Molecules capable of directing specific changes to nucleic acid sequences have become powerful tools for molecular biology and are promising candidates for the therapeutic correction of disease-causing mutations.  Different systems have been developed for directing both genome (DNA) and transcriptome (RNA) editing.  For each of these systems, unwanted reactions at off target sites can complicate their use.  Here we use knowledge of the high-resolution structure of a protein-RNA interface to develop highly selective combinations of mutant editing enzyme and directing oligonucleotide.  Mutations in a loop found in the deaminase domain of human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing activity.  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 in the complex.  Finally, we demonstrate efficient directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and oligonucleotide editing guide RNAs with reduced off target site activity.  <br/>

Bump/hole approach to oligonucleotide-directed RNA editing

2019 Spring National Meeting

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