Ligand Binding to Dynamically Populated G‐Quadruplex DNA

Abstract

Several small-molecule ligands specifically bind and stabilize Gquadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic.Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligandinduced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.

The different activities of RNA G-quadruplex structures are controlled by flanking sequences

“…In presence of PhenDC3 or PhenDH2, the CD intensity is slightly decreased indicating that interaction is occurring between the G4 conformation and the ligands ( Fig 1B ). Interestingly, when PhenDC3 was added before the annealing step, the shoulder at 300 nm was not observed ( Fig S1 ), thereby suggesting that the ligand shifts the equilibrium towards the parallel conformation as has been recently reported for other G4 sequences ( Aznauryan et al, 2021 ). Finally, we monitored the effects of PhenDH2 and PhenDC3 on the RNA sequences by CD melting.…”

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

“…In addition to the techniques here approached and used for investigating G4/ligand interactions, other robust and efficient biophysical, biochemical and molecular biology techniques are available to describe G4/ligand interactions, such as surface-enhanced Raman spectroscopy [ 223 ], single-molecule fluorescence imaging [ 97 , 224 ], equilibrium dialysis [ 225 ], gel electrophoresis [ 114 , 226 ], qPCR-stop assay [ 227 ], Taq polymerase stop assay [ 228 ] and TRAP assay [ 229 ]. Furthermore, other high-throughput methods are emerging, such as pull-down assays [ 230 ] and Affinity Selection-Mass Spectrometry (ALIS) [ 231 ].…”

Phen‐DC3 Induces Refolding of Human Telomeric DNA into a Chair‐Type Antiparallel G‐Quadruplex through Ligand Intercalation

“…[17,18] However, G4 polymorphism makes it challenging to understand and design specific recognition of particular structure(s) by ligands. [25][26][27] The polymorphism arises from differences in strand stoichiometry, position and conformation of the loops connecting Gs and mutual orientations of G-tracts in the core of the structure. [28] The archetypal polymorphic sequence is the human telomeric repeat sequence (TTAGGG) n , for which diverse (parallel, antiparallel and hybrid) intramolecular folding topologies have been identified (Figure 1).…”