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 language | English (US) |
|---|---|
| Pages (from-to) | 269-277.e5 |
| Journal | Cell Chemical Biology |
| Volume | 26 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 21 2019 |
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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
Cite this
A Bump-Hole Approach for Directed RNA Editing. / Monteleone, Leanna R.; Matthews, Melissa M.; Palumbo, Cody M.; Thomas, Justin M.; Zheng, Yuxuan; Chiang, Yao; Fisher, Andrew J; Beal, Peter A.
In: Cell Chemical Biology, Vol. 26, No. 2, 21.02.2019, p. 269-277.e5.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A Bump-Hole Approach for Directed RNA Editing
AU - Monteleone, Leanna R.
AU - Matthews, Melissa M.
AU - Palumbo, Cody M.
AU - Thomas, Justin M.
AU - Zheng, Yuxuan
AU - Chiang, Yao
AU - Fisher, Andrew J
AU - Beal, Peter A.
PY - 2019/2/21
Y1 - 2019/2/21
N2 - 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.
AB - 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.
KW - ADAR
KW - bump-hole
KW - epitranscriptome
KW - off-target sites
KW - site-directed RNA editing
UR - http://www.scopus.com/inward/record.url?scp=85061334300&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85061334300&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2018.10.025
DO - 10.1016/j.chembiol.2018.10.025
M3 - Article
C2 - 30581135
AN - SCOPUS:85061334300
VL - 26
SP - 269-277.e5
JO - Cell Chemical Biology
JF - Cell Chemical Biology
SN - 2451-9448
IS - 2
ER -