OXOPAP assay: For selective amplification of aminoacylated tRNAs from total cellular fractions

• Published in: Methods (San Diego, Calif.) Calif.), 2008-02, Vol.44 (2), p.170-175
• Main Authors: Gaston, Kirk W., Rubio, Mary Anne T., Alfonzo, Juan D.
• Format: Article
• Language: English
• Subjects: Aminoacylation; Editing; Nucleic Acid Amplification Techniques - methods; Oxidation-Reduction; Periodate; Polyadenylation; Polymerase Chain Reaction - methods; Polynucleotide Adenylyltransferase - metabolism; RNA, Transfer, Amino Acyl - isolation & purification; Synthetase; Transfer RNA Aminoacylation; tRNA
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2007.10.003

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.