ESI mass spectra show that up to five ruthenium molecules can bind non-covalently to double stranded 16mer DNA, and provide information on the relative affinity and DNA sequence selectivity of different ruthenium complexes

Aldrich-Wright, Janice

Beck, Jennifer L

Gupta, Rajesh

Ralph, Stephen F

Sheil, Margaret

Urathamakul, Thitima

Williamson, Nyree L

English

Research Online

University of Wollongong Research Online Faculty of Science - Papers (Archive) Faculty of Science, Medicine and Health 1-1-2003 Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry Jennifer L. Beck University of Wollongong, jbeck@uow.edu.au Rajesh Gupta University of Wollongong Thitima Urathamakul University of Wollongong, thitima@uow.edu.au Nyree L. Williamson University of Wollongong Margaret Sheil University of Wollongong, msheil@uow.edu.au See next page for additional authors Follow this and additional works at: https://ro.uow.edu.au/scipapers  Part of the Life Sciences Commons, Physical Sciences and Mathematics Commons, and the Social and Behavioral Sciences Commons Recommended Citation Beck, Jennifer L.; Gupta, Rajesh; Urathamakul, Thitima; Williamson, Nyree L.; Sheil, Margaret; Aldrich-Wright, Janice; and Ralph, Stephen F.: Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry 2003, 626-627. https://ro.uow.edu.au/scipapers/4238 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry Abstract ESI mass spectra show that up to five ruthenium molecules can bind non-covalently to double stranded 16mer DNA, and provide information on the relative affinity and DNA sequence selectivity of different ruthenium complexes. Keywords mass, esi, spectrometry, probing, ruthenium, dna, metallointercalators, selectivity, CMMB Disciplines Life Sciences | Physical Sciences and Mathematics | Social and Behavioral Sciences Publication Details Beck, J. L., Gupta, R., Urathamakul, T., Williamson, N. L., Sheil, M., Aldrich-Wright, J. & Ralph, S. F. (2003). Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry. Chemical Communications, (5), 626-627. Authors Jennifer L. Beck, Rajesh Gupta, Thitima Urathamakul, Nyree L. Williamson, Margaret Sheil, Janice Aldrich-Wright, and Stephen F. Ralph This journal article is available at Research Online: https://ro.uow.edu.au/scipapers/4238    Probing DNA selectivity of ruthenium metallointercalators using ESImass spectrometry†Jennifer L. Beck,a Rajesh Gupta,a Thitima Urathamakul,a Nyree L. Williamson,a Margaret M. Sheil,aJanice R. Aldrich-Wrightb and Stephen F. Ralph*aa Department of Chemistry, University of Wollongong, Northfields Avenue, Wollongong 2522, Australia.E-mail: stephen_ralph@uow.edu.au; Fax: (+61) 2 4221 4287; Tel: (+61) 2 4221 4286b Department of Chemistry, University of Western Sydney, Macarthur, PO Box 555, Campbelltown,AustraliaReceived (in Cambridge, UK) 6th December 2002, Accepted 13th January 2003First published as an Advance Article on the web 10th February 2003ESI mass spectra show that up to five ruthenium moleculescan bind non-covalently to double stranded 16mer DNA,and provide information on the relative affinity and DNAsequence selectivity of different ruthenium complexes.Metal compounds that bind non-covalently to DNA haveattracted considerable interest.1 This has been driven partly bya desire to understand these interactions, and partly by studiesthat have shown these compounds may be useful as nucleic acidprobes,2,3 synthetic restriction enzymes1,4 or DNA repairagents.1,5 In this paper we present recent results obtained usingElectrospray Ionisation Mass Spectrometry (ESI-MS) to char-acterise non-covalent complexes formed between duplex 16meroligonucleotides and racemic mixtures of the rutheniumcompounds shown in Fig. 1.‡ ESI-MS provided information onthe number, relative amounts, and metal/DNA stoichiometry ofindividual complexes. In addition ESI-MS was also used todetermine the relative affinities of the ruthenium compounds forDNA, and obtain information about their DNA sequenceselectivity. Although ESI-MS has been used widely to studynon-covalent interactions between biomolecules,6,7 or betweenbiomolecules and small organic molecules,7–10 there have beenrelatively few studies involving DNA and metal com-pounds.11Each of the ruthenium compounds in Fig. 1 has similarcharge, size and shape, and would therefore be expected to showa similar degree of electrostatic attraction towards the anionicphosphodiester backbone of DNA. Consequently any sig-nificant differences in DNA binding affinity between theruthenium compounds must be a result of variations in theirability to act as minor groove binders or intercalators. Theability of these compounds to act as intercalators variessignificantly as a consequence of the replacement of onephenanthroline ligand in [Ru(phen)3]2+ by larger ligands thatcan more readily insert into the DNA base stack. Three differentnon-self-complementary 16mer DNA duplexes, labelled D1,D2 and D3, with different numbers of GC and AT base pairs,were chosen for examination.§ The greater GC content of D1and D2 was expected to enhance binding by rutheniumcompounds that can intercalate,12 while D3 contains an AT basesequence favourable to compounds that prefer to bind along theDNA minor groove.13D1 d(CCTCGGCCGGCCGACC/GGTCGGCCGGCCGAGG)D2 d(CCTCATGGCCATGACC/GGTCATGGCCATGAGG)D3 d(CCTCAAAATTTTGACC/GGTCAAAATTTTGAGG)Fig. 2(a)–(d) shows ESI mass spectra of reaction mixturescontaining different ratios of [Ru(phen)2(dppz)]2+ and D2.¶When there was no metal in solution, the mass spectrum (Fig.2(a)) contained ions at m/z 1626.5 and 1952.1, assigned to[D226H]62 and [D225H]52, respectively. When the ratiometal+D2 was 1.5+1 the ESI mass spectrum (Fig. 2(b)) showedions of medium or high abundance attributable to unbound D2and a complex in which one [Ru(phen)2(dppz)]2+ was bound toD2. Fig. 2(b) also shows ions of weak to medium abundanceattributable to complexes containing two and three [Ru-(phen)2(dppz)]2+ bound to D2. As the ratio metal+D2 was† Electronic supplementary information (ESI) available: table of massspectral assignments. See http://www.rsc.org/suppdata/cc/b2/b212132h/Fig. 1 Structures of the ruthenium compounds used.Fig. 2 Negative ion ESI mass spectra of reaction mixtures containing D2and either [Ru(phen)2(dppz)]2+ or [Ru(phen)3]2+. (a) free D2. (b) [Ru-(phen)2(dppz)]2+:D2 = 1.5+1. (c) [Ru(phen)2(dppz)]2+:D2 = 3+1. (d)[Ru(phen)2(dppz)]2+:D2 = 6+1. (e) [Ru(phen)3]2+:D2 = 20+1.Th is journa l i s © The Roya l Soc ie ty of Chemist ry 2003626 CHEM. COMMUN. , 2003, 626–627DOI: 10.1039/b212132hDownloaded by University of Wollongong on 03 August 2012Published on 10 February 2003 on http://pubs.rsc.org | doi:10.1039/B212132HView Online / Journal Homepage / Table of Contents for this issueincreased the abundance of these ions also increased. When theratio metal+D2 was 6+1 (maximum ratio permitted by thesolubility of [Ru(phen)2(dppz)](PF6)2) the most abundant ion inthe spectrum (Fig. 2(d)) was from a complex containing four[Ru(phen)2(dppz)]2+ bound to D2. Also present in this spectrumwere ions attributable to complexes containing three and five[Ru(phen)2(dppz)]2+ bound to D2.Fig. 2(e) shows the spectrum of a solution containing a 20+1ratio of [Ru(phen)3]2+ and D2. Despite the high ratio, the mostabundant ion in the spectrum was assigned to free D2. Thespectrum also contained ions of medium to high abundancefrom a complex containing one [Ru(phen)3]2+ bound to D2, andions of low abundance from a complex containing two[Ru(phen)3]2+ bound to D2. These results are consistent withthose obtained using other techniques, that showed thatruthenium(II) compounds containing the dppz ligand have thehighest affinity for DNA of compounds of this type.14 Forexample, the binding constants for association of D and L-[Ru(phen)2(dppz)]2+ with calf thymus DNA are 3.2 3 106 and1.7 3 106 M21, respectively.15 These values are significantlygreater than those for binding of D and L-[Ru(phen)3]2+ to calfthymus DNA, which are 9 3 103 and 1.1 3 104 M21,respectively.15 Further information concerning the relativeaffinities of all the compounds shown in Fig. 1 for D2 ispresented in Fig. 3. This was constructed using relativeabundances from spectra of reaction mixtures containing a 6+1ratio of a single ruthenium compound and D2. Relativeabundances were obtained by dividing the combined ion currentfor 5-, 6- and 7- ions assigned to a specific DNA complex, bythe total ion current for all 5-, 6- and 7- ions.Fig. 3 shows that [Ru(phen)2(dppz)]2+ and [Ru-(phen)2(dpqC)]2+ formed the highest percentage of DNAcomplexes containing 4 and 5 molecules of ruthenium com-pound. This suggests that these complexes had the greatestaffinity for D2. Comparison of the data for the other metalcompounds shows that [Ru(phen)2(dpq)]2+ had the next highestbinding affinity, and [Ru(phen)3]2+ the lowest affinity towardsD2. By plotting relative abundances from reaction mixturescontaining the same ruthenium compound, but different oligo-nucleotides, it was also possible to obtain information aboutDNA sequence selectivity. For example, Fig. 4 shows that[Ru(phen)2(dpq)]2+ formed a higher percentage of complexescontaining 2, 3 or 4 molecules bound to DNA in experimentswith D2, compared to experiments with the other two duplexes.This shows that this ruthenium compound displayed a prefer-ence for binding to DNA sequences in D2. Previous NMRstudies showed that D-[Ru(phen)2(dppz)]2+ intercalates intoDNA from the minor groove, particularly at purine–purine/pyrimidine–pyrimidine sequences.16 One of the principalmechanisms of binding of this compound to D2 may thereforeinvolve intercalation into the GGCC/GGCC sequence in themiddle of the duplex. Intercalation was also expected to befavourable with D1 owing to its high GC content. The lowerdegree of interaction observed may be a result of closerproximity of binding sites in this duplex. Groove bindinginteractions between DNA and [Ru(phen)2(dpq)]2+ may alsooccur, however the lower level of reactivity towards the AT richduplex D3 suggests that these are less important than inter-calation. We are currently investigating reactions with longeroligonucleotides, and competition reactions involving organicDNA binding agents, in order to learn more about the DNAselectivity of these ruthenium compounds.Notes and references‡ [Ru(phen)3]Cl2 was obtained from the Aldrich Chemical Co. [Ru-(phen)2(dpq)]Cl2, [Ru(phen)2(dpqC)]Cl2 and [Ru(phen)2(dppz)]Cl2 wereprepared using literature methods.16,17§ Oligonucleotides were obtained from Geneworks, South Australia, as the‘trityl-on’ derivatives. These were deprotected by acid treatment andpurified by HPLC as previously described.18¶ Reaction mixtures were prepared by first annealing complementarystrands of DNA (1 mM), and then treating the resulting dsDNA with anappropriate amount of stock solution containing the desired rutheniumcompound at 20 °C. The final concentration of dsDNA in reaction mixtureswas 25 mM. Samples were diluted to 10 mM prior to analysis by ESI-MS.Negative ion ESI mass spectra were obtained using a Micromass Qtof2mass spectrometer (cone voltage 50 V; desolvation temperature 80 °C). Thesolvent used for annealing, complex formation and obtaining ESI massspectra was 0.1 M ammonium acetate, pH 8.5.1 K. E. Erkkila, D. T. Odom and J. K. Barton, Chem. Rev., 1999, 99,2777.2 C. S. Chow and J. K. Barton, J. Am. Chem. Soc., 1990, 112, 2839.3 A. Sitlani, C. M. Dupureur and J. K. Barton, J. Am. Chem. Soc., 1993,115, 12589.4 M. P. Fitzsimons and J. K. Barton, J. Am. Chem. Soc., 1997, 119,3379.5 P. J. Dandliker, R. E. Holmlin and J. K. Barton, Science, 1997, 275,1465.6 J. Loo, Mass Spectrom. Rev., 1997, 16, 1.7 J. L. Beck, M. L. Colgrave, S. F. Ralph and M. M. Sheil, Mass Spectrom.Rev., 2001, 20, 61.8 Q. Gao, X. Cheng, R. D. Smith, C. F. Yang and I. H. Goldberg, J. MassSpectrom., 1996, 31, 31.9 A. Triolo, F. M. Arcamone, A. Raffaelli and P. Salvadori, J. MassSpectrom., 1997, 32, 1186.10 A. Kapur, J. L. Beck and M. M. Sheil, Rapid Commun. Mass Spectrom.,1999, 13, 2489.11 K. X. Wan, T. Shibue and M. L. Gross, J. Am. Chem. Soc., 2000, 122,300.12 B. J. Geierstanger and D. E. Wemmer, Annu. Rev. Biophys. Biomol.Struct., 1995, 24, 463.13 A. Pullman and B. Pullman, Q. Rev. Biophys., 1981, 14, 289.14 C. M. Dupureur and J. K. Barton, J. Am. Chem. Soc., 1994, 116,10286.15 I. Haq, P. Lincoln, D. Suh, B. Norden, B. Z. Chowdhry and J. B.Chaires, J. Am. Chem. Soc., 1995, 117, 4788.16 J. G. Collins, A. D. Sleeman, J. R. Aldrich-Wright, I. Greguric and T. W.Hambley, Inorg. Chem., 1998, 37, 3133.17 C. M. Dupureur and J. K. Barton, Inorg. Chem., 1997, 36, 33.18 G. Wickham, P. Iannitti, J. Boschenok and M. M. Sheil, J. MassSpectrom., 1995, 30, S197.Fig. 3 Relative abundances of complexes in reaction mixtures containing a6+1 ratio of ruthenium compound and duplex D2.Fig. 4 Relative abundance of complexes in reaction mixtures containing a6+1 ratio of [Ru(phen)2(dpq)]2+ and oligonucleotides.627CHEM. COMMUN. , 2003, 626–627Downloaded by University of Wollongong on 03 August 2012Published on 10 February 2003 on http://pubs.rsc.org | doi:10.1039/B212132HView Online

Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry

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Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry

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