Methylene Blue as a G-Quadruplex Binding Probe for Label-Free Homogeneous Electrochemical Biosensing

Abstract

Herein, G-quadruplex sequence was found to significantly decrease the diffusion current of methylene blue (MB) in homogeneous solution for the first time. Electrochemical methods combined with circular dichroism spectroscopy and UV-vis spectroscopy were utilized to systematically explore the interaction between MB and an artificial G-quadruplex sequence, EAD2. The interaction of MB and EAD2 (the binding constant, K ≈ 1.3 × 10(6) M(-1)) was stronger than that of MB and double-stranded DNA (dsDNA) (K ≈ 2.2 × 10(5) M(-1)), and the binding stoichiometry (n) of EAD2/MB complex was calculated to be 1.0 according to the electrochemical titration curve combined with Scatchard analysis. MB was proved to stabilize the G-quadruplex structure of EAD2 and showed a competitive binding to G-quadruplex in the presence of hemin. EAD2 might mainly interact with MB, a positive ligand of G-quadruplex, through the end-stacking with π-system of the guanine quartet, which was quite different from the binding mechanism of dsDNA with MB by intercalation. A novel signal read-out mode based on the strong affinity between G-quadruplex and MB coupling with aptamer/G-quadruplex hairpin structure was successfully implemented in cocaine detection with high specificity. G-quadruplex/MB complex will function as a promising electrochemical indicator for constructing homogeneous label-free electrochemical biosensors, especially in the field of simple, rapid, and noninvasive biochemical assays.

Investigation of the interactions between methylene blue and intramolecular G-quadruplexes: an explicit distinction in electrochemical behavior

“…This agrees with the prediction in our previous work. 13 The mixed hybrid G-quadruplexes fall into the antiparallel and parallel regions, and the gap between parallel conformation zone and antiparallel conformation zone was not large. This phenomenon is not consistent with the terminal interaction rule observed from the interaction between Gquadruplexes and hemin completely, which can bind with Gtetrad plane of four guanines in G-quadruplex via end stacking.…”

“…Our results are not consistent with the previous assumption that the binding site of MB with G-quadruplex is the same as that of hemin, a porphyrin molecule, with G-quadruplex. Hemin shows a more obvious ability to distinguish G-quadruplexes with different conformations 13,24 We therefore propose that MB possibly stacks with intramolecular G-quadruplexes by interacting with up to two guanines because of its molecular size, which is different with intermolecular G-quadruplexes. 23 These results were validated by circular dichroism (CD), molecular docking and isothermal titration calorimetry (ITC).…”

“…11 In addition to the applications in oncology, quadruplex ligands are also implemented in the field of biosensors, fluorescence imaging and even assembly of nanostructures. [12][13][14] Therefore, the study of quadruplex ligands has been gaining in importance. Electrochemical method, a simple and fast technique, has been used as a good tool to study G-quadruplexes and screen of G-quadruplex ligands.…”

“…18 MB can also act as a G-quadruplex binding probe because of its stronger binding with G-quadruplex over DNA double strands. 13 Until now, the interaction studies of MB and Gquadruplexes are mainly focused on a limited number of specific Gquadruplex-forming sequences and the studies of G-quadruplexes interaction with small molecules were usually carried out by nuclear magnetic resonance (NMR), mass spectroscopy (MS) and X-ray diffraction that require expensive equipment and considerable experimental time. [19][20][21] Here, we made use of the intrinsic electrochemical property of MB to systematically explore its binding mechanisms with a series of common intramolecular G-quadruplex sequences by electrochemical method, which has distinct advantages of simplicity, miniaturization, rapid response, low cost and that o l μL solutio as e ui ed fo the e pe i e ts p ese ted here.…”

“…[19][20][21] Here, we made use of the intrinsic electrochemical property of MB to systematically explore its binding mechanisms with a series of common intramolecular G-quadruplex sequences by electrochemical method, which has distinct advantages of simplicity, miniaturization, rapid response, low cost and that o l μL solutio as e ui ed fo the e pe i e ts p ese ted here. Compared with other works, 13,22,23 we used electrochemical method to distinguish the difference for binding affinities of MB with different intramolecular Gquadruplexes, which greatly enriched the study range of Gquadruplexes. From the electrochemical results, we infer that the ability of MB interacting with intramolecular Gquadruplexes depends on conformations and sequence characteristics.…”