Specific molecular recognition of mixed nucleic acid sequences: an aromatic dication that binds in the DNA minor groove as a dimer - PubMed (original) (raw)

Specific molecular recognition of mixed nucleic acid sequences: an aromatic dication that binds in the DNA minor groove as a dimer

L Wang et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

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Figures

Figure 1

Figure 1

Structures for the minor-groove binding compounds netropsin, distamycin, Hoechst 33258, furamidine (DB75), DB270, and DB293; and sequences for oligo1, oligo2, oligo2-1, and oligo2-2.

Figure 2

Figure 2

Quantitative DNase I footprinting titration experiment with DB293 on the 265-bp DNA fragment. The _Eco_RI-_Pvu_II restriction fragment from plasmid pBS was 3′-end-labeled at the _Eco_RI site with [α-32P]dATP in the presence of AMV reverse transcriptase. (A) The products of the DNase I digestion were resolved on an 8% polyacrylamide gel containing 8M urea. Drug concentrations are (lanes 1–11) 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, and 3.0 μM for DB293 and (lanes 12–15) 0, 1, 2, and 5 μM for DB270. Tracks labeled G represent dimethyl sulfate-piperidine markers specific for guanines. The track labeled DNA contained no drug and no enzyme. Numbers at the right side of the gel refer to the numbering scheme of the fragment. The rectangles on the right side refer to the positions of an AT-rich (open box) and a GC-rich (filled box) binding site for DB293. (B) Footprinting plots for the binding of DB293 to the AT site 5′-AATTAA (open circles) and the GC-rich site 5′-ACCATG (filled squares). The relative band intensity R corresponds to the ratio _I_c/_I_o, where _I_c is the intensity of the band at the ligand concentration c and _I_o is the intensity of the same band in the absence of DB293. The differential cleavage plots in C compare the susceptibility of the DNA to cutting by DNase I in the presence of 5 μM DB270 (filled circles) or 1.5 μM DB293 (open squares). Deviation of points toward the lettered sequence (negative values) corresponds to a ligand-protected site and deviation away (positive values) represents enhanced cleavage. The vertical scale is in units of ln(_f_a) − ln(_f_c), where _f_a is the fractional cleavage at any bond in the presence of the drug and _f_c is the fractional cleavage of the same bond in the control. The results are displayed on a logarithmic scale for the sake of convenience. The rectangles below the sequence show the positions of the AT binding site (open box) and the GC-rich site (filled box). Footprinting reactions, separation of cleavage products, and data analysis were carried out as described (10).

Figure 3

Figure 3

Scatchard plots of the results for binding of DB293 and DB270 to oligo1 and oligo2-1 along with best fit binding curves are shown: closed and open triangles are for DB293 and DB270 binding to oligo1; and closed and open circles are for DB293 and DB270 binding to oligo2-1. Because of the weak binding of DB270 to oligo2-1, the results were fit with the assumption of a single DB270 binding to the duplex (13). Sensorgrams with the data for this plot are shown in Fig. 6, published as supplemental data.

Figure 4

Figure 4

Two-dimensional COSY spectra of the TH6-TCH3 spectral region are shown for free DNA (A); a 1:1 ratio sample of DB293 to oligo2-1 (B); and a 2:1 ratio (C). Signals for the free DNA and for the 2:1 complex in the 1:1 ratio sample are indicated by connecting lines to the top and bottom spectra.

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