Direct Immunodetection of Global A-to-I RNA Editing Activity with a Chemiluminescent Bioassay.
Direct Immunodetection of Global A-to-I RNA Editing Activity with a Chemiluminescent Bioassay.
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DOI：
10.1002/anie.202102762
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发表时间：
2021-07-26
期刊：
影响因子：
--
通讯作者：
Heemstra JM
中科院分区：
文献类型：
--
作者：
Knutson SD;Arthur RA;Johnston HR;Heemstra JM
腺苷到肌苷（A-to-I）编辑是一种保守的真核RNA修饰，有助于发育，免疫反应和整体细胞功能。RNA编辑模式可以在不同的细胞和组织类型之间显着变化，并且过度活跃的A-to-I签名指示几种疾病，包括癌症和自身免疫性疾病。由于这些差异的生物学和临床重要性，因此非常需要有效的方法来测量细胞RNA中的总体A-to-I编辑水平。目前的标准方法依赖于RNA-seq来间接检测编辑位点，这需要大量的时间和材料投资以及广泛的计算分析。在这里，我们利用内切核酸酶V（EndoV），它特异性地结合到RNA中的肌苷，开发一种基于蛋白质的荧光生物测定法，以直接分析A到I RNA编辑活性。我们以前表明，EndoV可以在RNA-seq之前结合和富集A-to-I编辑的转录本，我们现在利用这种活性构建EndoV连接的免疫吸附试验（EndoVLISA），作为一种快速，基于板的荧光方法，用于测量细胞RNA中的全局A-to-I编辑签名。我们首先优化和验证我们的测定与化学合成的寡核苷酸，说明高选择性和灵敏度的检测RNA中的肌苷。然后，我们证明了在处理的细胞系中快速检测肌苷含量，证明了与当前标准RNA-seq方法相当的性能。最后，我们部署了EndoVLISA来分析正常和患病人体组织中的差异A-I RNA编辑特征，说明了我们的平台作为诊断生物测定的实用性。总之，EndoVLISA方法具有成本效益，简单明了，并利用常见的实验室设备，为研究A-to-I编辑提供了一种高度可及的新方法。此外，多孔板形式使其成为第一种适用于疾病检测和药物开发中应用的A-to-I编辑的直接高通量定量的测定。腺苷到肌苷（A-to-I）编辑是一种关键的RNA修饰，控制着许多细胞途径。这一过程的失调也与自身免疫性疾病、神经退行性疾病和几种类型的癌症有关。尽管如此重要，检测这种修改既昂贵又耗时。在这里，我们开发了一种基于蛋白质的荧光生物测定法，以快速表征正常和患病人体组织中的全局A-to-I编辑签名，解决了研究RNA编辑及其与疾病关系的重大技术需求。
Adenosine-to-inosine (A-to-I) editing is a conserved eukaryotic RNA modification that contributes to development, immune response, and overall cellular function. RNA editing patterns can vary significantly between different cell and tissue types, and hyperactive A-to-I signatures are indicative of several diseases, including cancer and autoimmune disorders. Because of the biological and clinical importance of these differences, there is significant need for efficient methods to measure overall A-to-I editing levels in cellular RNA. The current standard approach relies on RNA-seq to indirectly detect editing sites, which requires significant investments in time and material as well as extensive computational analysis. Here, we utilize Endonuclease V (EndoV), which binds specifically to inosine in RNA, to develop a protein-based chemiluminescent bioassay to directly profile A-to-I RNA editing activity. We previously showed that EndoV can bind and enrich A-to-I edited transcripts prior to RNA-seq, and we now leverage this activity to construct an EndoV-linked immunosorbency assay (EndoVLISA) as a rapid, plate-based chemiluminescent method for measuring global A-to-I editing signatures in cellular RNA. We first optimize and validate our assay with chemically synthesized oligonucleotides, illustrating highly selective and sensitive detection of inosine in RNA. We then demonstrate rapid detection of inosine content in treated cell lines, demonstrating equivalent performance against current standard RNA-seq approaches. Lastly, we deploy our EndoVLISA for profiling differential A-to-I RNA editing signatures in normal and diseased human tissue, illustrating the utility of our platform as a diagnostic bioassay. Together, the EndoVLISA method is cost-effective, straightforward, and utilizes common laboratory equipment, offering a highly accessible new approach for studying A-to-I editing. Moreover, the multi-well plate format makes this the first assay amenable for direct high-throughput quantification of A-to-I editing for applications in disease detection and drug development. Adenosine-to-inosine (A-to-I) editing is a critical RNA modification and controls many cellular pathways. Dysregulation of this process is also linked with autoimmune disease, neurodegenerative disorders, and several types of cancer. Despite this importance, detecting this modification is both costly and time consuming. Here, we develop a protein-based chemiluminescent bioassay to rapidly characterize global A-to-I editing signatures in normal and diseased human tissue, addressing a significant technological need for studying RNA editing and its relationship with disease.
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