Simple and High-Throughput Fluorescence Assay Method for DNA Damage Analysis in Single-Stranded DNA-Encoded Library
Gyung A Kim1, Seungyoon Kang1, Myo Naing Win1
1Department of Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea.
Bioconjugate Chemistry
|July 8, 2025
Summary
A new fluorescent assay quantifies single-stranded DNA (ssDNA) damage during ssDNA-encoded library synthesis. This method enables rapid screening of reaction conditions to ensure DNA integrity and optimize library production.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Chemistry
Background:
- Single-stranded DNA (ssDNA) is crucial for encoded library synthesis.
- Assessing ssDNA integrity under various reaction conditions is vital for successful library construction.
- Existing methods may not be suitable for high-throughput screening of ssDNA stability.
Purpose of the Study:
- To develop a simple, high-throughput assay for quantifying DNA damage in single-stranded DNA-encoded library (ssDEL) synthesis.
- To evaluate the impact of common reaction conditions on ssDNA integrity.
- To facilitate the optimization of ssDEL synthesis protocols.
Main Methods:
- Development of a fluorescent dye-based assay using PicoGreen to measure ssDNA damage.
- Quantification of DNA hybridization changes as an indicator of ssDNA integrity.
- Evaluation of assay performance under varying pH, temperature, metal ions, and solvents.
- Cross-validation of results using high-performance liquid chromatography.
Main Results:
- The PicoGreen assay effectively quantifies ssDNA damage, reflecting ssDNA integrity.
- Certain conditions compatible with double-stranded DNA synthesis can cause significant damage to ssDNA.
- The assay's sensitivity to factors like pH, temperature, and chemical environment was demonstrated.
- ssDNA exhibits lower chemical stability compared to double-stranded DNA under tested conditions.
Conclusions:
- The developed fluorescent assay is a simple and rapid tool for assessing ssDNA integrity during ssDEL synthesis.
- This method allows for high-throughput screening of reaction conditions, crucial for optimizing ssDEL production.
- Understanding ssDNA stability under diverse conditions is essential for improving encoded library synthesis strategies.
