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Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

Brian R. McNaughton, James J. Cronican, David B. Thompson and David R. Liu

PNAS 106 (15): 6111-6116, 14 April 2009.
doi: 10.1073/pnas.0807883106

This week’s shrewd summary/analysis–impressively his third contribution to this blog–by David Weinberg:

In their 2009 PNAS paper, David Liu and colleagues demonstrate that a green fluorescent protein (GFP) variant that has been engineered to have a positively charged surface can penetrate mammalian cells and also chaperone nucleic acids into those same cells. The story began in 2007 when Liu’s lab published their initial characterization of so-called “supercharged” proteins. The motivation was to determine how changing the net charge of a protein can affect its stability. To do this, they began with an extra-stable GFP and changed as many surface-exposed residues as possible to positively charged residues (i.e., lysine and arginine). This resulted in a GFP variant with a net charge of +36 (herein referred to as +GFP) that folded and fluoresced similarly to the original GFP. Amazingly, however, +GFP was highly resistant to aggregation: boiling of the protein eliminated activity, but cooling the boiled protein restored most of the activity. This stabilizing effect of supercharging was not unique to GFP, as similarly supercharged variants of GST (a dimer) and streptavidin (a tetramer) showed similar properties.

So what does all of this have to do with RNA? In 2007, Liu’s lab also noted that +GFP could use its positively charge surface as “molecular Velcro” that can reversibly bind to RNA (tRNA) or DNA (plasmid dsDNA). Of course, +GFP-bound RNA is useless (to a first approximation) in a test tube. Where the PNAS paper begins is with the hypothesis that +GFP might be able to enter cells and thereby escort its bound nucleic acid cargo into the cell as well. The authors begin by conclusively showing that +GFP penetrate a variety of mammalian cells with an efficiency that varies with its charge. To satisfy the cell biologists in the audience, they provide an initial dissection of the mechanism of +GFP uptake using a variety of (mostly chemical) perturbations. From this, they conclude that the mechanism involves energy-dependent endocytosis that is dependent on actin polymerization and sulfated (positively charged) cell surface peptidoglycans but does not require caveolin or clathrin. Now focusing back on RNA, the authors demonstrate that +GFP can bind to siRNAs in vitro – not surprising given their previously published data showing that it can bind to tRNA. Moreover, +GFP-bound Cy3-labeled siRNAs can enter HeLa cells – their FACS data suggest that virtually all cells in the population take up the siRNAs to a similar degree, yielding a quite homogenous population of “transfected” cells. Although Lipofectamine (a standard transfection reagent) can also deliver Cy3-siRNA to HeLa cells, +GFP delivers ~100-fold more siRNA based on fluorescence. More impressively, in 4 other cell lines that are virtually resistant to Lipofectamine-mediated transfection, +GFP delivers huge amounts of Cy3-siRNA without any significant cytotoxicity. Not only do these siRNAs enter the cell, but they can interact with the RNA interference machinery and mediate gene silencing. Further characterization of +GFP-siRNA complexes reveals that +GFP enhances the stability of siRNA in serum and the protein is itself relatively stable in serum. Although less impressive in its efficiency, +GFP (with an HA tag) can also be used to transfect plasmid DNA that gets expressed in the nucleus.

The authors conclude that +GFP may provide an attractive alternative to nucleic acid delivery. Because it uses a general pathway (endocytosis) for delivery, it should in theory work in all cell types. In addition, it is relatively easy to use since it only requires mixing of recombinant +GFP (which can be readily made in E. coli) and the nucleic acid of interest, and adding this to cells. But beyond this immediate application as a transfection reagent, it seems that +GFP and supercharged proteins more generally may become a useful tool for all sorts of biology (which I have no doubt the Liu lab is already exploring).

Citation for researchblogging.org:

McNaughton BR, Cronican JJ, Thompson DB, & Liu DR (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), 6111-6 PMID: 19307578