Synthetic substrate analogs for the RNA-editing adenosine deaminase ADAR-2

• Published in: Nucleic acids research 1999-07, Vol.27 (14), p.2912-2917
• Main Authors: Yi-Brunozzi, Hye Young, Easterwood, LaHoma M., Kamilar, Gregg M., Beal, Peter A.
• Format: Article
• Language: English
• Subjects: Adenosine - analogs & derivatives; Adenosine - chemical synthesis; Adenosine - chemistry; Adenosine - metabolism; Adenosine Deaminase - metabolism; Animals; Base Pairing; Base Sequence; Kinetics; Methylation; Molecular Weight; Nucleic Acid Heteroduplexes - chemical synthesis; Nucleic Acid Heteroduplexes - chemistry; Nucleic Acid Heteroduplexes - genetics; Nucleic Acid Heteroduplexes - metabolism; Oligoribonucleotides - chemical synthesis; Oligoribonucleotides - chemistry; Oligoribonucleotides - genetics; Oligoribonucleotides - metabolism; Rats; Receptors, AMPA - genetics; Recombinant Proteins - metabolism; Regulatory Sequences, Nucleic Acid - genetics; Ribose - chemistry; Ribose - genetics; Ribose - metabolism; RNA Editing; RNA Precursors - chemical synthesis; RNA Precursors - chemistry; RNA Precursors - genetics; RNA Precursors - metabolism; RNA, Double-Stranded - chemical synthesis; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; RNA-Binding Proteins
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/27.14.2912

We have synthesized structural analogs of a natural RNA editing substrate and compared editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase. Deamination rates were shown to be sensitive to structural changes at the 2′-carbon of the edited adenosine. Methylation of the 2′-OH caused a large decrease in deamination rate, whereas 2′-deoxyadenosine and 2′-deoxy-2′-fluoroadenosine were deaminated at a rate similar to adenosine. In addition, a duplex containing as few as 19 bp of the stem structure adjacent to the R/G editing site of the GluR-B pre-mRNA supports deamination of the R/G adenosine by ADAR-2. This identification and initial characterization of synthetic RNA editing substrate analogs further defines structural elements in the RNA that are important for the deamination reaction and sets the stage for additional detailed structural, thermodynamic and kinetic studies of the ADAR-2 reaction.