title: A shRNA library constructed through the generation of loop-stem-loop DNA.
abstract: BACKGROUND
  Short hairpin RNA (shRNA) libraries are considered to comprise a powerful tool in genetic screening. Several library construction methods have been proposed; these methods require between four and nine tedious reaction steps to construct the library. In the present study, we describe a method of generating shRNA-coding DNA efficiently from randomized oligonucleotides based on four reaction steps.
  
  
  METHODS
  Blunt end 29 nucleotides (nt) stem-loop DNA was synthesized with a DNA polymerase I Klenow fragment from randomized oligonucleotides. The stem-loop DNA was ligated with a 5' phosphorylated hairpin linker to obtain novel loop-stem-loop DNA. Next, the complementary sequence, forming an 86 nt inverted repeat-containing stem-loop DNA, was synthesized using Klenow fragment. The stem-loop DNA was digested and cloned into a small RNA expression vector. Luciferase activity in cells co-transfected with a Renilla luciferase-targeting shRNA plasmid and the luciferase expression plasmid was analyzed for the purpose of evaluating their specificity and efficiency for RNA interference (RNAi).
  
  
  RESULTS
  shRNA-coding DNA was generated from randomized oligonucleotides. The average GC content in the random sequence was 46.6%. Each sequence was unique. A shRNA generated by this method suppressed luciferase activity at a lower dose compared to a typical shRNA that contain a sequence perfectly matched to the target.
  
  
  CONCLUSIONS
  We constructed a shRNA library through four reaction steps, including the generation of loop-stem-loop DNA, without the polymerase chain reaction. A shRNA generated by this method retained its specificity of RNAi; however, the shRNA contained a target-irrelevant 7 nt sequence adjacent to the loop. This method could be valuable in the area of functional genomics.
authors.affiliation: Graduate School of Bionics, Tokyo University of Technology, Hachioji, Tokyo, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nishikawa
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Sugiyama
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D055029:Inverted Repeat Sequences; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; D004247:DNA; D049410:Luciferases, Renilla