Introduction: Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes
consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD)
that converts adenosine (A) into inosine (I), which acts as guanosine (G) during translation. Using the
MS2 system, we engineered the DD of ADAR1 to direct it to a specific target. The aim of this work
was to compare the deaminase activities of ADAR1-DD and various isoforms of ADAR2-DD.
Materials and Methods: We measured the binding affinity of the artificial enzyme system on a Biacore
™ X100. ADARs usually target dsRNA, so we designed a guide RNA complementary to the target
RNA, and then fused the guide sequence to the MS2 stem-loop. A mutated amber (TAG) stop
codon at 58 amino acid (TGG) of EGFP was targeted. After transfection of these three factors into
HEK 293 cells, we observed fluorescence signals of various intensities.
Results: ADAR2-long without the Alu-cassette yielded a much higher fluorescence signal than
ADAR2-long with the Alu-cassette. With another isoform, ADAR2-short, which is 81 bp shorter at
the C-terminus, the fluorescence signal was undetectable. A single amino acid substitution of
ADAR2-long-DD (E488Q) rendered the enzyme more active than the wild type. The results of fluorescence
microscopy suggested that ADAR1-DD is more active than ADAR2-long-DD. Western blots
and sequencing confirmed that ADAR1-DD was more active than any other DD.
Conclusion: This study provides information that should facilitate the rational use of ADAR variants
for genetic restoration and treatment of genetic diseases.