Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid.
Chembiochem
; 23(4): e202100594, 2022 02 16.
Article
em En
| MEDLINE
| ID: mdl-34890095
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 â
M) reported wild-type SARS-CoV-2 nucleic acid at ≥1×10-9 â
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
SARS-CoV-2
Idioma:
En
Revista:
Chembiochem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Estados Unidos