Mammalian Cell Penetration,siRNA Transfection, and DNA Transfection by Supercharged Green Fluorescent Proteins

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Mammalian Cell Penetration,siRNA Transfection, and DNA Transfection by Supercharged Green Fluorescent Proteins

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Title: Mammalian Cell Penetration,siRNA Transfection, and DNA Transfection by Supercharged Green Fluorescent Proteins
Author: McNaughton, Brian R.; Cronican, James Joseph; Thompson, David Brandon; Liu, David Ruchien

Note: Order does not necessarily reflect citation order of authors.

Citation: McNaughton, Brian. R., James J. Cronican, David B. Thompson, and David R. Liu. 2009. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proceedings of the National Academy of Sciences of the United States of America 106(15): 6111-6116.
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Abstract: Nucleic acid reagents, including small interfering RNA (siRNA) and plasmid DNA, are important tools for the study of mammalian cells and are promising starting points for the development of new therapeutic agents. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. We recently described the extensive surface mutagenesis of proteins in a manner that dramatically increases their net charge. Here, we report that superpositively charged green fluorescent proteins, including a variant with a theoretical net charge of +36 (+36 GFP), can penetrate a variety of mammalian cell lines. Internalization of +36 GFP depends on nonspecific electrostatic interactions with sulfated proteoglycans present on the surface of most mammalian cells. When +36 GFP is mixed with siRNA, protein–siRNA complexes ≈1.7 μm in diameter are formed. Addition of these complexes to five mammalian cell lines, including four that are resistant to cationic lipid-mediated siRNA transfection, results in potent siRNA delivery. In four of these five cell lines, siRNA transfected by +36 GFP suppresses target gene expression. We show that +36 GFP is resistant to proteolysis, is stable in the presence of serum, and extends the serum half-life of siRNA and plasmid DNA with which it is complexed. A variant of +36 GFP can mediate DNA transfection, enabling plasmid-based gene expression. These findings indicate that superpositively charged proteins can overcome some of the key limitations of currently used transfection agents.
Published Version: http://dx.doi.org/10.1073/pnas.0807883106
Other Sources: http://evolve.harvard.edu/51-gfp.pdf
Terms of Use: This article is made available under the terms and conditions applicable to Open Access Policy Articles, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#OAP
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:3426893

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  • FAS Scholarly Articles [7374]
    Peer reviewed scholarly articles from the Faculty of Arts and Sciences of Harvard University
 
 

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