Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid and Potential Utility for SARS-CoV-2 Detection

Abstract

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 ^-12 M) reported wild-type SARS-CoV-2 nucleic acid at ≥1 × 10 ^-9 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.

FIGURE 1.

Concept and biocatalytic preparation of prototype E-beacon (A) Scheme showing HhC catalyzed bioconjugation of Nluc (grey) to a hairpin-forming mono-sterylated oligonucleotide (steramer) equipped with a 3’ dark quencher (Q). Conjugation involves formation of an internal thioester (step 1), followed by steramer binding and acyl transfer to the sterol hydroxyl group (step 2). (B) General mechanism for turn-on nucleic acid detection by E-beacon, whereby hybridization of hairpin oligonucleotide with complementary nucleic acid displaces the quencher (Q) from the Nluc enzyme, enhancing bioluminescence signal. (C) E-beacon preparation monitored by SDS-PAGE. A fusion of Nluc with HhC is purified first by Ni-NTA chromatography, followed by size exclusion chromatography (SEC), then reacted with steramer (conjugation). E-beacon is isolated by agarose gel extraction. Nucleic acid was visualized by UV with GelRed staining.

FIGURE 2.. E-beacon proof of concept and sequence specific nucleic acid detection*

(A) Bioluminescence from Eb.1 increases in the presence of complementary oligonucleotide (green, n=48) compared to E-beacon samples mixed with noncomplementary oligonucleotide (red, n=48). (B) Results of “blinded test” of Eb.1 specificity indicate that Eb.1 distinguishes complementary oligonucleotide (green) from oligonucleotides containing 1–3 base mismatches. See Table 1 for sequence information. (C) Comparison of E-beacons with different quenchers: IowaBlack, dabcyl, and ONYX-A. Bioluminescence was measured after 10-minute incubation with complementary oligonucleotide (green, n=48) or noncomplementary oligonucleotide (red, n=48). *In A, B, and C, the E. beacon was present at 2 × 10⁻⁹ M final; oligonucleotide at 25 × 10⁻⁹ M; temperature, 25 °C; substrate, furimazine.

FIGURE 3.

E-beacons for SARS-CoV-2 (A) Bioluminescence readings of samples with Eb.19 (WT) after 10 min or 3 hr incubation with increasing concentration of complementary oligonucleotide. The curves show hyperbolic binding isotherms with calculated half maximum emission (EC₅₀) at 2.54 × 10⁻⁹ M of oligonucleotide using the 10 min incubation, and 0.11 × 10⁻⁹ M for the 3 incubation. (B) Single base-pair mismatch discrimination by E-beacon, Eb.19 (WT). Bioluminescence from Eb.19(WT), at 80 × 10⁻¹² M, is plotted as a function of increasing oligonucleotide concentration. E484 is the complementary target, E484K is single base pair mismatch, “random” is non-complementary (see Table 1). (C) Bioluminescence spectrum showing the selective detection of E484K oligonucleotide by E-beacon, Eb.19 (E484K). Oligonucleotides E484 and E484Q represent single base pair variants of wild type SARS-CoV-2 and Kappa SARS-CoV-2, respectively.