Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid**
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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.
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SciScore for 10.1101/2021.08.30.458287: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: …
SciScore for 10.1101/2021.08.30.458287: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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