Identification, cleavage, and amplification (ICA): A versatile strategy for highly sensitive detection of miRNA

Abstract

MicroRNAs (miRNAs) are small RNA molecules that can play important roles as diagnostic/prognostic biomarkers and therapeutic targets for cancers and other diseases. Herein, an identification-cleavage-amplification (ICA) strategy for highly sensitive and versatile detection of miRNA has been proposed, and successfully applied to miR-155 and miR-21 assays. It combines an aligner-target mediated cleavage with strand displacement amplification (ATMC-SDA) to achieve the ICA process. During the identification process, a DNA-aligner (DA) and a DNA-amplicon (DM) can bind together with the help of target miRNA, forming a T-junction structure. Then, a nicking endonuclease (NEase), binding on the recognition sequence at the stem part of DA, can make a cleavage on DM, and the cleaved DM (CDM) can serve as an initiator to trigger the SDA reaction for signal amplification. Sharing the same set of enzymes and primers, the proposed ATMC-SDA can serve as a versatile ICA strategy for highly sensitive detection of various miRNAs, without the requirement of reverse transcription. Results show that the limits of detection (LOD) for miR-155 and miR-21 are 5.4 aM and 6.8 aM, respectively, with a dynamic range from 10.0 aM to 10.0 pM. The compatibility of ATMC-SDA with biological samples has also been tested by using human serum, indicating a promising potential for a wide variety of applications.

Keywords: Aligner-target mediated cleavage; MicroRNA; Nucleic acid detection; Strand displacement amplification.