Objective 1:
To study in vitro the interplay between 8-oxoG, DNA topology (such as G-quadruplex or G4) and protein interactions to modulate gene expression, we have developed a label-free single-molecule assay to follow the dynamics of G4s and their interaction with proteins in double-stranded DNA (dsDNA) to get closer to their biological context (Tran PLT et al, Nucleic Acids Research, 2021 and Valle-Orero J, Tran PLT, et al, in preparation).
In our recent publication (Tran PLT, et al, Nucleic Acids Research, 2021), we have shown that G4s are much more persistent in the promoters and the replication origin than in human telomere repeats. In addition, we revealed that commonly used G4-specific-ligands and antibodies increase both the folding rate and the persistence of the G4 structures in this context. Our novel assay opens new perspectives for the measurement of G4 dynamics in dsDNA, which is critical to understand the role of G4 in gene regulation.
Besides their regulatory function, persistent G4s can be a roadblock for replication and transcription. Using magnetic tweezer system, we manage to visualize in real-time and calculate the time (~15s) for G4 structure resolution by Pif1 helicase (Valle-Orero J, Tran PLT, et al, in preparation).
Using the recently developed assays, we studied the effect of oxidative lesions on G4 formation and how 8-oxoG can affect protein binding. We showed that 8-oxoG disturbed G4-cMYC folding differently according to the position of the modification (Tran PLT et al., in preparation). Next, we will study how these modifications could favor or disturb protein binding (such as transcription factors or base excision repair (BER) enzymes). These results will enable to better understand the mechanism of how 8-oxoG modulates gene expression (its epigenetic function).
Objective 2:
To capture a specific genomic DNA fragment from yeast, we have designed and assembled in vitro CRISPR/Cas9 complexes guided to specifically cut away a given locus from the rest of the genome. We have successfully captured the inserted CEB25 fragment (a synthetic G-rich DNA fragment mimicking a G-rich human minisatellite repeat) from yeast genomic DNA and ligated it to the adapters to manipulate it under magnetic tweezers. Next, we will optimize it for higher organisms, including the human genome.
Objective 3:
We have already screened all existing commercial 8-oxoG antibodies for this objective and we have selected the best one that gives accurate results. 8-oxoG, as well as other modifications, such as 5-methyl cytosines or 6-methyl adenines, can be systematically and unambiguously identified on synthetic DNA molecules at single-nucleotide resolution using SIMDEQ (SIngle Molecule DEtection and Quantification) sequencing platforms (from Depixus) based on magnetic tweezers (Wang Z et al., Communications Biology, 2021). Next, we will use this technology to map genome-wide 8-oxoG at single-nucleotide resolution.
