Journal
TALANTA
Volume 217, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2020.121022
Keywords
Aptamer; Photochrome aptamer switch assay; Fluorescence anisotropy; Stilbene; NMR spectroscopy; UV melt
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Funding
- Ministry of Education [RG54/13]
- Natural Sciences and Engineering Research Council of Canada (NSERC)
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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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