Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid** |
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Authors: | Xiaoyu Zhang Dr Venubabu Kotikam Prof Eriks Rozners Prof Brian P Callahan |
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Affiliation: | Department of Chemistry, Binghamton University, The State University of New York, 4400 Vestal Parkway East Binghamton, New York, 13902 USA |
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Abstract: | Enzymatic beacons, or E-beacons, are 1 : 1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming ssDNA equipped with a dark quencher. We prepared E-beacons biocatalytically using HhC, the promiscuous Hedgehog C-terminal protein-cholesterol ligase. HhC attached nanoluciferase site-specifically to mono-sterylated hairpin oligonucleotides, called steramers. Three E-beacon dark quenchers were evaluated: Iowa Black, Onyx-A, and dabcyl. Each quencher enabled sensitive, sequence-specific nucleic acid detection through enhanced E-beacon bioluminescence upon target hybridization. We assembled prototype dabcyl-quenched E-beacons specific for SARS-CoV-2. Targeting the E484 codon of the virus Spike protein, E-beacons (80×10−12 M) reported wild-type SARS-CoV-2 nucleic acid at ≥1×10−9 M by increased bioluminescence of 8-fold. E-beacon prepared for the SARS-CoV-2 E484K variant functioned with similar sensitivity. Both E-beacons could discriminate their target from the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with mismatch specificity, E-beacons are two to three orders of magnitude more sensitive than synthetic molecular beacons. |
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Keywords: | beacons biosensors hedgehog nucleic acids SARS-Cov-2 |
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