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.