A Transition State Analogue for an RNA-Editing Reaction

• Published in: Journal of the American Chemical Society 2004-09, Vol.126 (36), p.11213-11219
• Main Authors: Haudenschild, Brittany L, Maydanovych, Olena, Véliz, Eduardo A, Macbeth, Mark R, Bass, Brenda L, Beal, Peter A
• Format: Article
• Language: English
• Subjects: Adenosine Deaminase - chemistry; Adenosine Deaminase - metabolism; Aza Compounds - chemical synthesis; Aza Compounds - chemistry; Aza Compounds - metabolism; Base Sequence; Biological and medical sciences; Biomimetic Materials - chemical synthesis; Biomimetic Materials - chemistry; Biomimetic Materials - metabolism; Fundamental and applied biological sciences. Psychology; Interactions. Associations; Intermolecular phenomena; Kinetics; Molecular biophysics; Molecular Sequence Data; Organophosphorus Compounds - chemical synthesis; Organophosphorus Compounds - chemistry; Organophosphorus Compounds - metabolism; RNA Editing; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; RNA-Binding Proteins
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0472073

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 B 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.