A Bump-Hole Approach for Directed RNA Editing

Leanna R. Monteleone, Melissa M. Matthews, Cody M. Palumbo, Justin M. Thomas, Yuxuan Zheng, Yao Chiang, Andrew J. Fisher, Peter A Beal

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity. Systems developed for genome and transcriptome editing have unwanted off-target reactions. Monteleone et al. used a bump-hole strategy to develop highly selective combinations of mutant ADARs and directing oligonucleotides. SDRE is shown in vitro and in human cells using mutant ADAR2 proteins and guide RNAs with reduced off-target activity.

Original languageEnglish (US)
Pages (from-to)269-277.e5
JournalCell Chemical Biology
Volume26
Issue number2
DOIs
StatePublished - Feb 21 2019

Keywords

  • ADAR
  • bump-hole
  • epitranscriptome
  • off-target sites
  • site-directed RNA editing

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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  • Cite this

    Monteleone, L. R., Matthews, M. M., Palumbo, C. M., Thomas, J. M., Zheng, Y., Chiang, Y., Fisher, A. J., & Beal, P. A. (2019). A Bump-Hole Approach for Directed RNA Editing. Cell Chemical Biology, 26(2), 269-277.e5. https://doi.org/10.1016/j.chembiol.2018.10.025