Rational design of a triple helix-specific intercalating ligand

Abstract

DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3, 4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteen-paired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson-Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and/or induce triple helix formation under physiological conditions.

Figure 1

(a) Structure of different triplex-specific ligands shown to intercalate into triple helices. In the present study, R₁ = OCH₃ and R₂ = NH(CH₂)₃NH₂. (b) Structure of a T⋅A×T base triplet, with the names given to the different grooves.

Figure 2

Energy-minimized models of BfPQ (a) or BQQ (b) intercalated in a triple helix made of Hoogsteen T⋅A×T base triplets. The planar ring system is shown stacked with the T⋅A×T base triplet on its 5′ side (with respect to the purine strand) (Upper) and on its 3′ side (Lower). The Watson strand (pyrimidine strand) is shown in blue, the Crick strand (purine strand) is in red, and the Hoogsteen strand (third strand) is in yellow. The intercalated molecule is colored using standard colors for each atom (C in green, H in white, N in blue, and O in red).

Figure 3

Melting temperature as a function of pH for the mixture of oligonucleotides (1 μM each), which can form either a 14-mer parallel Hoogsteen duplex in the presence of 15 μM BfPQ (1), or a 10-bp antiparallel Watson–Crick duplex in the absence of BfPQ (2). Experiments were carried out in a cacodylate buffer (10 mM) containing 0.1 M NaCl.

Figure 4

First derivatives of the melting curves for the mixture of a 26-bp duplex with increasing third-strand concentrations in the presence of 10 μM BQQ (sequences are shown at the top of the figure). The duplex concentration was 1.5 μM, and the third-strand-to-duplex ratio was 0:1 (•), 0.33:1 (○), 0.66:1 (▪), and 1:1 (□). Experiments were carried out in a 10 mM cacodylate buffer (pH 6.2) containing 0.1 M NaCl.

Figure 5

Inhibition of DraI restriction endonuclease cleavage by oligonucleotides in the presence of BQQ or BePI. (a) The plasmid (pLTR-HIV) contains four DraI cleavage sites that generate DNA fragments of 19, 692, 1,386, and 2,403 bp. One of these four sites corresponds to the junction of the 16-bp oligopyrimidine⋅oligopurine sequence (polypurine tract, PPT) of the HIV-1 provirus contained within the nef gene. (b) Agarose gel reveals the extent of DraI cleavage inhibition by showing the decreased 1,386- and 2,403-bp fragments and the increased 3,789-bp fragment. The intensity of the 692-bp fragment is not affected. The concentration of oligonucleotides and that of ligands are indicated (see Materials and Methods for experimental conditions).