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Direct Immunodetection of Global A‐to‐I RNA Editing Activity with a Chemiluminescent Bioassay
Author(s) -
Knutson Steve D.,
Arthur Robert A.,
Johnston H. Richard,
Heemstra Jennifer M.
Publication year - 2021
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.202102762
Subject(s) - bioassay , chemiluminescence , rna , chemistry , computational biology , biology , chromatography , biochemistry , genetics , gene
Abstract Adenosine‐to‐inosine (A‐to‐I) editing is a conserved eukaryotic RNA modification that contributes to development, immune response, and overall cellular function. Here, we utilize Endonuclease V (EndoV), which binds specifically to inosine in RNA, to develop an EndoV ‐ l inked i mmuno s orbency a ssay (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. 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.