Branched DNA switchable CRISPR-Cas12a system for sensing FEN1 activity

费斯特共振能量转移 清脆的 DNA 计算生物学 生物 生物物理学 DNA损伤 化学 分子生物学 细胞生物学 荧光 生物化学 基因 物理 量子力学
作者
Xingrong Li,Decai Zhang,Xiaoying Cai,Xiaojia Shu,Zijie Zeng,Shijia Ding,Yurong Yan
出处
期刊:Chemical Engineering Journal [Elsevier BV]
卷期号:470: 144407-144407 被引量:15
标识
DOI:10.1016/j.cej.2023.144407
摘要

FEN1 plays a crucial role in tumor progression and proliferation, and it has been considered a prospective cancer biomarker and druggable target. However, its detection is compromised by expenditure consumption and challenged by the lack of straightforward and high-efficiency strategies both in vitro and intracellular applications. Herein, we describe an original technique for hypersensitive detection of FEN1 by incorporating structure-specific branched DNA (BD) blocked activator with CRISPR-Cas12a methodology. Given the presence of overhanging structure at the 5′ end region of the tripartite BD, it is difficult for the BD to activate Cas12a protein which could be ascribed to the larger steric hindrance effect at the 5′ terminal regions and interferes with the approachability of the constituent elements for crRNA to fully hybridize with the complementary sequences of BD. However, the appearance of FEN1 can cleave the overhanging branch of BD to form an identifiable split DNA activator, thus successfully activating the cis/trans-cleavage activity of Cas12a. By introducing a fluorescence resonance energy transfer (FRET)-based single-stranded DNA reporter, consecutive degradation events lead to an immediately perceptible intensification in fluorescence intensity, realizing highly efficient detection of FEN1. The strategy has also been successfully applied to complex biological sample analysis and intracellular imaging, demonstrating its potential application in biochemical and molecular biology research as well as clinical diagnosis. In addition, we preliminarily verified the feasibility of integrating the established strategy with an electrochemiluminescent (ECL) platform, and confirmed that this strategy can be further expanded to other miniaturized sensing devices and has great prospects for point-of-care applications.
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