Isolation of peptide aptamers that inhibit intracellular processes

We have developed a method for isolation of random peptides that inhibit in tracellular processes in bacteria. A library of random peptides expressed a s fusions to Escherichia coil thioredoxin (aptamers) were expressed under t he tight control of the arabinose-inducible P-BAD promoter. A selection was applied to the library to isolate aptamers that interfered with the activi ty of thymidylate synthase (ThyA) in vivo. Expression of an aptamer isolate d by this method resulted in a ThyA(-) phenotype that was suppressed by sim ultaneous overexpression of ThyA. Two-hybrid analysis showed that this apta mer is likely to interact with ThyA in vivo. The library also was screened for aptamers that inhibited growth of bacteria expressing them, and five su ch aptamers were characterized. Four aptamers were bacteriostatic when expr essed, whereas one showed a bactericidal effect. Introduction of translatio nal stop codons into various aptamers blocked their activity, suggesting th at their biological effects were likely to be due to protein aptamer rather than RNA. Combinatorial aptamers provide a new genetic and biochemical too l for identifying targets for antibacterial drug development.