Using capillary electrophoresis to study the RNA-acting adenosine deaminase reaction

CHED 930

Juan E. Kusnir, jkusnir@rollins.edu and Darren G. Stoub, dstoub@rollins.edu. Department of Chemistry, Rollins College, 1000 Holt Ave, Box 2743, Winter Park, FL 32789-4499
RNA-editing adenosine deaminase (ADAR2) is an enzyme that catalyzes the site-specific conversion of an adenosine to inosine in a glutamate neurotransmitter receptor pre-mRNA, a post-transcriptional modification that results in a codon change. Without this modification, the receptor is ineffective at binding glutamate. The mechanism for catalysis by ADAR2 is currently under investigation; however, the enzymatic assay is cumbersome and time-intensive. We have investigated Capillary Electrophoresis (CE) as a plausible method for the analysis of the ADAR2 enzymatic reaction. We report our efforts towards validating a CE method that resolves the RNA substrate from the point mutated (A to I) RNA product. Experimental variables that have been investigated include use of coated and/or filled capillaries, various buffers, and modification of instrumental parameters. With this new method, the ADAR2 enzymatic reaction could be efficiently assayed so that further mechanistic studies can be more easily conducted.