期刊论文详细信息
TALANTA 卷:217
Analysis of the role played by ligand-induced folding of the cocaine-binding aptamer in the photochrome aptamer switch assay
Article
Shoara, Aron A.1,2  Churcher, Zachary R.1,2  Steele, Terry W. J.3  Johnson, Philip E.1,2 
[1] York Univ, Dept Chem, 4700 Keele St, Toronto, ON M3J 1P3, Canada
[2] York Univ, Ctr Res Biomol Interact, 4700 Keele St, Toronto, ON M3J 1P3, Canada
[3] Nanyang Technol Univ NTU, Sch Mat Sci & Engn MSE, Div Mat Technol, Singapore 639798, Singapore
关键词: Aptamer;    Photochrome aptamer switch assay;    Fluorescence anisotropy;    Stilbene;    NMR spectroscopy;    UV melt;   
DOI  :  10.1016/j.talanta.2020.121022
来源: Elsevier
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【 摘 要 】

The Photochrome Aptamer Switch Assay (PHASA) relies on ligand binding by an aptamer to alter the local environment of a stilbene compound covalently attached to the 5' end of the aptamer. We used the PHASA with both structure switching and non-structure switching versions of the cocaine-binding aptamer. We show that the largest change in fluorescence intensity and the lowest concentration limit of detection (C(Lo)o(D)) is obtained using the structure-switching cocaine-binding aptamer. Fluorescence anisotropy measurements were used to quantify the affinity of the conjugated aptamer to cocaine. We also used thermal melt analysis and Nuclear Magnetic Resonance (NMR) spectroscopy to show that the addition of the stilbene to the aptamer increases the melt temperature of the cocaine-bound structure-switching aptamer by (6.4 +/- 0.3) degrees C compared to the unconjugated aptamer while the free form of the structure-switching aptamer-stilbene conjugate remains unfolded.

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