Abstract：
:Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a U A: G premature termination codon (PTC) to tryptophan (U G: G) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Montiel-González MF,Vallecillo-Viejo IC,Rosenthal JJdoi
10.1093/nar/gkw738subject
Has Abstractpub_date
2016-12-01 00:00:00pages
e157issue
21eissn
0305-1048issn
1362-4962pii
gkw738journal_volume
44pub_type
杂志文章abstract：:Aryldiazonium salts are shown to be useful as phototriggered structural probes for RNA mapping as well as for footprinting of RNA/protein interaction. In particular the yeast tRNA(Asp)/aspartyl-tRNA synthetase complex is shown to involve the variable loop face and the concave side of the L-shaped nucleic acid bound to...
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更新日期：2014-07-01 00:00:00
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更新日期：1983-05-25 00:00:00
