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  • Competitive binding sites of a ruthenium arene anticancer complex on oligonucleotides studied by mass spectrometry: ladder-sequencing versus top-down.

Competitive binding sites of a ruthenium arene anticancer complex on oligonucleotides studied by mass spectrometry: ladder-sequencing versus top-down.

Journal of the American Society for Mass Spectrometry (2013-02-14)
Kui Wu, Wenbing Hu, Qun Luo, Xianchan Li, Shaoxiang Xiong, Peter J Sadler, Fuyi Wang
ABSTRACT

We report identification of the binding sites for an organometallic ruthenium anticancer complex [(η (6)-biphenyl)Ru(en)Cl][PF6] (1; en = ethylenediamine) on the 15-mer single-stranded oligodeoxynucleotides (ODNs), 5'-CTCTCTX7G8Y9CTTCTC-3' [X = Y = T (I); X = C and Y = A (II); X = A and Y = T (III); X = T and Y = A (IV)] by electrospray ionization mass spectrometry (ESI-MS) in conjunction with enzymatic digestion or tandem mass spectrometry (top-down MS). ESI-MS combined with enzymatic digestion (termed MS-based ladder-sequencing), is effective for identification of the thermodynamically-favored G-binding sites, but not applicable to determine the thermodynamically unstable T-binding sites because the T-bound adducts dissociate during enzymatic digestion. In contrast, top-down MS is efficient for localization of the T binding sites, but not suitable for mapping ruthenated G bases, due to the facile fragmentation of G bases from ODN backbones prior to the dissociation of the phosphodiester bonds. The combination of the two MS approaches reveals that G8 in each ODN is the preferred binding site for 1, and that the T binding sites of 1 are either T7 or T11 on I and IV, and either T6 or T11 on II and III, respectively. These findings not only demonstrate for the first time that T-bases in single-stranded oligonucleotides are kinetically competitive with guanine for such organoruthenium complexes, but also illustrate the relative merits of the combination of ladder-sequencing and top-down MS approaches to elucidate the interactions of metal anticancer complexes with DNA.

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Ethylenediamine dihydrochloride, 98%
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₩147,784
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Ethylenediamine, puriss. p.a., absolute, ≥99.5% (GC)
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250 mL
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2.5 L
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250 mL
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₩123,526
1 L
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Supelco
Ethylenediamine, analytical standard
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Ethylenediamine solution, technical, 75-80%
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Ethylenediamine, purified by redistillation, ≥99.5%
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₩280,749
100 mL
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Ethylenediamine, ReagentPlus®, ≥99%
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