title: A purine at +2 rather than +1 adjacent to the human U6 promoter is required to prepare effective short hairpin RNAs.
abstract: 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 (PKACalpha) was used as material for library construction. We then used luciferase reporter cell lines to screen shRNAs which effectively reduced PKACalpha 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.
authors.affiliation: GenoFunction, Inc., 1-25-14 Kannondai, Tsukuba, Ibaraki 305-085, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Iijima
authors.forename: Hajime
authors.initials: H
authors.lastname: Fukano
authors.forename: Harumi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Shirai
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Suzuki
mesh_terms: D002460:Cell Line; D020868:Gene Silencing; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011687:Purines / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000096:biosynthesis* / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D011687:Purines; D016337:RNA, Catalytic; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; C030985:purine