Cellular siRNA delivery using TatU1A and photo-induced RNA interference

Methods Mol Biol. 2010:623:271-81. doi: 10.1007/978-1-60761-588-0_17.

Abstract

RNA interference (RNAi)-mediated silencing of specific genes represents a powerful tool for analyzing protein function. It also has profound biotechnological applications for cellular engineering and therapeutics. However, it is necessary to have a method that controls RNAi in response to artificially regulated stimulation. We designed a fluorescently labeled carrier protein to deliver short hairpin RNA (shRNA) with activity that could be regulated via photostimulation. We constructed a cell-permeable RNA-binding protein (RBP) by fusing the U1A RBP and a HIV-1 Tat peptide, which was labeled with an Alexa Fluor 546 fluorophore (TatU1A-Alexa). TatU1A-Alexa bound specifically to shRNA, which contains a U1A-binding sequence. The TatU1A-Alexa/shRNA complex was then internalized into cells via an endocytotic pathway and redistributed from endosomes to the cytosol by photostimulation, which induced RNAi-mediated gene silencing. This successive strategy was termed CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Endocytosis
  • Photobiology / methods*
  • RNA Interference*
  • RNA, Small Interfering / genetics*
  • RNA-Binding Proteins / genetics
  • tat Gene Products, Human Immunodeficiency Virus / genetics*
  • tat Gene Products, Human Immunodeficiency Virus / isolation & purification

Substances

  • RNA, Small Interfering
  • RNA-Binding Proteins
  • tat Gene Products, Human Immunodeficiency Virus