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Research Summary• Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability.• Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization.• An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or β -galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis.• The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo . This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.

Protein transduction domains comprised of basic amino acid-rich peptides, can efficiently deliver covalently fused macromolecules into cells. Quantum dots (QDs) are luminescent semiconductor nanocrystals that are finding increasing application in biological imaging. Previous studies showed that protein transduction domains mediate the internalization of covalently attached QDs. In this study, we demonstrate that arginine-rich intracellular delivery peptides (cell-penetrating peptides; CPPs), analogs of naturally-occuring protein transduction domains, deliver noncovalently associated QDs into living cells; CPPs dramatically increase the rate and efficiency of cellular uptake of QD probes. The optimal molecular ratio between arginine-rich CPPs and QD cargoes for cellular internalization is approximately 60:1. Upon entry into cells, the QDs are concentrated in the perinuclear region. There is no cytotoxicity following transport of QDs present at concentrations up to 200 nM. The mechanism for arginine-rich CPP/QD complexes to traverse cell membrane appears to involve a combination of internalization pathways. These results provide insight into the mechanism of arginine-rich CPP delivery of noncovalently attached cargoes, and may provide a powerful tool for imaging in vivo.

The delivery and expression of exogenous genes in plant cells have been of particular interest for plant research and biotechnology. Here, we present results demonstrating a simple DNA transfection system in plants. Short arginine-rich intracellular delivery peptide, a protein transduction domain, was capable of delivering plasmid DNA into living plant cells non-covalently. This peptide-mediated DNA delivery conferred several advantages, such as nuclear targeting, non-toxic effect, and ease of preparation without protoplast formulation. Thus, this novel technology shall provide a powerful tool to investigate gene function in vivo, and lay the foundation for the production of transgenic plants in future.

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