The variations of microRNA (miRNA)
expression can be valuable biomarkers
in disease diagnosis and prognosis. However, current miRNA detection
techniques mainly rely on reverse transcription and template replication,
which suffer from slowness, contamination risk, and sample loss. To
address these limitations, here we introduce a cascade toehold-mediated
strand displacement reaction (CTSDR) and CRISPR/Cas12a trans-cleavage
for highly sensitive fluorescent miRNA sensing, namely CTSDR-Cas12a.
In this work, the target miRNA hybridizes with the terminal toehold
site of a rationally designed probe and subsequently initiates dynamic
CTSDR, leading to enzyme-free target recycling and the production
of multiple programmable DNA duplexes. The obtained DNA duplex acts
as an activator to trigger Cas12a trans-cleavage, generating significantly
amplified fluorescence readout for highly sensitive detection of the
miRNA target. Under the optimal conditions, the developed sensing
method can detect target miRNA down to 70.28 fM with a wide linear
range from 100 fM to 100 pM. In particular, by designing a set of
probes and crRNAs, we demonstrate its broad applicability for the
detection of six kinds of miRNAs with high sequence specificity. Furthermore,
the method can be satisfactorily applied to monitor miR-21 in total
RNA extracted from cells and clinical serum samples. Considering the
high sensitivity, specificity, universality, and ease of handling,
this strategy provides a great potential platform for the detection
of miRNA biomarkers in molecular diagnostic practice.
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