A turn-on fluorescent nucleoside enabling sequence-insensitive DNA labeling

Fluorescent nucleobase analogs (FBAs) are valuable tools for studying nucleic acid structure and dynamics. However, their utility is often limited by substantial fluorescence quenching upon incorporation into oligonucleotides and variable brightness influenced by neighboring bases. In this study, we present a novel turn-on nucleoside, 3b, a thiazolyl-dU analog (hereinafter referred as TzdU), engineered to overcome these limitations and enable reliable DNA fluorescence imaging. Compared to its nearly nonfluorescent free form, TzdU shows approximately a 10-fold increase in brightness in single-stranded DNA (ssDNA) and up to a 50-fold enhancement in double-stranded DNA (dsDNA). Importantly, it maintains relatively stable brightness regardless of surrounding bases by evading common quenching pathways, including solvent-induced collisional quenching and excited-state proton transfer (ESPT). The triphosphate derivative of TzdU is efficiently utilized by various DNA polymerases, including Deep Vent and KOD XL, facilitating real-time, intensity-based monitoring of critical enzymatic processes such as PCR and primer extension without external labels. Furthermore, TzdU can illuminate DNA in a gradient manner, enabling the visualization and encryption of information. As the first FBA to achieve universal turn-on characteristics, sequence insensitivity, and compatibility with enzymatic reactions, TzdU serves as a novel tool for investigating nucleic acid dynamics and advancing fluorescence-based methodologies.