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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Caplen, N. J., Parrish, S., Imani, F., Fire, A., and Morgan, R. A. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA. 98, 9742–9747.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.
Zhang, S., Zhao, B., Jiang, H., Wang, B., and Ma, B. (2007) Cationic lipids and polymers mediated vectors for delivery of siRNA. J. Control Release 123, 1–10.
Endoh, T., Sisido, M., and Ohtsuki, T. (2008) Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference. Bioconjug. Chem. 19, 1017–1024.
Murriel, C. L., and Dowdy, S. F. (2006) Influence of protein transduction domains on intracellular delivery of macromolecules. Expert Opin. Drug Deliv. 3,739–746.
Wadia, J. S., and Dowdy, S. F. (2002) Protein transduction technology. Curr. Opin. Biotechnol. 13, 52–56.
Brooks, H., Lebleu, B., and Vives, E. (2005) Tat peptide-mediated cellular delivery: back to basics. Adv. Drug Deliv. Rev. 57, 559–577.
Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315.
Abes, S., Williams, D., Prevot, P., Thierry, A., Gait, M. J., and Lebleu, B. (2006) Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. J. Control Release 110, 595–604.
Michiue, H., Tomizawa, K., Wei, F. Y., Matsushita, M., Lu, Y. F., Ichikawa, T., et al. (2005) The NH2 terminus of influenza virus hemagglutinin-2 subunit peptides enhances the antitumor potency of polyarginine-mediated p53 protein transduction. J. Biol. Chem. 280, 8285–8289.
Maiolo, J. R., 3rd, Ottinger, E. A., and Ferrer, M. (2004) Specific redistribution of cell-penetrating peptides from endosomes to the cytoplasm and nucleus upon laser illumination.J. Am. Chem. Soc. 126, 15376–15377.
Matsushita, M., Noguchi, H., Lu, Y. F., Tomizawa, K., Michiue, H., Li, S. T., et al. (2004) Photo-acceleration of protein release from endosome in the protein transduction system. FEBS Lett. 572, 221–226.
Acknowledgments
We thank Prof. M. Sisido (Okayama University) for valuable discussions. This work was supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO) (05A02707a).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Endoh, T., Ohtsuki, T. (2010). Cellular siRNA Delivery Using TatU1A and Photo-Induced RNA Interference. In: Min, WP., Ichim, T. (eds) RNA Interference. Methods in Molecular Biology, vol 623. Humana Press. https://doi.org/10.1007/978-1-60761-588-0_17
Download citation
DOI: https://doi.org/10.1007/978-1-60761-588-0_17
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-587-3
Online ISBN: 978-1-60761-588-0
eBook Packages: Springer Protocols