MicroRNAs (miRNAs) are endogenous RNAs of about 20-25 nucleotides that play important roles in gene regulation. Based on miRNA backbones, artificial miRNAs, amiRNAs, can be constructed and expressed exogenously. AmiRNAs can function as natural miRNAs or siRNAs. AmiRNAs display higher expression levels and more potent silencing efficiency than simple shRNAs. In practice, an amiRNA library is usually needed to screen for functional amiRNAs. Here we describe a novel, convenient, and cost-effective method for enzymatically constructing an amiRNA library. A novel strategy was applied to ensure the correct orientation of the anti-sense strand and also render less thermo-stability to the 5' end of antisense strand to facilitate its entry into the RISC complex, which can largely increase the silencing efficiency. Furthermore, a "Padlock probe" method was also incorporated to ensure the fidelity of the sequence and stem-loop structure. To test the feasibility of the method, an amiRNA library targeting mouse p53 ORF was constructed. Through screening of 82 amiRNAs in the library, 7 amiRNAs which displayed potent p53-silencing efficiency were identified and verified. This method also has the potential to construct an amiRNA library that targets a whole transcriptome for genome-wide RNAi screening, or a randomized amiRNA library to search for functional amiRNAs. (C) 2009 Elsevier Inc. All rights reserved.