Abstract Accurate single-nucleotide discrimination of miRNA is clinically vital because small sequence variations can have significant phenotypic and clinical consequences, yet existing techniques can only detect single nucleotide variations (SNVs) at specific loci. Here, we present a generalized peptide nucleic acid ( P NA) mediated C RI SPR/ C as13a syst e m (PRICE), enabling detection of SNVs in miRNA sequence without sacrificing the sensitivity. PRICE utilizes PNA blockers fully complementary to non-target miRNAs (e.g., miRNAs containing SNVs at loci of no interest) but not to the target miRNA. These blockers selectively hybridize with and inhibit non-target sequences in samples (serum, cells, or tissues). Only the unhybridized target miRNA then binds to crRNA within the Cas13a complex, activating Cas13a to cleave a fluorescent reporter-quencher linker, generating a detectable signal (~10 fM limit). By designing a panel of PNAs against SNVs, PRICE provides a versatile, amplification-free platform for precise miRNA analysis, advancing cancer diagnosis, prognosis, and biology.
Wang et al. (Thu,) studied this question.