Cavity hairpin ThT-light nucleic acid switches: the construction of label- and enzyme-free sensing and imaging platforms

Abstract

Thioflavin T (ThT) is a classical fluorescent dye gaining prominence in current research regarding nucleic acid conformations (NACs). However, most NACs with the ability to excite ThT fluorescent are unique or form in demanding conditions, limiting the extensiveness and depth of ThT application in sensing and imaging. Therefore, this study proposed CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS) with excellent fluorescence excitation over 500-fold higher than spontaneous, 17∼20-fold higher than ssDNA and 2.5∼5-fold higher than complementary duplex. Based on the excellent fluorescence excitation, convenient conformation formation, good sequence programmability, and flexible allosteric ability (known as the Worm-crack pod mechanism mediated by the target), it achieved the label- and enzyme-free detection of tetracycline (TET) and berberine (BB) at the pM level within 10 min. Moreover, it was found enable to realize the sensitive tracking of intracellular carriers at the nM level of ThT entry concentration, and prolongated its cell nuclear-entry time of ThT over 8 h, overcoming the non-specific high background signal interference of ThT in the nuclear region, and expanding the diversified application of ThT in cell biology research. Therefore, CHTLNAS is a more universal, practical tool than G-quadruplex or other kinds of NACs for ThT development and utilization in sensing and imaging platforms.

Topological effect of an intramolecular split G-quadruplex on thioflavin T binding and fluorescence light-up

“…26 In the last decade, ThT whose weak fluorescence at 492 nm also can be intensified upon binding to specific nucleic acids, 27–29 has been applied for detecting potassium ions, imaging intracellular nucleic acids and monitoring RNA metabolism in vivo . 29–31 For example, ThT triggered G4 folding in the 22AG human telomeric DNA, producing 2100-fold enhanced fluorescence, and exhibited high selectivity toward distinct G4 structures. 32 Although much progress has been made on nucleic acid recognition by ThT, it is still essential and significant to systematically investigate the topological effect of G4 on ThT binding and its fluorescence response.…”

An Effective Docking-Guided Strategy for Rational Tailoring of Fluorescent Aptamer Switches of Dimethylindole Red Analogue

“…Besides, the interaction mechanism of the DIR-aptamer pair is so far uncertain. Nowadays, bioinformatics tools such as molecular docking provide a cost-effective, time-saving, and reliable strategy for modeling aptamer structures and predicting aptamer binding ability to targets, which provide valuable information for the discovery and optimization of high-performance aptamers. − Thus, supported by bioinformatics approaches, a feasible and efficient strategy for synchronously and rationally adjusting the DIR chemical structure and the DIR aptamer performance is urgent needed for strengthening the availability and filling in the gaps of performance upgrades.…”