Hammerhead ribozyme-based U-insertion and deletion RNA editing assays for multiplexing in HTS applications

Untranslatable mitochondrial transcripts in kinetoplastids are decrypted post-transcriptionally through an RNA editing process that entails uridine insertion/deletion. This unique stepwise process is mediated by the editosome, a multiprotein complex that is a validated drug target of considerable in...

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Bibliographic Details
Published in:RNA (Cambridge) 2023-02, Vol.29 (2), p.252-261
Main Authors: Rostamighadi, Mojtaba, Mehta, Vaibhav, Hassan Khan, Rufaida, Moses, Daniel, Salavati, Reza
Format: Article
Language:English
Subjects:
DNA probes
Editing
Fluorescence resonance energy transfer
Gene deletion
Insertion
Method
Mitochondria
Nucleotide sequence
Post-transcription
RNA Editing
RNA, Catalytic - genetics
RNA, Catalytic - metabolism
RNA, Protozoan - genetics
Therapeutic targets
Trypanosoma brucei brucei - genetics
Uridine
Uridine - genetics
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Summary:Untranslatable mitochondrial transcripts in kinetoplastids are decrypted post-transcriptionally through an RNA editing process that entails uridine insertion/deletion. This unique stepwise process is mediated by the editosome, a multiprotein complex that is a validated drug target of considerable interest in addressing the unmet medical needs for kinetoplastid diseases. With that objective, several in vitro RNA editing assays have been developed, albeit with limited success in discovering potent inhibitors. This manuscript describes the development of three hammerhead ribozyme (HHR) FRET reporter-based RNA editing assays for precleaved deletion, insertion, and ligation assays that bypass the rate-limiting endonucleolytic cleavage step, providing information on U-deletion, U-insertion, and ligation activities. These assays exhibit higher editing efficiencies in shorter incubation times while requiring significantly less purified editosome and 10,000-fold less ATP than the previously published full round of in vitro RNA editing assay. Moreover, modifications in the reporter ribozyme sequence enable the feasibility of multiplexing a ribozyme-based insertion/deletion editing (RIDE) assay that simultaneously surveils U-insertion and deletion editing suitable for HTS. These assays can be used to find novel chemical compounds with chemotherapeutic applications or as probes for studying the editosome machinery.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.079454.122