Simple and High-Throughput Fluorescence Assay Method for DNA Damage Analysis in Single-Stranded DNA-Encoded Library Synthesis.

The fluorescent dye-based simple and high-throughput DNA damage assay method was developed for the single-stranded DNA-encoded library (ssDEL) synthesis. The extent of DNA hybridization between single-stranded DNA (ssDNA) and its complementary DNA is influenced by the damage ratio of ssDNA. This damage can be quantified by measuring the change in the fluorescence of PicoGreen, providing valuable insights into the overall ssDNA integrity and facilitating the screening of DNA-compatible conditions for ssDEL synthesis. The assay performance under various conditions, such as pH, temperature, metal ions, and solvents commonly used in ssDEL synthesis, was evaluated, and the results were cross-checked using high-performance liquid chromatography. While certain conditions have been identified as compatible with double-stranded DNA-encoded library synthesis using headpiece DNA, they may still cause considerable DNA damage during ssDEL synthesis due to the low chemical stability of ssDNA. Given its simplicity and speed, this assay can facilitate high-throughput screening of multiple reaction conditions during the early stages of ssDEL synthesis.