School of Public Health, Hangzhou Medical College, Hangzhou, 310013, China.
Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, 310023, China.
Anal Chim Acta. 2023 Oct 2;1276:341592. doi: 10.1016/j.aca.2023.341592. Epub 2023 Jul 12.
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.
微 RNA（miRNA）是一种小 RNA 分子，可以作为癌症和其他疾病的诊断/预后生物标志物和治疗靶点发挥重要作用。在此，提出了一种用于 miRNA 高灵敏和多功能检测的识别-切割-扩增（ICA）策略，并成功应用于 miR-155 和 miR-21 的检测。它结合了与靶标 miRNA 结合的对齐器靶向介导切割与链置换扩增（ATMC-SDA），以实现 ICA 过程。在鉴定过程中，DNA 对齐器（DA）和 DNA 扩增子（DM）可以在靶标 miRNA 的帮助下结合在一起，形成 T 型结构。然后，结合在 DA 茎部识别序列上的切口内切酶（NEase）可以在 DM 上进行切割，而切割的 DM（CDM）可以作为引发物触发用于信号放大的 SDA 反应。所提出的 ATMC-SDA 共享相同的酶和引物组，可以作为用于各种 miRNA 高灵敏检测的多功能 ICA 策略，而无需逆转录。结果表明，miR-155 和 miR-21 的检测限（LOD）分别为 5.4 aM 和 6.8 aM，动态范围为 10.0 aM 至 10.0 pM。ATMC-SDA 与生物样品的兼容性也已通过使用人血清进行了测试，表明其具有广泛应用的巨大潜力。
