Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid

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.

Keywords: SARS-Cov-2; beacons; biosensors; hedgehog; nucleic acids.

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) modified 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 the Nluc conjugated hairpin oligonucleotide with complementary nucleic acid displaces the quencher (Q) from Nluc, 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 stain.

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 (Eb.1), dabcyl (Eb.2), and ONYX-A (Eb.3). 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. 1 hr or 3 hr incubation with increasing concentration of complementary target 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, 0.2 × 10⁻⁹ M for the 1 hr incubation and 0.11 × 10⁻⁹ M for the 3 hr 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). Samples were incubated for 3 hrs followed by bioluminescence mesurent. (C) Bioluminescence spectrum measured by RLU/sec 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.