An exceptional fluorescence turn-on nucleoside: Lighting up single-stranded DNA with constant brightness regardless neighboring bases

Fluorescent nucleobase analogs (FBAs) have proven valuable for studying nucleic acid structure and dynamics. Regrettably, most FBAs exhibit reduced quantum yields when incorporated into DNA, particularly when neighboring residues are present. In this study, we introduce a turn-on nucleoside (thieno cyclopenta -dU, 3b) that increases the brightness of single-stranded oligonucleotides by approximately 10-fold compared to the free nucleoside, regardless of neighboring bases. Furthermore, an up to 50-fold increase in brightness is observed during duplex formation. To the best of our knowledge, compound 3b is the only turn-on type fluorescent nucleoside known to maintain a stable quantum yield after incorporation, and it can be well-accepted by DNA polymerases. These findings highlight the potential of turn-on FBAs for fluorescence sensing applications in enzymatic DNA synthesis and in vivo strand hybridization.