A sensitive fluorescent DNA hydrogel aptasensor based
on the self-assembly
of rolling circle amplification (RCA) products was developed for ochratoxin
A (OTA) detection in beer. A competitive binding mode of aptamer,
complementary sequence, and target was integrated into the DNA hydrogel
for OTA detection. The OTA aptamer first combined with the primer
to form the hybridized product. Then, in the presence of OTA, the
aptamer combined with OTA, which released the primer. The released
primer hybridized with the padlock probe to form a circular template,
and the RCA reaction was initiated by adding ligase, polymerase, and
dNTPs. The fluorescent DNA hydrogel was obtained by adding Cy3-dUTP
together with dNTPs, and the fluorescence (FL) intensity of the DNA
hydrogel was positively correlated with OTA concentration. Under the
optimal experimental conditions, the linear range of the relationship
varied from 0.05 ng/mL to 100 ng/mL with a detection limit for OTA
of 0.01 ng/mL. The fluorescent DNA hydrogel aptasensor showed good
specificity and stability in beer samples. Therefore, the fabricated
DNA hydrogel aptasensor shows considerable potential applications
in detecting OTA for food safety.
Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.
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