title: Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein.
abstract: RNA interference (RNAi) induced by short interfering RNA (siRNA) allows for discovery research and large-scale screening; however, owing to their size and anionic charge, siRNAs do not readily enter cells. Current approaches do not deliver siRNAs into a high percentage of primary cells without cytotoxicity. Here we report an efficient siRNA delivery approach that uses a peptide transduction domain-double-stranded RNA-binding domain (PTD-DRBD) fusion protein. DRBDs bind to siRNAs with high avidity, masking the siRNA's negative charge and allowing PTD-mediated cellular uptake. PTD-DRBD-delivered siRNA induced rapid RNAi in a large percentage of various primary and transformed cells, including T cells, human umbilical vein endothelial cells and human embryonic stem cells. We observed no cytotoxicity, minimal off-target transcriptional changes and no induction of innate immune responses. Thus, PTD-DRBD-mediated siRNA delivery allows efficient gene silencing in difficult-to-transfect primary cell types.
authors.affiliation: Howard Hughes Medical Institute, La Jolla, California, USA.
authors.forename: Akiko
authors.initials: A
authors.lastname: Eguchi
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Meade
authors.forename: Yung-Chi
authors.initials: YC
authors.lastname: Chang
authors.forename: Craig T
authors.initials: CT
authors.lastname: Fredrickson
authors.forename: Karl
authors.initials: K
authors.lastname: Willert
authors.forename: Nitin
authors.initials: N
authors.lastname: Puri
authors.forename: Steven F
authors.initials: SF
authors.lastname: Dowdy
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D053595:Embryonic Stem Cells; D004847:Epithelial Cells; D005434:Flow Cytometry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes; D014471:Umbilical Veins; D000097573:Red Fluorescent Protein
chemical_list: D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C043912:peptide permease