Fluorescence-Based Simple and Practical Assay Method for DNA Damage Analysis in DNA-Encoded Library Synthesis.

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• DNA-encoded libraries (DELs) are powerful tools for small molecule drug discovery.
• Assessing DNA integrity during DEL synthesis is critical for library quality and screening accuracy.
• Existing methods for DNA damage detection may not be suitable for DEL synthesis conditions.

Purpose of the Study:

• To develop and validate a novel fluorescent dye-based assay for detecting double-stranded DNA (dsDNA) damage.
• To apply this assay to identify DNA-damaging conditions during DEL synthesis.
• To confirm the assay's applicability in optimizing DEL construction protocols.

Main Methods:

• Development of a dsDNA-selective fluorescent dye assay.
• Validation using high-performance liquid chromatography (HPLC) analysis.
• Application to screen on-DNA amide coupling and N-arylation reactions.

Main Results:

• The assay accurately detects DNA damage under various DEL synthesis conditions.
• Specific ratios of organic solvents were identified as critical inducers of DNA damage.
• The assay successfully screened DNA-damaging conditions in amide coupling and N-arylation reactions.

Conclusions:

• The developed fluorescent dye assay is a reliable tool for monitoring DNA integrity in DEL synthesis.
• Understanding and controlling DNA damage is essential for robust DEL library generation.
• This assay facilitates the optimization of chemical reactions used in DEL construction, improving library quality.