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

Authors

Ruo-Yao Cui, Yu-Chao Cao, Yi-Xuan Gu, Ling-Li Zhao, Xu-Yang Shen, Ying Liu, Ying-Lin Zhou, Xin-Xiang Zhang

Published in

Analytica chimica acta. Volume 1421. Pages 346015. Nov 01, 2026. Epub Jul 31, 2026.

Abstract

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.
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.
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.

PMID:
42702446
Bibliographic data and abstract were imported from PubMed on 07 Sep 2026.

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