Progressive fluorescence amplification of molecular rotors via G-run-length engineering of G-rich DNA scaffolds

Background G-quadruplexes (G4s) act as versatile scaffolds for biosensing, yet a generalizable strategy to systematically amplify the fluorescence of G-rich scaffold-bound molecular rotors remains lacking. Results Herein, we propose a G-run-length engineering strategy to enhance the “light-up” performance of fluorogenic ligands using Thioflavin T (ThT) as a model molecular rotor. Through rational design of a sequence library derived from EAD4 with variable G-run lengths (N = 2–6, where N denotes the number of consecutive guanines per G-tract), we show that increasing N progressively amplified ThT fluorescence by up to ∼1000-fold under metal-free conditions. Mechanistic investigations—including induced circular dichroism, thermal melting analysis, Job plots, isothermal titration calorimetry and hydroxylation kinetics—support that G-run extension increases ligand-accessible binding environments and shields bound ThT from the aqueous phase, thereby restricting intramolecular rotation and suppressing non-radiative decay. The enhanced shielding likely arises from strengthened terminal stacking and/or possible cavity-associated binding, although the present data do not define a single G4 topology or a unique microscopic binding mode. The G-run-length-dependent enhancement trend was further observed in derivative libraries based on different G-rich DNA scaffolds and with other molecular rotors, including Thiazole Orange and Auramine O, suggesting functional generalizability. By contrast, macrocyclic planar ligands such as hemin and N-Methyl Mesoporphyrin IX, which are dominated by terminal G-tetrad association rather than intramolecular rotation restriction, did not show the same trend. Significance These findings establish G-run-length engineering as a practical strategy for regulating G-rich scaffold–fluorophore interactions and provide mechanistic guidance for nucleic-acid-based fluorescence signal amplification.