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For efficient gene delivery in plant systems, nonviral vector and DNA complexes require extracellular stability, cell wall/membrane translocation capability, and the ability to mediate both endosomal escape and intracellular DNA release. Peptides make appealing gene delivery vectors due to their DNA-binding, cell-penetrating, and endosome escape properties. However, DNA release within cells has so far been inefficient, which results in poor and delayed gene expression, while the lack of understanding of both internalization and trafficking mechanisms is a further obstacle to the design of efficient peptide gene delivery vectors. Here, we report successful gene delivery into plants using a cellular environment-responsive vector, BPCH7, which is an efficient cell-penetrating peptide with a cyclic DNA-binding domain that is formed by a disulfide bond between two cysteines. The cyclic structure of BPCH7 confers high avidity attachment to DNA in vitro. Following endocytosis into cells, disulfide bond cleavage facilitated by intracellular glutathione induces structural changes within BPCH7 that enable the release of the associated DNA cargo. Comparative studies with BPKH, a cell-penetrating peptide with a linear DNA-binding domain, show that BPCH7 maximized and expedited gene transfer in cells and unveil for the first time the crucial role of plant stomata in the internalization of peptide-DNA complexes.

Stimulus-Responsive Peptide for Effective Delivery and Release of DNA in Plants