A tunable assay for modulators of genome-destabilizing DNA structures

Area of Science:

• Genetics and Molecular Biology
• Biochemistry
• Cancer Research

Background:

• Genomic instability hotspots frequently co-localize with non-B DNA structures like H-DNA.
• Non-B DNA sequences are prevalent at translocation breakpoints in human cancer, indicating their role in genetic instability.
• Understanding the mechanisms of non-B DNA-induced instability is crucial for developing targeted therapies.

Purpose of the Study:

• To develop a novel assay for detecting small molecules that modulate non-B DNA (specifically triplex/H-DNA) formation and stability.
• To utilize this assay as a tool for studying DNA structure-induced genetic instability.
• To discover novel ligands that can regulate genetic instability by targeting non-B DNA structures.

Main Methods:

• Development of a tunable Förster resonance energy transfer (FRET)-based assay.
• Incorporation of a fluorophore-quencher pair into an H-DNA-forming sequence from the human c-MYC oncogene.
• Dual-reporter functionality achieved by tuning triplex stability via buffer composition to simultaneously identify stabilizers and destabilizers.
• Assay validation using known ligands (BePI, coralyne, MCRa2) and screening of 320 custom compounds in a 384-well plate format.

Main Results:

• Successfully demonstrated the FRET assay's principle for detecting triplex/H-DNA stabilizers and destabilizers.
• Validated the assay's performance with known compounds and through a high-throughput screen of 320 custom molecules.
• Identified potential novel stabilizers and destabilizers of H-DNA structures.

Conclusions:

• The developed FRET assay is an effective tool for identifying ligands that modulate non-B DNA structures.
• This assay facilitates the study of DNA structure-induced genetic instability.
• Discovery of novel non-B DNA-targeting molecules holds potential for regulating genetic instability in human cancer genomes.