Programmable oligomers for minor groove DNA recognition

Abstract

The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im), pyrrole (Py), and hydroxypyrrole (Hp), four different ways. On the basis of the paradigm of unsymmetrical paired edges of aromatic rings for minor groove recognition, a second generation set of heterocycle pairs, imidazopyridine/pyrrole (Ip/Py) and hydroxybenzimidazole/pyrrole (Hz/Py), revealed that recognition elements not based on analogues of distamycin could be realized. A new set of end-cap heterocycle dimers, oxazole-hydroxybenzimidazole (No-Hz) and chlorothiophene-hydroxybenzimidazole (Ct-Hz), paired with Py-Py are shown to bind contiguous base pairs of DNA in the minor groove, specifically 5'-GT-3' and 5'-TT-3', with high affinity and selectivity. Utilizing this technology, we have developed a new class of oligomers for sequence-specific DNA minor groove recognition no longer based on the N-methyl pyrrole carboxamides of distamycin.

Figure 1

Structures of dimers (a) imidazole-hydroxybenzimidazole (Im-Hz), (b) oxazole-hydroxybenzimidazole (No-Hz), and (c) chlorothiophene-hydroxybenzimidazole (Ct-Hz) dimer caps in comparison with their respective five membered ring systems. Hydrogen-bonding surfaces to the DNA minor-groove floor are bolded.

Figure 2

Postulated hydrogen-bonding models for the 1:1 polyamide-DNA complexes with their matched sequence and their ball and stick representations. a) Im-Hz-Py-Py-γ-Im-Py-Py-Py-β-Dp (1), b) No-Hz-Py-Py-γ-Im-Py-Py-Py-β-Dp (2), and c) Ct-Hz-Py-Py-γ-Im-Py-Py-Py-β-Dp (3).

Scheme 1

Representative solid-phase synthesis of polyamide 2 and 3 along with a table of the amino acid building blocks used. Reaction conditions: (i) 80% TFA/DCM; (ii) Boc-Py-OBt, DIEA, DMF; (iii) Ac₂O, DIEA, DMF; (iv) repeat i – iii × 2; (v) 80% TFA/DCM; (vi) Boc-Im-OH, HBTU, DIEA, DMF; (vii) Ac₂O, DIEA, DMF; (viii) 80% TFA/DCM; (ix) Boc-γ-OH, HBTU, DIEA, DMF; (x) Ac₂O, DIEA, DMF; (xi) repeat i – iii × 2; (xii) 80% TFA/DCM; (xiii) No-HzOMe-OH, HBTU, DIEA, DMF; (xiv) 3-dimethylamino-1-propylamine (Dp), 80 ºC 2 h; (xv) preparative HPLC; (xvi) thiophenol, NaH, DMF; (xvii) preparative HPLC; (xviii) Ct-HzOMe-OH, HBTU, DIEA, DMF; (xix) same as steps xiv – xvii.

Figure 3

Quantitative DNase I footprinting experiments in the hairpin motif for polyamides 1, 2, and 3 respectively, on the 278 bp, 5'-end-labelled PCR product of plasmid CW15: lane 1, intact DNA; lane 2, A reaction; lane 3, DNase I standard; lanes 4−14, 1 pM, 3 pM, 10 pM, 30 pM, 100 pM, 300 pM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM polyamide, respectively. Each footprinting gel is accompanied by the following: (top) Chemical structure of the pairing of interest; and (bottom) Binding isotherms for the four designed sites. θ_norm values were obtained according to published methods. A binding model for the hairpin motif is shown centered at the top as a ball and stick model with the polyamide bound to its target DNA sequence. Imidazoles and pyrroles are shown as filled and non-filled circles, respectively; β-alanine is shown as a diamond; the γ-aminobutyric acid turn residue is shown as a semicircle connecting the two subunits.

Figure 4

a) Postulated hydrogen-bonding model and structure of oligomer 9. b) Ball and stick representation of 9 and the 6-base-pair binding site with variable region (W = A or T) shown. c) Quantitative DNase I footprint titration experiment on the 5'-³²P-labeled PCR product shown with an illustration and complete sequence of the 285 bp EcoRI/PυuII restriction fragment from plasmid pDEH10. Binding affinities are shown next to their respective binding sites and the match site is designated.