Department of Chemistry & Centre for Research on Biomolecular Interactions, York University, 4700 Keele St., Toronto, Ontario, M3J 1P3, Canada.
School of Materials Science and Engineering (MSE), Division of Materials Technology, Nanyang Technological University (NTU), Singapore, 639798, Singapore.
Talanta. 2020 Sep 1;217:121022. doi: 10.1016/j.talanta.2020.121022. Epub 2020 Apr 11.
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 (Co) 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) °C compared to the unconjugated aptamer while the free form of the structure-switching aptamer-stilbene conjugate remains unfolded.
光致变色适体开关分析（PHASA）依赖于适体与配体的结合，从而改变与适体 5' 端共价连接的二苯乙烯化合物的局部环境。我们使用具有结构切换和非结构切换可卡因结合适体的 PHASA。我们表明，使用结构切换可卡因结合适体可获得最大的荧光强度变化和最低的检测限（Co）。荧光各向异性测量用于定量共轭适体与可卡因的亲和力。我们还使用热融解分析和核磁共振（NMR）光谱来表明，与未共轭适体相比，二苯乙烯的添加使与可卡因结合的结构切换适体的融解温度升高了 (6.4 ± 0.3) °C，而结构切换适体-二苯乙烯的游离形式仍然未折叠。
