The C-10–C-4a bond cleavage
of anthraquinone is believed
to be a crucial step in fungal seco-anthraquinone biosynthesis and
has long been proposed as a classic Baeyer–Villiger oxidation.
Nonetheless, genetic, enzymatic, and chemical information on ring
opening remains elusive. Here, a revised questin ring-opening mechanism
was elucidated by in vivo gene disruption, in vitro enzymatic analysis, and 18O chasing
experiments. It has been confirmed that the reductase GedF is responsible
for the reduction of the keto group at C-10 in questin to a hydroxyl
group with the aid of NADPH. The C-10–C-4a bond of the resultant
questin hydroquinone is subsequently cleaved by the atypical cofactor-free
dioxygenase GedK, giving rise to desmethylsulochrin. This proposed
bienzyme-catalytic and dioxygenation-mediated anthraquinone ring-opening
reaction shows universality.
Clustered regularly interspaced short palindromic repeats
(CRISPR)-based
assays have been an emerging diagnostic technology for pathogen diagnosis.
In this work, we developed a polydisperse droplet digital CRISPR-Cas-based
assay (PddCas) for the rapid and ultrasensitive amplification-free
detection of viral DNA/RNA with minimum instruments. LbaCas12a and
LbuCas13a were used for the direct detection of viral DNA and RNA,
respectively. The reaction mixtures were partitioned with a common
vortex mixer to generate picoliter-scale polydisperse droplets in
several seconds. The limit of detection (LoD) for the target DNA and
RNA is approximately 100 aM and 10 aM, respectively, which is about
3 × 104–105 fold more sensitive
than corresponding bulk CRISPR assays. We applied the PddCas to successfully
detect severe acute respiratory syndrome coronavirus (SARS-CoV-2)
and human papillomavirus type 18 (HPV 18) in clinical samples. For
the 23 HPV 18-suspected cervical epithelial cell samples and 32 nasopharyngeal
swabs for SARS-CoV-2, 100% sensitivity and 100% specificity were demonstrated.
The dual-gene virus detection with PddCas was also established and
verified. Therefore, PddCas has potential for point-of-care application
and is envisioned to be readily deployed for frequent testing as part
of an integrated public health surveillance program.
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