A 2'-O-methyl oligonucleotide complementary to 18 nucleotides in the dihydrouridine stemloop of Escherichia coli tRNA(Cys) has been shown to stably bind to the tRNA. The binding inhibits aminoacylation of the tRNA by cysteine tRNA synthetase. The same oligonucleotide sequence but with the DNA deoxy backbone does not bind to the tRNA. This provides the basis for the design and test of a series of 2'-O-methyl oligonucleotides for their ability to bind to E. coli tRNA(Cys) and inhibit aminoacylation. We show here that different regions of the tRNA have different sensitivities to oligonucleotides. A 10-mer that targets G15 forms a stable complex with the tRNA. The Kd of the complex is several orders of magnitude lower than that of the tRNA-synthetase complex. Measurements of dissociation rate constants indicate that the stronger affinity of the 10-mer to tRNA(Cys) is due to a significantly slower rate of dissociation (by a factor of 10(6)) than that of the synthetase from the tRNA. Only a stoichiometric amount of the 10-mer is necessary to completely inhibit aminoacylation. Because tRNA aminoacylation is fundamental to cell growth, these results provide the rationale for the 10-mer and its derivatives as pharmaceutical agents that target specific cell growth.