Functional shRNA expression system with reduced off-target effects

The capability to induce off-target effects by small interfering RNA (siRNA) is highly correlated with the calculated melting temperature (Tm) of the protein-free seed-target duplex, indicating that its thermodynamic stability is a major factor in determining the degree of the off-target effect. In this study, we analyzed changes in microarray expression profiles following transfection of short hairpin RNAs (shRNAs). In a similar manner to siRNA, no or little off-target effects were observed by shRNA with a low Tm value in the seed-target duplex, whereas a high degree of off-target effect was induced by shRNA with a high Tm. Furthermore, shRNAs were often cleaved by Drosha and/or Dicer at the shifted sites in their primary transcripts, resulting in unexpected siRNA products with shifted sequences. Based on these results, the following conditions should be satisfied to ensure a functional shRNA with reduced off-target effects: the presence of A/U residues at nucleo tide positions 1 and −1 from the 5' end of the guide strand, three to six A/Us in nucleotide positions 2–7, G/C at positions 19 and 18, and no GC stretch of more than nine nucleotides. In addition, the Tm values of the seed regions in both strands should be low.