Improving the stability of short hairpin RNA against fetal bovine serum using the third double-stranded RNA-binding domain from staufen protein

Abstract

RNA interference (RNAi) is a technology that specifically inhibits gene expression and is carried out by small 21-27-nucleotide double-stranded small interfering RNA (siRNA) or short hairpin RNA (shRNA). RNAi has become a very promising technology for genetic research, however problems remain with the delivery of siRNA into cells. SiRNA and shRNA are easily degraded by RNases in body fluids and are hardly able to permeate cell membranes because of hydrophilic, polyanionic macromolecules which make their bioavailability very low. We have focused on the third double-stranded RNA-binding domain (dsRBD3) from the Mus musculus Staufen protein in order to develop a non-viral, multifunctional, artificial virus-like delivery system. We constructed a dsRBD3 expression vector and the recombinant dsRBD3 was expressed as a fusion protein with affinity tags. Purified dsRBD3 was mixed with siRNA or shRNA at various ratios and then added to fetal bovine serum (FBS) to evaluate the inhibition of the degradation of double-stranded RNA (dsRNA) by RNase. Unexpectedly, dsRBD3 was not able to protect the siRNA against degradation by FBS, but shRNA was stabilized to some degree by dsRBD3. © 2009 Pharmaceutical Society of Japan.