A gene delivery method mediated by three arginine-rich cell-penetrating peptides in plant cells

Abstract

The transient gene delivery systems are generally mediated by viral infection, particle bombardment, electroporation, and microinjection for transferring exogenous DNAs into host cells. Recently, the peptide-mediated DNA delivery system became a novel tool for gene transfer, and these peptides, such as cell-penetrating peptides (CPPs), contained the ability of permeating plasma membranes and carrying cargoes to enter cells. In this study, we use three different arginine-rich CPPs, synthetic nona-arginine (SR9), histidine-rich nona-arginine (HR9), and Pas nona-arginine (PR9), for gene delivery into root-tip cells of mung bean and rice. We demonstrate that these arginine-rich CPPs have the ability to transfer Cyanine 3 (Cy3)-labeled DNAs into plant cells. Moreover, the CPP-mediated DNA transfer in cells was confirmed by polymerase chain reaction (PCR) analysis. Finally, our results of the functional gene assay indicated that DNA exerts its biological activity after intracellular delivery via CPPs. This CPP-mediated gene delivery method is characterized by the simplicity, noninvasion, and efficiency. Three arginine-rich CPPs may be ideal tools for the research of plant gene transformation.

Endocytic Trafficking of Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a Penetration Accelerating Sequence

“…TEM analysis revealed the endocytic trafficking of PR9/QD complexes in cells. A reporter gene assay confirmed that plasmid DNA delivered by PR9s can be actively expressed by cells [49]. Zeta-potentials of CPP/cargo complexes correlated with transduction efficiency [48], emphasizing the importance of electrostatic interactions of PR9/QD complexes with plasma membranes.…”

Intracellular Delivery of Nanoparticles and DNAs by IR9 Cell-penetrating Peptides

“…CPPs can deliver cargoes with sizes up to 200 nm in diameter [17]. Our laboratory has used arginine-rich CPPs to deliver proteins [18]–[26], DNAs [27]–[33], RNAs [34] and nanoparticles [35]–[39] into cells from various species. The internalization kinetics of CPPs is rapid, with a first-order rate constant of 0.007 sec −1 [40].…”

Cell-Penetrating Peptides

“…Still the majority of CPPs known and actively used for the delivery of bioactive cargoes obey the rules postulated two decades ago. The family of protein-derived CPPs quickly expanded including novel highly cationic peptides deduced from vascular epithelial cadherin (pVEC) [ 42 ], herpes (VP22) [ 43 ] and other viruses [ 44 ], and homeoproteins Islet-1 [ 45 ], Engrailed-2 [ 46 ], and others [ 47 ]. The sequences from human calcitonin [ 48 , 49 ] and lactoferrin [ 50 ] have also been intensely investigated and developed as well as peptides from toxins crotamine [ 51 ] and maurocalcine [ 52 , 53 ].…”

“…It remains that, in contrast with Tat, endocytosis is not a prerequisite for penetratin transfer into the cytoplasm and nucleus. Among modifi ers of penetratin uptake are the highly negatively charged carbohydrates that surround most cells, in particular glycosaminoglycans (GAGs) [ 46 ]. The complex sugars could restrict penetratin access to the membrane, promote penetratin…”

“…First, in 1988, two independent research groups demonstrated the shuttling properties for an HIV tat trans-activator protein [2,3]. Second, in 1991, the group of Alain Prochiantz reported [4] on cellular internalization of the 60 aa homeodomain of Antennapedia (a Drosophila homeoprotein), followed in 1994 by the report on a short 16 aa peptide, pAntp (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), later named penetratin, which was necessary and suffi cient for this translocation [5].…”