An innovative “double-locked” CRISPR/Cas12a system based on DNAzyme for the precise imaging of microRNAs in living cells

IF 7.7 1区化学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

International Journal of Biological Macromolecules Pub Date : 2025-04-28 DOI:10.1016/j.ijbiomac.2025.143605

Lu Lu, Shihua Gan, Shixiu Xiao, Xiaomei Mu, Shulin Zhao, Jianniao Tian

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引用次数: 0

Abstract

We constructed a “double-locked” CRISPR/Cas12a system based on DNAzyme for miRNA detection and precise imaging. One lock function to close the catalytic activity of DNAzyme, while the other lock serves to inhibit the cleavage function of CRISPR-induced RNA (crRNA). This “double-lock” mechanism ensures that the system is inhibited in the absence of the target molecule miRNA-141, effectively reducing the background signal. When the target miRNA-141 is present, the “lock” of DNAzyme is opened, and DNAzyme further opens the “lock” of crRNA, which activates the trans-cleavage ability of Cas12a on F-Q probe, and the fluorescence signal is restored. The linear range of miRNA-141 was 50 pmol/L ~ 15 nmol/L, and the detection limit was 47 pmol/L (S/N = 3). The system has been successfully applied to detect miRNA-141 expression levels in cell lysates. Meanwhile, this method can be applied for intracellular miRNA-141 fluorescence imaging and fluctuations in intracellular miRNA-141 expression. Overall, this strategy not only offers new prospects for programmable Cas12a detection systems, but also provides new insights for early diagnosis and screening of diseases by combining this in vitro assay with live cell imaging analysis for the detection of cancer markers.

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基于DNAzyme的创新“双锁”CRISPR/Cas12a系统，用于活细胞中microrna的精确成像

我们构建了基于DNAzyme的“双锁”CRISPR/Cas12a系统，用于miRNA检测和精准成像。一个锁的功能是关闭DNAzyme的催化活性，而另一个锁的功能是抑制crispr诱导RNA （crRNA）的切割功能。这种“双锁”机制确保了系统在缺乏靶分子miRNA-141的情况下被抑制，有效地降低了背景信号。当目标miRNA-141存在时，DNAzyme的“锁”被打开，DNAzyme进一步打开crRNA的“锁”，激活F-Q探针上Cas12a的反式切割能力，荧光信号恢复。miRNA-141的线性范围为50 pmol/L ~ 15 nmol/L，检测限为47 pmol/L (S/N = 3)，该系统已成功应用于细胞裂解物中miRNA-141的表达水平检测。同时，该方法可用于细胞内miRNA-141的荧光成像和细胞内miRNA-141表达的波动。总的来说，该策略不仅为可编程Cas12a检测系统提供了新的前景，而且通过将该体外试验与活细胞成像分析相结合，检测癌症标志物，为疾病的早期诊断和筛查提供了新的见解。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

International Journal of Biological Macromolecules 生物-生化与分子生物学

CiteScore

13.70

自引率

9.80%

发文量

2728

审稿时长

64 days

期刊介绍： The International Journal of Biological Macromolecules is a well-established international journal dedicated to research on the chemical and biological aspects of natural macromolecules. Focusing on proteins, macromolecular carbohydrates, glycoproteins, proteoglycans, lignins, biological poly-acids, and nucleic acids, the journal presents the latest findings in molecular structure, properties, biological activities, interactions, modifications, and functional properties. Papers must offer new and novel insights, encompassing related model systems, structural conformational studies, theoretical developments, and analytical techniques. Each paper is required to primarily focus on at least one named biological macromolecule, reflected in the title, abstract, and text.