexes in the gas phase. [1]

Figure 4. Monoisotopic masses of the theoretically predicted fragments resulting from the cleavage of the phosphodiester backbone of ssODN1 and MS/MS spectrum of the nominal m/z = 504.1 which corresponds to 6- ionic state.

Since HPLC was previously demonstrated to have potential in the determination of the stoichiometry of non-covalent complexes [4], we have employed a microLC system coupled with a TripleTOF® 5600 mass spectrometer to assess the possibility of a quantitative study of the thermodynamics of Diuron complexation with the ODNs in solution. The approach for measuring binding stoichiometry is based on the idea that upon injection of the incubation mixture non-covalent complexes may dissociate due to the hydrophobic environment of the chromatographic column. Once dissociated from the complex, the ligand can be quantified using the previously generated calibration curve if the following criteria are met: the ligand and ODN elute at different times, ligand does not display a strong affinity for the column material and it is possible to remove the ligand excess (if any) from the reaction mixture.