Intra-molecular G-quadruplex structure generated by DNA-templated click chemistry: "turn-on" fluorescent probe for...

A novel homogenous fluorescent sensor for signal-on detection of Cu(2+) has been developed based on intra-molecular G-quadruplex formed by DNA-templated click reaction and crystal violet (CV) as label-free signal reporter. The clickable DNA probe consists of two G-rich strands (A and B) bearing azide and alkyne group, respectively, and a template strand (C) locating two proximate reactants by pairing with A and B. The sequences of A and B are derived from asymmetric split of the G-quadruplex sequence (TTAGGG)4. In the presence of Cu(2+), the whole G-quadruplex sequence A-B is generated by chemical ligation of A and B via copper ion-catalyzed alkyne-azide cycloaddition, then released from template by toehold strand displacement, and consequently forming a stable intra-molecular G-quadruplex, which binds with CV to generate a strong fluorescent signal. Oppositely, weak fluorescence was obtained without Cu(2+) because of unstable intermolecular G-quadruplex formed by A and B and lack of lateral loop connection. Therefore, the Cu(2+) can be sensitively and specifically detected by the fluorescence of the CV-stained G-quadruplex with a low detection limit of 65nM and a linear range of 0.1-3µM. This method rationally integrated the DNA-templated synthesis and G-quadruplex structure-switch, presenting a simple and promising approach for biosensor development.