title: A Fluorescent Adenosine Analogue as a Substrate for an A-to-I RNA Editing Enzyme.
abstract: Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine ((th)A). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that (th)A is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give (th)I. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the (th)A-modified RNA. The observed high (th)A reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.
authors.affiliation: Department of Chemistry, University of California, Davis, One Shields Ave, Davis, CA 95616 (USA).
authors.forename: Rena A
authors.initials: RA
authors.lastname: Mizrahi
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 (USA).
authors.forename: Dongwon
authors.initials: D
authors.lastname: Shin
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 (USA).
authors.forename: Renatus W
authors.initials: RW
authors.lastname: Sinkeldam
authors.affiliation: Department of Chemistry, University of California, Davis, One Shields Ave, Davis, CA 95616 (USA).
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Phelps
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 (USA).
authors.forename: Andrea
authors.initials: A
authors.lastname: Fin
authors.affiliation: Department of Chemistry, University of California, Davis, One Shields Ave, Davis, CA 95616 (USA).
authors.forename: Dean J
authors.initials: DJ
authors.lastname: Tantillo
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 (USA). ytor@ucsd.edu.
authors.forename: Yitzhak
authors.initials: Y
authors.lastname: Tor
authors.affiliation: Department of Chemistry, University of California, Davis, One Shields Ave, Davis, CA 95616 (USA). pabeal@ucdavis.edu.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Beal
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000243:Adenosine Deaminase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017393:RNA Editing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D016601:RNA-Binding Proteins; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; D000241:Adenosine
keywords: ADAR enzymes; RNA editing; adenosine deaminase; fluorescent nucleosides