Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor 8 subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR-RNA complexes.
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US Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Univ Calif Berkeley, Dept Plant & Microbial Biol, Berkeley, CA 94720 USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Min, Byoungnam
Wu, Baojun
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Clark Univ, Biol Dept, Worcester, MA 01610 USA
Massey Univ, Sch Fundamental Sci, Stat & Bioinformat Grp, Palmerston North, New ZealandUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Wu, Baojun
Gaskell, Jill
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USDA Forest Prod Lab, Madison, WI USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Gaskell, Jill
Zhang, Jiwei
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Univ Minnesota, Dept Bioprod & Biosyst Engn, St Paul, MN USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Zhang, Jiwei
Toapanta, Christina
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Univ Minnesota, Dept Plant Pathol, St Paul, MN USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Toapanta, Christina
Ahrendt, Steven
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US Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Ahrendt, Steven
Blanchette, Robert A.
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Univ Minnesota, Dept Plant Pathol, St Paul, MN USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Blanchette, Robert A.
Master, Emma
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Univ Toronto, Dept Chem Engn & Appl Chem, Toronto, ON, CanadaUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Master, Emma
Cullen, Daniel
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USDA Forest Prod Lab, Madison, WI USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Cullen, Daniel
Hibbett, David S.
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Clark Univ, Biol Dept, Worcester, MA 01610 USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Hibbett, David S.
Grigoriev, Igor V.
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US Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA
Univ Calif Berkeley, Dept Plant & Microbial Biol, Berkeley, CA 94720 USAUS Dept Energy Joint Genome Inst, Lawrence Berkeley Natl Lab, Berkeley, CA 94720 USA