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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Cell Line
-
Cell Line, Tumor
-
Cell Survival / drug effects
-
Embryonic Stem Cells
-
Epithelial Cells
-
Flow Cytometry
-
Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
-
Green Fluorescent Proteins / genetics
-
Green Fluorescent Proteins / metabolism
-
Humans
-
Jurkat Cells
-
Luminescent Proteins / genetics
-
Luminescent Proteins / metabolism
-
Membrane Transport Proteins / genetics
-
Membrane Transport Proteins / metabolism*
-
Mice
-
Protein Structure, Tertiary
-
RNA Interference*
-
RNA, Small Interfering* / genetics
-
RNA, Small Interfering* / metabolism
-
RNA-Binding Proteins / genetics
-
RNA-Binding Proteins / metabolism*
-
Recombinant Fusion Proteins / genetics
-
Recombinant Fusion Proteins / metabolism
-
Red Fluorescent Protein
-
T-Lymphocytes
-
Umbilical Veins
Substances
-
Luminescent Proteins
-
Membrane Transport Proteins
-
RNA, Small Interfering
-
RNA-Binding Proteins
-
Recombinant Fusion Proteins
-
Green Fluorescent Proteins
-
peptide permease
-
Glyceraldehyde-3-Phosphate Dehydrogenases