期刊
TALANTA
卷 200, 期 -, 页码 424-431出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2019.03.080
关键词
Thrombin binding aptamer; Jatrorrhizine; Electrospray ionization mass spectrometry; Fluorescence spectroscopy; Noncovalent interaction
资金
- Natural Sciences and Engineering Research Council (NSERC) of Canada
- National Natural Science Foundation of China [21475061]
- Priority Academic Program Development of Jiangsu Higher Education Institution of China
A 15-mer thrombin-binding aptamer (TBA) was discovered with specificity for thrombin. It forms a unique G-quadruplex (G4), which is postulated to be the molecular basis for its binding specificity. Many analytical methods make use of affinity binding between the thrombin and TBA as they form a very stable complex. We develop a strategy to stabilize TBA/G4's structure by introducing G4-interactive molecules, which may enhance its ability to recognize the target. Herein, a fast screening ESI-MS assay was employed to determine potential binding of natural products molecules with the TBA/G4 complex. The experimental results showed that four investigated natural alkaloids had apparent binding affinities. One of them, jatrorrhizine (L1), has been shown to bind strongly to the TBA/G4 mainly in 1:2 M ratio. Once the working conditions were established, the interaction of the jatrorrhizine with the TBA/G4 was explored using a combination of ESI-MS and spectroscopic techniques. Ligand-induced effects on TBA/G4 structure and its stability were examined by means of circular dichroism (CD). Jatrorrhizine inducing the G4 formation seems also to be the more effective in terms of thermal stabilization under the experimental conditions used. Both results of UV and fluorescence experiments undoubtedly showed a good binding affinity with the binding constant around 10(5) L mol(-1). The stacking interactions of jatrorrhizine with the G-tetrads in TBA/G4 were further confirmed by competition experiment. ESI-MS was carried out to determine the coexistence of 1:1 and 1:2 complexes in TBA/G4-L1 system, and showed a dynamical shift from 1:1 to 1:2 complex in minutes.
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