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 Ebeacons specific for SARS-CoV-2. Targeting the E484 codon of the virus Spike protein, E-beacons (80 × 10 À 12 M) reported wildtype SARS-CoV-2 nucleic acid at � 1 × 10 À 9 M by increased bioluminescence of 8-fold. E-beacon prepared for the SARS-CoV-2 E484K variant functioned with similar sensitivity. Both Ebeacons 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.

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

“…Detect adenosine [163] /distinguish classical rabies virus (genotype 1) from European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real-time [164] /detect BRAF mutation from circulating free DNA (cfDNA) [165] OQ A � 515 450-550 -/-FAM/DABCYL Study the stability of LVs [167] /detect E484Q mutation in the SARS-CoV-2 variant [168] OQ B Cy3/fluorescein Monitor ATP in the mitochondria [171] /detect aptamer ligands and DNAs by the Boolean logic functions and DNA logic gates [172] Iowa Black RQ 645 500-800 44 506 Cy5.5/TYE 665 (293, 800; 665) Monitor MB structural changes [170] /evaluate anti-CMV and anti-HSV-2 antibodies [173] ZEN-Iowa Black FQ -/-480-580 -/-FAM Detect deoxyribonucleoside triphosphates (dNTP) [182] /SARS-CoV-2 [54b] and Porphyromonas gingivalis (Pg, Gram-negative, pathogenic bacterium, mainly causes periodontosis) [183] ZEN-Iowa Black RQ -/-520-680 -/--/--/-sensing platforms. In addition to small-molecule quenchers, there are various materials that can be used as donors and acceptors in FRET systems, which significantly extend the selection of quenchers and expand the applicability of FRET-based assays.…”

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

“…Detect adenosine [163] /distinguish classical rabies virus (genotype 1) from European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real-time [164] /detect BRAF mutation from circulating free DNA (cfDNA) [165] OQ A � 515 450-550 -/-FAM/DABCYL Study the stability of LVs [167] /detect E484Q mutation in the SARS-CoV-2 variant [168] OQ B Cy3/fluorescein Monitor ATP in the mitochondria [171] /detect aptamer ligands and DNAs by the Boolean logic functions and DNA logic gates [172] Iowa Black RQ 645 500-800 44 506 Cy5.5/TYE 665 (293, 800; 665) Monitor MB structural changes [170] /evaluate anti-CMV and anti-HSV-2 antibodies [173] ZEN-Iowa Black FQ -/-480-580 -/-FAM Detect deoxyribonucleoside triphosphates (dNTP) [182] /SARS-CoV-2 [54b] and Porphyromonas gingivalis (Pg, Gram-negative, pathogenic bacterium, mainly causes periodontosis) [183] ZEN-Iowa Black Moreover, a specific biological event may occur simultaneously with two or more biomolecules. Therefore, the detection of multiple biochemical reactions by one probe is expected to provide a better understanding of complex biological processes.…”

“…This sophisticated design can accurately control DNA release and be used to expand the availability of DNA nanotechnology, improving its application in the fields of bioimaging, biosensing, and diagnosis. OQs have also been paired with FAM, DABCYL, and Cy3 in various biological applications, such as optimization for lentiviral vectors (LVs), [167] detecting E484Q mutation in the SARS‐CoV‐2 variant, [168] and real‐time monitoring of MB conformational changes [169] …”

Synthesis of Protein-Oligonucleotide Conjugates

“…Other classes of enzymes used for protein oligonucleotide conjugation rely on the transfer of post-translational modification. For example, a protein farnesyl transferase (PFTase) can transfer an oligonucleotide labelled with farnesyl pyrophosphate to the cysteine residue of a CVIA consensus sequence [ 37 ], while Hedgehog autoprocessing domains can transfer a steroid labelled oligonucleotide to a fused protein of interest, followed by self-cleavage [ 44 , 45 ].…”

“…The authors were able to hijack this reactivity to allow for the C-terminal functionalization of proteins with so called “steramers” (sterylated DNA), thereby providing an enzymatic means of conjugating nucleic acids to proteins. The authors exploit this system in a subsequent publication [ 44 ] for the detection of mutations within the spike protein of SARS-CoV2. This is facilitated through the production of an enzymatic beacon (E-beacon), through the conjugation of a conventional molecular beacon architecture bearing a quencher moiety (Q), to the ATP-independent bioluminescent nanoluciferase Nluc.…”