It has been of great interest to design new, precise systems that allow for directing specific changes in nucleic acid sequences. This has great potential for therapeutic applications and acts as a biotechnology tool. However, for the systems thus far developed there has been undesired side reactions at off-target sites. To develop an orthogonal site-directed RNA editing system, the protein RNA interface from a high-resolution crystal structure of human ADAR2 (adenosine deaminase acting on RNA 2) bound to double stranded RNA was utilized. From the crystal structure, a key intercalating residue at position 488 interacts with the orphan base, the base across from the editing site. If the 488 residue is mutated to a bulky aromatic residue it is suggested that a steric clash would occur with the orphan base therefore reducing editing activity. However, to restore editing activity with the bulky ADAR mutants the nucleobase of the orphan base can be replaced by a hydrogen (a reduced abasic site) resulting in removal of the clash. Using the bulky ADAR mutants in combination with a reduced abasic guide RNA we have demonstrated efficient site-directed RNA editing in vitro and in human cells with reduced off-target editing. A crystal structure of the E488Y mutant bound to double stranded RNA containing the reduced abasic site demonstrated the incorporation of the tyrosine side chain in the duplex RNA. This approach for site-directed RNA editing has great promise for correcting deleterious mutations with minimal reactions at off-target sites.

Orthogonal site-directed RNA editing system utilizing structure-guided engineering of a protein-RNA interface

256th National Meeting (2018)

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