Ligation-recognition triggered RPA-Cas12a cis-cleavage fluorogenic RNA aptamer for one-pot and label-free detection of MicroRNA in breast cancer

Abstract

"One-pot" assays which combine amplification with CRISPR/Cas12a system are in constant attracted for biosensors development. Herein, we present a one-pot isothermal assay that Ligation-recognition triggered Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a cis-cleavage (LRPA-CRISPR) fluorescent biosensor for sensitive, specific, and label-free miRNA detection. Firstly, we reveal the programmed double-stranded DNA amplicons, which utilized the ligation-recognition and polymerization to form and amplified by the RPA system. Meanwhile, we enabled exponential ligation-recognition triggered recombinase polymerase amplification of miRNA-21 sequences and exploited the cis-cleavage mechanism of Cas12a with transcription to generate functional Mango RNA for signal output. This assay can be completed within 40 min and can allow a limit of detection of 3.43 aM for miRNA-21 detection, owing to the RPA with transcription amplification and enables to product the functional Mango RNA aptamer by in vitro transcription that binds to the TO1-Biotin fluorogenic dye. Moreover, our method exhibits the advantages of self-supply crRNA, label-free, excellent specificity, and universal detection platform via the design of one-pot detection in serum and cell samples, showing tremendous potential in biomarkers diagnostics of breast cancer.

Keywords: Cas12a cis-cleavage; In vitro transcription; Label-free; Mango RNA aptamer; One-pot; miRNA-21.