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The dynamic binding status between the thrombin and its G-quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence-coupled capillary electrophoresis method. A 29-nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G-quadruplex from unstructured 29-nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6-carboxyfluorescein labeled G-quadruplex aptamer was measured. Third, the stability of G-quadruplex aptamer-thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29-nucleic acid thrombin binding aptamer could compete with G-quadruplex aptamer and thus disassociated the G-quadruplex structure into an unstructured aptamer. These data suggest that our in-house established fluorescence-coupled capillary electrophoresis assay could be applied to binding studies of the G-quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

HAT tag, a natural His affinity tag, is one of the most popular fusion tags. However, HAT tag containing three positive charges limited its self-assembly with quantum dots (QDs). Herein, ATTO 590-labeled HAT peptide was synthesized to self-assemble with QDs inside the capillary. QDs and ATTO 590-labeled HAT peptide were sequentially injected into the capillary and probed by fluorescence-coupled CE (CE-FL), showing an obvious Förster resonance energy transfer signal. Online self-assembly, separation, and detection were achieved within 10 min. CE-FL further revealed that imidazole and H 6 G 6 peptide could partially outcompete with HAT tag on the QDs surface inside the capillary. The displacement intermediates were separated clearly using CE-FL. Our study demonstrates the power of online CE-FL in analyzing the binding interaction between ligands containing positive charges and QDs., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)