A chemical labeling method for selective conjugation to DNA 3′-phosphoglycolate termini

DNA strand breaks with chemically modified termini are a critical form of damage caused by ionizing radiation, environmental genotoxins, and therapeutic agents. Among them, 3′-phosphoglycolate (3′-PG) termini are particularly problematic, as they obstruct DNA repair and contribute to genome instability. Several indirect detection strategies have provided valuable insights into 3′-PG lesions; however, the lack of a method for direct and specific labeling of 3′-PG termini continues to limit detailed analyses of their distribution and repair in biological systems. We report a novel chemical labeling method that enables the selective conjugation of biotin to the 3′-PG terminus of synthetic oligonucleotides. The method involves carbodiimide-mediated activation of the 3′-PG carboxyl group using EDC·HCl (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), followed by coupling with Biotin-PEG11-Amine to form a stable amide bond. This reaction proceeds specifically at the 3′-PG terminus without modifying 3′-phosphate ends. Under the conditions used, the labeling efficiency was approximately 50%. Our results demonstrate the feasibility of chemically tagging 3′-PG ends with high specificity in vitro, providing a foundation for future studies aimed at visualizing and quantifying 3′-PG lesions in more complex biological systems.