Efficient siRNA Delivery Into Primary Cells by a Peptide Transduction domain-dsRNA Binding Domain Fusion Protein

Nat Biotechnol. 2009 Jun;27(6):567-71. doi: 10.1038/nbt.1541. Epub 2009 May 17.

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
  • T-Lymphocytes
  • Umbilical Veins

Substances

  • Luminescent Proteins
  • Membrane Transport Proteins
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • red fluorescent protein
  • Green Fluorescent Proteins
  • peptide permease
  • Glyceraldehyde-3-Phosphate Dehydrogenases