Abstract
G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level.
Highlights
Nucleic acid sequences with four or more stretches of multiple guanines (GnNxGnNxGnNxGn; n ≥ 2, x ≤ 7) can form non-canonical four-stranded structures called Gquadruplexes (G4s)
To decipher G4 folding/unfolding dynamics at the molecular level, in a context where G4 structure exists in competition with a dsDNA structure, we developed a single molecule assay based on manipulation of DNA hairpin under a magnetic tweezer setup [54]
To previous single-molecule measurements that were performed at constant forces and without the presence of a complementary strand [39,40,41,42,43,44,45], our assay allows in vitro measurement of G4 folding and persistence times in a context that simulates the competitive environment of G4 structures formation in vivo
Summary
Nucleic acid sequences with four or more stretches of multiple guanines (GnNxGnNxGnNxGn; n ≥ 2, x ≤ 7) can form non-canonical four-stranded structures called Gquadruplexes (G4s). These structures are assembled by stacking of coplanar guanine’s quartets (G-quartets) that are stabilized by monovalent cations, such as K+ or Na+.
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