Synthesis and evaluation of an RNA editing substrate bearing 2′-deoxy-2′-mercaptoadenosine

The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in double stranded structure found in various RNA substrates, including mRNAs. Here we describe the synthesis of a phosphoramidite of 2′-deoxy-2′-mercaptoadenosine and its incorporation into an ADAR substrate. Surprisingly, no deamination product was observed with this substrate indicating replacing the 2′-OH with a 2′-SH at the editing site is highly inhibitory. Modeling of nucleotide binding into the active site suggests the side chain of T375 of human ADAR2 to be in proximity of the 2 ′-substituent. Mutation of this residue to cysteine caused a greater that 100-fold reduction in deamination rate with the 2 ′-OH substrate.