A purine at +2 rather than +1 adjacent to the human U6 promoter is required to prepare effective short hairpin RNAs

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 (PKACα) was used as material for library construction. We then used luciferase reporter cell lines to screen shRNAs which effectively reduced PKACα 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.