ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A·C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A·U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 ± 1.5 nM·min-1 at 30 nM ADAR2 for a k(ss) = 0.43 ± 0.05 min-1. In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.
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