OXOPAP assay: for selective amplification of aminoacylated tRNAs from total cellular fractions.
Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA.
Abstract
Transfer
RNA (tRNA) plays a pivotal role in protein synthesis within cells,
where it is recognized by one cognate aminoacyl-tRNA synthetase, in
competition with the remaining non-cognate synthetases, and esterified
with an amino acid. For many years the levels of tRNA aminoacylation, in
a given population of cellular RNA, have been analyzed using methods
that include northern analysis and/or oxidation techniques to separate
aminoacylated from non-aminoacylated species. In the present report we
describe an approach recently developed by us that combines
oxidation-protection with polyadenylation and PCR. The OXOPAP approach
permits the amplification of tRNA species that are nearly identical and
that evade differential identification by more classical northern
hybridization methods. Our approach also allows the identification of
aminoacylatable "naïve" species, where no prior knowledge of sequence
content is necessary for amplification.
