A fluorescence biosensor for detecting LncRNA MALAT1 based on isothermal amplification by cyclic extension

文献类型： 外文期刊

第一作者： Xue, Shixing

作者： Xue, Shixing;Zhang, Xinyue;Han, Leng;Dai, Enyong;Bu, Shengjun;Jiayu, Wan;Sun, He;Zhang, Xingdong;Zhang, Xiaoying

作者机构：

关键词： Novel isothermal amplification; Biosensor; CRISPR-Cas12a; Non-coding RNA; LncRNA MALAT1; Tumor biomarkers

期刊名称：ANALYTICA CHIMICA ACTA （ 影响因子：6.0； 五年影响因子：5.7 ）

ISSN： 0003-2670

年卷期： 2025 年 1357 卷

页码：

收录情况： SCI

摘要： Background: Long non-coding RNA (lncRNA) Metastasis-Associated Lung Adenocarcinoma Transcript (MALAT1), a crucial regulator of gene expression, has emerged as a highly promising biomarker in the progression of various cancers. The clinical detection of lncRNA MALAT1 primarily relies on Reverse TranscriptionPolymerase Chain Reaction (RT-PCR), which requires skilled operators and large, expensive thermal cycling equipment. These limitations have restricted the application of RT-PCR, particularly in resource-constrained settings. Results: In this study, we developed a novel signal amplification method, termed Isothermal Amplification by Cyclic Extension (IACE), based on the linear extension of a single-stranded DNA probe. IACE operates through the continuous extension of Probe 1 (a) into long single-stranded DNA with multiple repetitive sequences, facilitated by Probe 2 (a*a*) and Bst DNA polymerase. We found that the single-stranded DNA product of IACE could directly activate the CRISPR-Cas12a system without requiring a protospacer adjacent motif (PAM). By integrating IACE with a three-way junction structure and a nicking enzyme, we established a one-step signal amplification strategy for the detection of lncRNA MALAT1, achieving a detection limit as low as 37.5 fM using the CRISPR-Cas system. Significance: The biosensor developed in the present study simplifies workflows, minimizes contamination risks, and demonstrates exceptional detection performance in tumor patient samples, highlighting its potential advance clinical tumor diagnostic approaches.

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