DNA self-assembly-boosted transcription amplification coupled with CRISPR/Cas13a system for plant microRNA analysis

IF 5.6 1区化学 Q1 CHEMISTRY, ANALYTICAL

Talanta Pub Date : 2024-09-14 DOI:10.1016/j.talanta.2024.126890

Gaoting Wang, Zhengping Li

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引用次数: 0

Abstract

MicroRNAs (miRNAs) play important roles in the growth process of plants, and some food-originated plant miRNAs have potential impacts on human health, which makes the detection of plant miRNAs of great significance. However, plant miRNAs are naturally modified with 2′-O-methyl at the 3′-terminal, which is difficult to be directly quantified by enzyme-catalyzed terminal polymerization protocols. Herein, we have proposed a simple strategy by coupling DNA self-assembly-boosted transcription amplification with CRISPR/Cas13a platform (termed as Cas13a-SATA) for the specific and sensitive detection of plant miRNA. In the Cas13a-SATA, the plant miRNA will mediate DNA self-assembly on the surface of microbeads and then trigger efficient transcription amplification to yield numerous single-stranded RNA (ssRNA) molecules, which can effectively activate the Cas13a trans-cleavage activity to generate intense fluorescence signal in a plant miRNA dosage-responsive manner. Using the Cas13a-SATA, we have realized the sensitive detection of plant miR156a with the limit of detection (LOD) down to 3.8 fM. Furthermore, Cas13a-SATA has been successfully applied to the accurate quantification of miR156a in Arabidopsis and maize, demonstrating its feasibility in analyzing plant miRNAs in real biological samples.

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DNA 自组装增强转录扩增与 CRISPR/Cas13a 系统结合用于植物微 RNA 分析

微RNA（miRNA）在植物的生长过程中发挥着重要作用，一些来源于食物的植物miRNA对人类健康有潜在影响，因此检测植物miRNA具有重要意义。然而，植物miRNA的3′末端被2′-O-甲基天然修饰，很难通过酶催化末端聚合方案直接定量。在此，我们提出了一种简单的策略，将DNA自组装增强转录扩增与CRISPR/Cas13a平台（称为Cas13a-SATA）相结合，用于特异灵敏地检测植物miRNA。在Cas13a-SATA中，植物miRNA会在微珠表面介导DNA自组装，然后触发高效转录扩增，产生大量单链RNA（ssRNA）分子，从而有效激活Cas13a的反式裂解活性，以植物miRNA剂量响应的方式产生强烈的荧光信号。利用 Cas13a-SATA，我们实现了对植物 miR156a 的灵敏检测，检测限（LOD）低至 3.8 fM。此外，Cas13a-SATA 还成功应用于拟南芥和玉米中 miR156a 的精确定量，证明了它在分析真实生物样本中植物 miRNA 的可行性。

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来源期刊

Talanta 化学-分析化学

CiteScore

12.30

自引率

4.90%

发文量

861

审稿时长

29 days

期刊介绍： Talanta provides a forum for the publication of original research papers, short communications, and critical reviews in all branches of pure and applied analytical chemistry. Papers are evaluated based on established guidelines, including the fundamental nature of the study, scientific novelty, substantial improvement or advantage over existing technology or methods, and demonstrated analytical applicability. Original research papers on fundamental studies, and on novel sensor and instrumentation developments, are encouraged. Novel or improved applications in areas such as clinical and biological chemistry, environmental analysis, geochemistry, materials science and engineering, and analytical platforms for omics development are welcome. Analytical performance of methods should be determined, including interference and matrix effects, and methods should be validated by comparison with a standard method, or analysis of a certified reference material. Simple spiking recoveries may not be sufficient. The developed method should especially comprise information on selectivity, sensitivity, detection limits, accuracy, and reliability. However, applying official validation or robustness studies to a routine method or technique does not necessarily constitute novelty. Proper statistical treatment of the data should be provided. Relevant literature should be cited, including related publications by the authors, and authors should discuss how their proposed methodology compares with previously reported methods.