A Bump-Hole Approach for Directed RNA Editing. Issue 2 (21st February 2019)

• Record Type: Journal Article
• Authors: Monteleone, Leanna R.; Matthews, Melissa M.; Palumbo, Cody M.; Thomas, Justin M.; Zheng, Yuxuan; Chiang, Yao; Fisher, Andrew J.; Beal, Peter A.
• Is Part Of: Cell chemical biology. Volume 26:Issue 2(2019)
• Page Start: 269
• Page End: 277.e5
• Publication Date: 2019-02-21
• Subjects: site-directed RNA editing -- bump-hole -- ADAR -- off-target sites -- epitranscriptome; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2018.10.025
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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. Graphical Abstract: Highlights: Bump-hole strategy developed for selective site-directed RNA editing (SDRE) system SDRE in vitro and in human cells with bulky mutant ADAR2 proteins and guide RNAs Directed editing on endogenous targets with reduced off-target activity Crystal structure of ADAR2-D E488Y with RNA duplex containing reduced abasic site Abstract : 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.