Specific inactivation of Escherichia coli tRNAPhe by antisense DNA-treatment under Mg2+-deficient conditions Original Research Article

The preparation of an Escherichia coli tRNA mixture lacking several specific species may be useful for applications ranging from cell-free protein preparation to protein engineering. We have already demonstrated that tRNAAsp can be inactivated, or ‘knocked out’, with practical specificity by an antisense strategy. In the present study, we synthesized five tRNAPhe-targeted antisense oligonucleotides and tested if this tRNA can also be inactivated specifically. The salt conditions used previously for the tRNAAsp inactivation were not applicable to tRNAPhe. Instead, Mg2+-deficient conditions were found to be useful for the inactivation of tRNAPhe by the antisense oligonucleotides. These conditions were also applicable to the inactivation of tRNAAsp. The susceptibility to the antisense DNAs can change drastically, depending on the concentration of Mg2+.