T4 RNA Ligase Mediated Preparation of Novel “Chemically Misacylated” tRNA^phes

T4 RNA ligase was employed for the condensation of Escherichia coli tRNA^Phe missing cytidine-75 and adenosine-76 (tRNA^Phe-C_OH; the acceptor “oligomer”) with each of several chemically acylated derivatives of pCpA”(the donor oligomer). The resulting chemically misacylated tRNA s were obtained in 20-65% yields following chromatographic workup on DEAE-cellulose and benzoylated DEAE-cellulose. Characterization of the chemically misacylated tRNAs was accomplished by (i) enzymatic reaminoacylation of chemically misacylated tRNA with phenylalanine by E. coli phenylalanyl-tRNA synthetase following chemical deacylation of the “incorrect” amino acid, (ii) comparison of the hydrolytic effects of Cu²⁺ solutions on chemically and enzymatically prepared samples of N-acetyl-L-phenylalanyl-tRNA^Phes, and (iii) measurement of the chromatographic behavior of the tRNA species derived from chemical misacylation.