The human U6 (hU6) promoter is widely used to express short hairpin RNAs (shRNAs) in mammalian cells. To verify the validity of the generalized concept-the hU6 promoter essentially requires a purine (usually guanine) at +1 for transcription, we enzymatically constructed an arbitrary shRNA library with the following features: (1) to have any one of adenine, cytosine, guanine, and thymine at the site; (2) to comprise shRNAs of 25-30 nucleotides in stem length which are transcribed through the promoter. cDNA of the catalytic subunit of cAMP-dependent protein kinase (PKAC alpha) was used as material for library construction. We then used luciferase reporter cell lines to screen shRNAs which effectively reduced PKAC alpha activity. Consequently, a purine was mostly present at +2, not at +1, of the clones isolated, suggesting that a purine at +2 rather than +1 adjacent to the hU6 promoter provides effective shRNAs for target gene silencing. (c) 2006 Elsevier Inc. All rights reserved.