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Nucleic Acids Research, 2002, Vol. 30, No. 16 e82
© 2002 Oxford University Press

Determination of affinity, stoichiometry and sequence selectivity of minor groove binder complexes with double-stranded oligodeoxynucleotides by electrospray ionization mass spectrometry

Frederic Rosu, Valérie Gabelica*,1, Claude Houssier and Edwin De Pauw1

Biospectroscopy Laboratory and 1 Mass Spectrometry Laboratory, University of Liège, Chemistry Institute, Bâtiment B6c, B-4000 Liège, Belgium

*To whom correspondence should be addressed. Tel: +32 4 366 34 32; Fax: +32 4 366 34 13; Email: v.gabelica{at}ulg.ac.be

Electrospray mass spectrometry was evaluated regarding the reliability of the determination of the stoichiometries and equilibrium association constants from single spectra. Complexes between minor groove binders (Hoechst 33258, Hoechst 33342, DAPI, netropsin and berenil) and 12mer oligonucleotide duplexes with a central sequence (A/T)4 flanked by G/C base pairs were chosen as model systems. To validate the electrospray ionization mass spectrometry (ESI-MS) method, comparisons were made with circular dichroism and fluorescence spectroscopy measurements. ESI-MS allowed the detection of minor (2 drug + DNA) species for Hoechst 33258, Hoechst 33342, DAPI and berenil with duplex d(GGGG(A/T)4GGGG)· d(CCCC(A/T)4CCCC), which were undetectable with the other techniques. Assuming that the duplexes and the complexes have the same electrospray response factors, the equilbrium association constants of the 1:1 and 2:1 complexes were determined by ESI-MS, and the values show a good quantitative agreement with fluorescence determined constants for Hoechst 33258 and Hoechst 33342. It is also shown that ESI-MS can quickly give reliable information on the A/T sequence selectivity of a drug: the signal of a complex is directly related to the affinity of the drug for that particular duplex. The potential of ESI-MS as a qualitative and quantitative affinity screening method is emphasized.


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