Nucleic Acids Research Advance Access originally published online on November 28, 2006
Nucleic Acids Research 2006 34(22):6561-6573; doi:10.1093/nar/gkl941
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2006, Vol. 34, No. 22 6561-6573
© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
RNA |
Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect
Institut für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Ratzeburger Allee 160 23538 Lübeck, Germany
*To whom correspondence should be addressed. Tel: +49 451 500 2745; Fax: +49 451 500 2729; Email: restle{at}imm.uni-luebeck.de
Received October 9, 2006. Accepted October 18, 2006.
Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPG
, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPG-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPG/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPG/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPG/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPG-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Crombez, M. C. Morris, S. Dufort, G. Aldrian-Herrada, Q. Nguyen, G. Mc Master, J.-L. Coll, F. Heitz, and G. Divita Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth Nucleic Acids Res., August 1, 2009; 37(14): 4559 - 4569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R. McNaughton, J. J. Cronican, D. B. Thompson, and D. R. Liu Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins PNAS, April 14, 2009; 106(15): 6111 - 6116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Juliano, Md. R. Alam, V. Dixit, and H. Kang Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides Nucleic Acids Res., July 1, 2008; 36(12): 4158 - 4171. [Abstract] [Full Text] [PDF]
|
|