Target recycling-triggered polymerization/isomerization amplification cascades for sensitive microRNA assay

Abstract

Highly sensitive quantification of microRNA biomarkers is useful for the prediction and diagnosis of various diseases. Here, a powerful and cascaded signal amplification strategy by integrating target recycling and polymerization/isomerization cyclic amplification (PICA) with the CRISPR/Cas12a system is established for sensitive fluorescent microRNA-215 detection. The elaborately designed variable primer hybridizes with microRNA-215 to trigger the target recycling process for the formation of many hairpins with the assistance of the reverse transcriptase. Aided by the DNA polymerase and betaine, subsequent PICA reaction of the hairpins is initiated for the yield of many long ssDNAs with multiple repeated sequences, which bind and activate the trans-cleavage activity of Cas12a/crRNA to digest fluorescently quenched ssDNA reporter sequences for the production of drastically amplified signals, enabling the detection of microRNA-215 with a low detection limit of 2.1 pM. In addition, the sensing method can selectively distinguish microRNA-215 against other interfering substances and achieve the monitoring of low levels microRNA-215 in diluted serums, indicating its promising potential for sensing various microRNA biomarkers at trace levels.