Zhang Xiaoyu, Kotikam Venubabu, Rozners Eriks, Callahan Brian P
Department of Chemistry, Binghamton University, the State University of New York, 4400 Vestal Parkway East, Binghamton, New York 13902, United States.
bioRxiv. 2021 Aug 31:2021.08.30.458287. doi: 10.1101/2021.08.30.458287.
Enzymatic beacons, or E-beacons, are 1:1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming DNA oligonucleotides equipped with a dark quencher. We prepared E-beacons biocatalytically using the promiscuous "hedgehog" protein-cholesterol ligase, HhC. Instead of cholesterol, HhC attached nanoluciferase site-specifically to mono-sterylated hairpin DNA, prepared in high yield by solid phase synthesis. We tested three potential E-beacon dark quenchers: Iowa Black, Onyx-A, and dabcyl. Prototype E-beacon carrying each of those quenchers provided sequence-specific nucleic acid sensing through turn-on bioluminescence. For practical application, we prepared dabcyl-quenched E-beacons for potential use in detecting the COVID-19 virus, SARS-CoV-2. Targeting the E484 codon of the SARS-CoV-2 Spike protein, E-beacons (80 × 10 M) reported wild-type SARS-CoV-2 nucleic acid at ≥1 × 10 M with increased bioluminescence of 8-fold. E-beacon prepared for the E484K variant of SARS-CoV-2 functioned with similar sensitivity. These E-beacons could discriminate their complementary target from nucleic acid encoding the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with specificity, detection sensitivity with E-beacons is two to three orders of magnitude better than synthetic molecular beacons, rivaling the most sensitive nucleic acid detection agents reported to date.
酶标信标，即E-信标，是纳米荧光素酶与在其C端共价连接的发夹状DNA寡核苷酸的1:1生物共轭物，该DNA寡核苷酸带有暗猝灭剂。我们使用混杂的“刺猬”蛋白-胆固醇连接酶HhC通过生物催化制备了E-信标。HhC不是连接胆固醇，而是将纳米荧光素酶位点特异性地连接到通过固相合成高产制备的单酯化发夹DNA上。我们测试了三种潜在的E-信标暗猝灭剂：爱荷华黑、玛瑙-A和达可昔。携带这些猝灭剂的原型E-信标通过开启生物发光提供序列特异性核酸传感。为了实际应用，我们制备了用于检测新冠病毒SARS-CoV-2的达可昔猝灭的E-信标。靶向SARS-CoV-2刺突蛋白的E484密码子，E-信标（80×10⁻⁶M）在≥1×10⁻⁶M时报告野生型SARS-CoV-2核酸，生物发光增加8倍。为SARS-CoV-2的E484K变体制备的E-信标具有相似的灵敏度。这些E-信标可以将其互补靶标与编码SARS-CoV-2卡帕变体E484Q突变的核酸区分开来。除了特异性，E-信标的检测灵敏度比合成分子信标高两到三个数量级，可与迄今为止报道的最灵敏的核酸检测试剂相媲美。
