Aminoacyl-tRNA synthetases (ARSs) recognize a specific sequence or structural characteristics of their cognate tRNAs. To contribute to the understanding how these recognition sites were selected, we generated two different RNA libraries containing either 42mer or 70mer random sequence and used them to select RNA aptamers that specifically bound to methionyl-tRNA synthetase (MRS) of Mycobacterium tuberculosis. The aptamer pools selected from the two RNA libraries showed strong binding affinity and selectivity to M. tuberculosis MRS compared to that of the homologous Escherichia coil MRS. The RNA aptamers selected from the two completely unrelated RNA pools shared the octamer sequence including CAU and the anticodon sequence of tRNA(Met). The secondary structure prediction suggested that the octamer motif in the selected aptamers would form a loop similar to the anticodon loop of tRNA(Met). The results suggest that the RNA loop containing CAU triplet could selected as a major recognition site for MRS during evolution more or less regarding, and also showed that species-specific ARS inhibitors can be obtained by in vitro evolution. (C) 2013 Elsevier Inc. All rights reserved.