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Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells

Nature Biotechnology volume 17, pages 873–877 (1999)Cite this article

Abstract

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9–ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9–ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

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Figure 1: (A) Two-step reaction to conjugate a cationic peptide (ScT) to the M9 sequence of hnRNP A1.
Figure 2: Endosomal escape and detection of lipofected plasmid in the cytoplasm.
Figure 3: The effect of M9 sequence of hnRNP on transfection efficiency of confluent BAECs.
Figure 4: Lipofection of confluent BAECs with pCMVβgal in the presence or absence of peptides.
Figure 5: Total expression of β-galactosidase (β-gal) activity after complexation of pCMVβgal with peptides containing the M9 sequence.
Figure 6: Localization of rhodamine–plasmid complexed with M9–ScT in digitonin-permeabilized BAECs.

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Acknowledgements

S.L.D. is a recipient of a National Science Foundation National Young Investigator Award and National Institutes of Health Grant no. R21-14014 Award.

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  1. Department of Chemical Engineering, Institute for Medicine and Engineering, 1010 Vagelos Research Laboratories, University of Pennsylvania, Philadelphia, 19104, PA

    Ajit Subramanian, P. Ranganathan & Scott L. Diamond

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  1. Ajit Subramanian
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  3. Scott L. Diamond
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Correspondence to Scott L. Diamond.

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Subramanian, A., Ranganathan, P. & Diamond, S. Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells. Nat Biotechnol 17, 873–877 (1999). https://doi.org/10.1038/12860

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