Resolution of mixed site DNA complexes with dimer-forming minor-groove binders by using electrospray ionization mass spectrometry: compound structure and DNA sequence effects

Abstract

Small-molecule targeting of the DNA minor groove is a promising approach to modulate genomic processes necessary for normal cellular function. For instance, dicationic diamindines, a well-known class of minor groove binding compounds, have been shown to inhibit interactions of transcription factors binding to genomic DNA. The applications of these compounds could be significantly expanded if we understand sequence-specific recognition of DNA better and could use the information to design more sequence-specific compounds. Aside from polyamides, minor groove binders typically recognize DNA at A-tract or alternating AT base pair sites. Targeting sites with GC base pairs, referred to here as mixed base pair sequences, is much more difficult than those rich in AT base pairs. Compound 1 is the first dicationic diamidine reported to recognize a mixed base pair site. It binds in the minor groove of ATGA sequences as a dimer with positive cooperativity. Due to the well-characterized behavior of 1 with ATGA and AT rich sequences, it provides a paradigm for understanding the elements that are key for recognition of mixed sequence sites. Electrospray ionization mass spectrometry (ESI-MS) is a powerful method to screen DNA complexes formed by analogues of 1 for specific recognition. We also report a novel approach to determine patterns of recognition by 1 for cognate ATGA and ATGA-mutant sequences. We found that functional group modifications and mutating the DNA target site significantly affect binding and stacking, respectively. Both compound conformation and DNA sequence directionality are crucial for recognition.

Keywords: DNA recognition; dimerization; mass spectrometry; minor groove binder; mixed DNA sequence.

Figure 1

Structures of dicationic diamidine minor groove binding compounds used to investigate dimer formation in mixed sequence sites. Compound 1 is a reference compound known to dimerize in the mixed sequence site ATGA. Compound 2 is a classical minor groove binding compound known to recognize AT rich sites. Compound 3 to 8 are analogs of 1. Molecular weights are listed below the respective structures.

Figure 2

Mixed hairpin DNA sequences used to screen interactions for monomer and dimer-forming complex interactions with multiple sequences. Top row: ATATAT, AAATTT, and ATGA test sequences; bottom row: R1 and R2 as reference DNA sequences.

Figure 3

Example ESI-MS spectra of 1 titrated with multiple DNA sequences. Free DNA sequences are apparent by the sequence “name” above the corresponding peak (e.g. AAATTT m/z 7,921.5) and ligand-DNA complex as “name + (n ligands bound) ligand name” (e.g. ATGA + (2) 1, m/z 7,375). Concentrations of 1 are expressed as a mole to mole ratio for 1 to DNA and range [0:1] to [2:1]. Note that the positive cooperative nature of 1 binding to ATGA is indicated by increasing peak for the dimer species and no detectable 1:1 species. (A) [0:1], (B) [1:1], (C) [2:1].

Figure 4

Spectra of DNA sequences titrated with compounds 3 to 8 analogs. Unbound DNAs are indicated by the sequence “name” above the respective peak (e.g. AAATTT, m/z 7,921.5) and ligand-DNA complex as “name + (n ligands bound) ligand name” (e.g. AAATTT + (1) 3, m/z 8,265.5). Molar ratios are expressed as [4:1] where ligand is to DNA. (A) 7, (B) 8, (C) 4, (D) 5, (E) 6, and (F) 3.

Figure 5

Mixed DNA sequence results with 3 are expanded between the range m/z 7,250 to 7,950 to highlight the unexpected dimerization of two molecules of 3 bound to R1. The molar ratio shown is [4:1].

Figure 6

Comparison of 1 and analogs 3, 4, 5, and 6. Left illustrates the electrostatic potential map for the compounds. The right column shows a side view of the twists experienced in the overall structures. Molecules were minimized and electrostatic potential maps calculated using Spartan.

Figure 7

Models of compound 3 recognizing the mixed sequences ATGA as a dimer. (A) The spaced-filled model illustrates the stacked dimer formation of 3 in the minor groove of ATGA. (B) Side view of the stacked compounds. The curvature of the bottom molecule (orange) turns in towards the floor of the minor groove whereas the top molecule (green) faces out toward the solvent. (C) The stacked 3 dimer interactions with the base pairs 5′-ATGA-3′ and 3′-TCAT-5′. H-bond interactions between the base pairs are shown having dashed lines with distances in Å.

Figure 8

ATGA cognate and ATGA sequence variants used to examine the sequence specificity of 1. Base pairs flanking the target sites were maintained to allow similar response. Loops were modified for distinguishability using ESI-MS.

Figure 9

Spectra of ATGA cognate and ATGA mutant sequences with 1. Free DNA sequences range m/z 6,650-7,750 (left) and compound 1-DNA complexes m/z 7,250-7,975 (right). Both spectra belong to the same titration sample having a molar ratio of [4:1]. Peak intensities for the complexes are relative to the peak for ATGA + (2) 1.

Figure 10

(A) Comparison of the relative peak intensities (± 3%) for complexes and ΔT_m values (± 0.5 °C) for mixed DNA sequences with 1 and the dimer-forming analogs 3 – 6. ΔT_m values (secondary y-axis) are for dimer-complexes formed between ligands and ATGA at a [4:1] molar ratio. (B) Structural variability and spatial arrangement for dimer-forming compounds are shown by superimposing the molecules over their mutual phenyl-amidines. 1 (tan), 3 (blue), 4 (green), 5 (orange), and 6 (pink).

Figure 11

Comparison of the relative peak intensities (± 3%) of complexes and ΔT_m values (± 0.5 °C) for ATGA cognate and ATGA mutant sequences with 1. Relative abundances (primary y-axis) display the peak intensities for both 1:1 and 2:1 binding of 1 with DNA as monomer and dimer complexes, respectively. ΔT_m values (secondary y-axis) from studies performed using a [4:1] molar ratio of compound 1 to DNA sequence.