A variation in thiophene-extended nucleosides as microenvironment-sensitive fluorescent probes

ABSTRACT Fluorescence-coded nucleoside analogues, in which photophysical properties are rationally encoded into the nucleobase scaffold, offer powerful tools for probing nucleic acid structure and function in complex biological systems. However, the development of structurally tunable, environment-sensitive fluorescent nucleosides that retain high biocompatibility and versatility remains limited. To address this, we present a tunable platform of Thex T -based fluorescent nucleosides, developed by introducing electronically distinct donor groups. This modular design enables fine-tuning of their photophysical behavior, resulting in donor-dependent variations in solvatochromism, viscosity sensitivity, and quantum yield—hallmarks of fluorescent molecular rotors. aniThex T and mopThex T were synthesized and site-selectively incorporated into DNA without disrupting base-pairing fidelity or duplex stability, preserving their rotor-like characteristics within oligonucleotide strands. When embedded into the G4-forming aptamer AS1411, these probes exhibited fluorescence turn-on effects upon G4 folding and selective nucleolin binding. This enhancement was further evaluated using gel-shift assays, and live-cell imaging, where Thex T derivatives-labeled AS1411 enabled targeted visualization of nucleolin-overexpressing MDA-MB-231 cancer cells. Collectively, this work showcases our Thex T derivatives as versatile fluorogenic probes with broad applicability in nucleic acid structure analysis, biomolecular interaction mapping, and functional cellular imaging.