RESUMEN
Most G-quadruplex (G4)-targeting ligands reported so far contain planar heteroaromatic groups and can intercalate into adjacent base pairs of double-stranded DNA (dsDNA). However, quantitative data on the binding number γ (ligands/bp) of G4 ligands that intercalate into long dsDNA remain lacking, which are essential for understanding the selectivity of G4 ligands. Here, using a single-molecule stretching assay based on the lengthening of dsDNA, we analyzed the dissociation constants and the binding number of eight most commonly used G4 ligands that intercalate into dsDNA. We showed that five ligands (CX-5461, BRACO-19, RHPS4, TrisQ, and Phen-DC3) intercalate into dsDNA avidly (Kd = 0.5-2.1 µM, saturated γ > 0.2 ligands/bp), which was similar to the typical dsDNA intercalator EB. Two bisquinolines, PDS and 360A, showed moderate intercalation ability (Kd = 22.5 and 48.7 µM) and γ < 0.01 ligands/bp in the presence of 1 µM ligands. Porphyrin NMM showed no intercalative binding even at 200 µM. Molecular docking and molecular dynamics simulations were carried out to further evaluate the intercalative binding of these G4 ligands with dsDNA by calculating the binding energies and π-π stacking probability.
Asunto(s)
ADN , G-Cuádruplex , Simulación del Acoplamiento Molecular , Ligandos , ADN/química , Simulación de Dinámica MolecularRESUMEN
Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.