Probing of G-Quadruplex Structures via Ligand-Sensitized Photochemical Reactions in BrU-Substituted DNA

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.

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes

“…First, a single BrU residue was introduced into the central loop of each CEB25 sequence (Figure A). As previously reported, T to BrU replacement does not affect significantly the G4 structure; it was nonetheless verified by CD measurement of the BrU-modified CEB25-L191 sequence (Figure S5). On the basis of the EMSA titration, the protein:DNA ratio was fixed at 50:1 for photoreaction to ensure formation of a significant amount of complexes while avoiding saturation that could minimize differences between the sequences (see Figure F).…”

“…Therefore, an alternative approach was applied, which is based on a wellestablished workflow for analyzing RNA protein complexes directly from solution samples. 53,54 Then direct trypsinization without DNA digestion should generate bulky DNA peptide adducts unlikely to be detected by LC-MS/MS. Under these conditions, comparison of the ratio of all peptides in the UV-irradiated solution versus the nonirradiated control solution should show specific peak depletion corresponding to cross-linked peptides.…”

Nucleic Acid Conformation Influences Postsynthetic Suzuki–Miyaura Labeling of Oligonucleotides

“…UV irradiation of 5-halouracil-containing ONs results in the formation of a uracil-5-yl radical, which in an atom-specific and conformation-dependent manner induces hydrogen abstraction and intrastand cross-links . Further, ligand-sensitized photochemical reactions with 5-bromouracil-modified GQ-forming ONs enabled the “photofootprinting” of GQ folding topologies . Per se this is not a labeling approach, and the end products including the deoxyribonolactone and side reactions such as strand cleavage limit the downstream utility of the ON products.…”

“…31 Further, ligandsensitized photochemical reactions with 5-bromouracil-modified GQ-forming ONs enabled the "photofootprinting" of GQ folding topologies. 32 Per se this is not a labeling approach, and the end products including the deoxyribonolactone and side reactions such as strand cleavage limit the downstream utility of the ON products. GQ-forming sequences have also been used as scaffolds for carrying out template-assisted azide− alkyne cycloaddition reactions.…”