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

Abstract

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.

Figure 1

Structure of the minor groove binders used in this study. Masses are noted in parentheses.

Figure 2

ESI-MS full scan spectra of equimolar mixtures (C₀ = 10 µM) of Hoechst 33258 with the four duplexes studied: (A) AAAA, (B) ATAT, (C) AATT and (D) the Dickerson dodecamer (DK). ss, single strands remaining; the complexes are noted with the same conventions as in the text.

Figure 3

ESI-MS full scan spectra of equimolar mixtures (C₀ = 10 µM) of the duplex d(GGGGATATGGGG)·d(CCCCATATCCCC) with four drugs: (A) Hoechst 33342, (B) DAPI, (C) netropsin and (D) berenil.

Figure 4

Titration of the ATAT duplex with Hoechst 33258 monitored by circular dichroism. The band centered at 355 nm is attributed to the complex with the drug and the change in ellipticity at this wavelength as a function of the drug/duplex fraction r is plotted in the inset. An inflexion point is observed for r = 1 (1:1 complex).

Figure 5

Job plot for Hoechst 33258 binding to the ATAT duplex. The fluorescence F is measured as a function of the molar fraction of drug (X_drug). The two straight lines intersect at X_drug = 0.485, which indicates the presence of a 1:1 complex.

Figure 6

Fluorescence titration of the duplex ATAT with Hoechst 33258 (excitation wavelength = 354 nm, fluorescence emission collected at 480 nm). (A) Results of the fitting with equation 6 and (B) results of the fitting with equation 7 (a = 1).

Figure 7

Comparison between the constants K₁ determined by fluorescence titration and fitting of the data with equation 6 (black bars) and the equilibrium association constants K₁ determined by electrospray mass spectrometry (hatched bars). Data are shown for each of the fluorescent drugs (A) Hoechst 33258, (B) Hoechst 33342 and (C) DAPI complexed with each of the duplexes.