Hybridization chain reaction (HCR) is a DNAbased target-induced cascade reaction. Due to its unique enzymefree amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.
A two-dimensional nanoparticle–single-stranded
DNA (ssDNA)
array has been assembled for the detection of bacterial species using
machine-learning (ML) algorithms. Out of 60 unknowns prepared from
bacterial lysates, 54 unknowns were predicted correctly. Furthermore,
the nanosensor array, supported by ML algorithms, was able to distinguish
wild-type Escherichia coli from its mutant by a single
gene difference. In addition, the nanosensor array was able to distinguish
untreated wild-type E. coli from those treated with
antimicrobial drugs. This work demonstrates the potential of nanoparticle–ssDNA
arrays and ML algorithms for the discrimination and identification
of complex biological matrixes.
The CRISPR-Cas12a nuclease shreds short single-stranded DNA (ssDNA) substrates indiscriminately through transcleavage upon activation with a specific target DNA. This shredding activity offered the potential for development of ssDNA-templated probes with fluorescent dye (F) and quencher (Q) labels. However, the formulations of double-stranded DNA (dsDNA)-templated fluorescent probes have not been reported possibly due to unknown (or limited) activity of Cas12a against short dsDNAs. The ssDNA probes have been shown to be powerful for diagnostic applications; however, limiting the probe selections to short ssDNAs could be restrictive from an application and probe diversification standpoint. Here, we report a dsDNA substrate (probe-full) for probing Cas12a trans-cleavage activity upon target detection. A diverse set of Cas12a substrates with alternating dsDNA character were designed and studied using fluorescence
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