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. Copyright © 2010 John Wiley & Sons, Ltd.