TY - JOUR
T1 - Bioluminescent Protein-Inhibitor Pair in the Design of a Molecular Aptamer Beacon Biosensing System
AU - Moutsiopoulou, Angeliki
AU - Broyles, David
AU - Joda, Hamdi
AU - Dikici, Emre
AU - Kaur, Avinash
AU - Kaifer, Angel
AU - Daunert, Sylvia
AU - Deo, Sapna K.
N1 - Funding Information:
The authors would like to thank NIGMS (Grants R01GM114321 and R01GM127706) and the National Science Foundation (Grants CHE-1506740 and CBET-1841419) for funding support. S.D. thanks the Miller School of Medicine of the University of Miami for the Lucille P. Markey Chair in Biochemistry and Molecular Biology.
PY - 2020/6/2
Y1 - 2020/6/2
N2 - Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. In this work, we designed a conjugated anti-interferon-γ(IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, Gaussia luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γand provided an optimized, physiologically relevant detection limit of 1.0 nM. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems.
AB - Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. In this work, we designed a conjugated anti-interferon-γ(IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, Gaussia luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γand provided an optimized, physiologically relevant detection limit of 1.0 nM. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems.
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U2 - 10.1021/acs.analchem.0c00518
DO - 10.1021/acs.analchem.0c00518
M3 - Article
AN - SCOPUS:85087591167
VL - 92
SP - 7393
EP - 7398
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 11
ER -