Abstract
We studied photochemical reactions of BrU-substituted G-quadruplex (G4) DNA substrates with two pyrene-substituted polyazamacrocyclic ligands, M-1PY and M-2PY. Both ligands bind to and stabilize G4-DNA structures without altering their folding topology, as demonstrated by FRET-melting experiments, fluorimetric titrations and CD spectroscopy. Notably, the bis-pyrene derivative (M-2PY) behaves as a significantly more affine and selective G4 ligand, compared with its mono-pyrene counterpart (M-1PY) and control compounds. Upon short UVA irradiation (365 nm) both ligands, in particular M-2PY, efficiently sensitize photoreactions at BrU residues incorporated in G4 structures and give rise to two kinds of photoproducts, namely DNA strand cleavage and covalent ligand–DNA photoadducts. Remarkably, the photoinduced strand cleavage is observed exclusively with G4 structures presenting BrU residues in lateral or diagonal loops, but not with parallel G4-DNA structures presenting only propeller loops. In contrast, the formation of fluorescent photoadducts is observed with all BrU-substituted G4-DNA substrates, with M-2PY giving significantly higher yields (up to 27%) than M-1PY. Both ligand-sensitized photoreactions are specific to BrU-modified G4-DNA structures with respect to double-stranded or stem-loop substrates. Thus, ligand-sensitized photoreactions with BrU-substituted G4-DNA may be exploited (i) as a photochemical probe, allowing “photofootprinting” of G4 folding topologies in vitro and (ii) for covalent trapping of G4 structures as photoadducts with pyrene-substituted ligands.
Highlights
NH2 PY-NH2 respectively, represent promising chemical biology tools for identification, isolation, and mapping of these motifs in the genome
In the context of G4 DNA structures, Sugiyama et al performed photochemical probing of IU-modified G4-DNA formed by the human telomeric sequence, d[AGGG(TTAGGG)3], and demonstrated that only the antiparallel G4 structure formed in Na+ conditions efficiently underwent the photoreaction
We investigated the ligand-sensitized photochemical reactions of BrU-modified G4-DNA belonging to different G-quadruplex folding topologies
Summary
NH2 PY-NH2 respectively, represent promising chemical biology tools for identification, isolation, and mapping of these motifs in the genome. Besides direct photoexcitation of 5-halouracil residues with UVB light, generation of the uracil-5-yl radical may be achieved through an electron transfer reaction of BrU with a photo-excited chromophore, typically pyrene, serving as electron donor, or “injector”[28] Such ligand-sensitized photochemical probing is advantageous, since the chromophore can be selectively and efficiently excited with a lower-energy UVA light (e.g., λ = 365 nm for pyrene), thereby minimizing the non-specific DNA damage. We studied ligand-sensitized photochemical reactions of BrU-substituted G4-DNA structures belonging to different folding topologies (antiparallel, parallel, or hybrid) Towards this end, we employed two pyrene-substituted derivatives of the bis-naphthalene macrocycle 2,6-BisNP (hereafter M), namely M-1PY and M-2PY (Fig. 1)[31], previously documented as affine and selective ligands for G4-DNA formed by the human telomeric sequence[32]. Using various DNA sequences, we show that these photoreactions may lead to different products (strand cleavage and covalent photoadduct), whose distribution is specific to the topology of the G4 substrate
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