在我们的身体里，有一种叫做8 - 氧代鸟嘌呤（o8G）的物质，它是RNA氧化修饰的一种典型代表。这种修饰和衰老、癌症以及心血管疾病等密切相关。然而，检测o8G却不是一件容易的事。o8G结构不稳定，还会因为所在序列不同产生诱变效应，这让现有的检测方法很难快速又灵敏地找到被o8G修饰的微小RNA（miRNA），而且操作起来也不便捷。为了解决这个难题，科学家们开发了一种叫做“精氨酸蛋白整合检测（AID）”的系统。这个系统把中温精氨酸蛋白（Ago）具有的序列特异性切割活性和滚环扩增（RCA）技术结合起来。Ago蛋白有一个特别的本事，当遇到被o8G修饰的miRNA时，它的切割会停下来；而对于没有被修饰的RNA，它就会进行选择性切割。这样一来，被o8G修饰的miRNA就能被保留下来，然后通过RCA技术进行信号放大。AID系统非常厉害，它能达到皮摩尔级别的灵敏度，还能分辨出单个核苷酸的差异。这个系统的反应在恒温条件下就能进行，还可以集成到便于携带到野外使用的设备中。我们用肉眼或者智能手机就能看到检测结果，在几个小时内就能检测出皮摩尔级别的被o8G修饰的RNA。这个便携式系统既保证了分子检测的特异性，操作又很简单。它不仅有可能在临床诊断中快速检测出氧化RNA生物标志物，而且不需要昂贵的设备，还能为相关疾病的表观遗传调控机制研究提供一个好用的工具。

期刊

ACS Sensors

标题

Argonaute-Coupled Rolling Circle Amplification Enables the Rapid and Sensitive Detection of 8-Oxoguanine-Modified miRNA

作者

Shuze Peng, Jinghan Liu, Wenqi Ge, Ran Tu, Jiaying Hu, Haoyang Wu, Bo Cao, Xiang Yu, Tianbao Yao, Xingyu Ye, Yan Feng, Qian Liu

摘要

8-Oxoguanine (o8G), a prominent oxidative RNA modification linked to aging, cancer, and cardiovascular pathologies, presents significant detection challenges due to its structural lability and sequence-dependent mutagenic effects. Current methodologies struggle to achieve rapid and sensitive identification of o8G-modified microRNAs (miRNAs), particularly in user-friendly operations. To address this limitation, an argonaute-integrated detection (AID) system was developed that synergizes the sequence-specific cleavage activity of mesophilic Argonaute (Ago) with rolling circle amplification (RCA) for the o8G-modified target selective amplification and quantification. Our strategy exploits the unique property of Ago to stall cleavage within o8G-modified miRNAs, enabling the selective cleavage of unmodified RNAs while preserving o8G-modified miRNA for subsequent RCA-mediated signal amplification. The AID platform achieved picomolar sensitivity with a single-nucleotide resolution. The isothermal reaction of this workflow permitted integration into a field-deployable device capable of the visual readout by the naked eye or smartphone, demonstrating a detection limit of pM for o8G-modified RNAs within hours. This portable system combines molecular specificity with operational simplicity, offering potential for clinical diagnostics through the rapid detection of oxidative RNA biomarkers without the need of expensive equipment, while also providing a versatile tool for mechanistic studies of epigenetic regulation in related diseases.

原文链接

https://pubs.acs.org/doi/10.1021/acssensors.5c02475

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