Targeted downregulation of select endogenous plant genes is known to confer disease or pest resistance in crops and is routinely accomplished via transgenic modification of plants for constitutive gene silencing. An attractive alternative to the use of transgenics or pesticides in agriculture is the use of a ‘green’ alternative known as RNAi, which involves the delivery of siRNAs that downregulate endogenous genes to confer resistance. However, siRNA is a molecule that is highly susceptible to enzymatic degradation and is difficult to deliver across the lignin-rich and multi-layered plant cell wall that poses the dominant physical barrier to biomolecule delivery in plants. We have demonstrated that DNA nanostructures can be utilized as a cargo carrier for direct siRNA delivery and gene silencing in mature plants. The size, shape, compactness and stiffness of the DNA nanostructure affect both internalization into plant cells and subsequent gene silencing efficiency. Herein, we provide a detailed protocol that can be readily adopted with standard biology benchtop equipment to generate geometrically optimized DNA nanostructures for transgene-free and force-independent siRNA delivery and gene silencing in mature plants. We further discuss how such DNA nanostructures can be rationally designed to efficiently enter plant cells and deliver cargoes to mature plants, and provide guidance for DNA nanostructure characterization, storage and use. The protocol described herein can be completed in 4 d.

已知选择性下调植物内源基因可赋予作物抗病或害虫的能力，并且通常通过对植物进行组成型基因沉默的转基因修饰来实现。在农业中使用转基因或杀虫剂的一个有吸引力的替代方案是使用称为 RNAi 的“绿色”替代方案，它涉及递送 siRNA，下调内源基因以赋予抗性。然而，siRNA 是一种非常容易被酶降解的分子，并且难以穿过富含木质素的多层植物细胞壁，而细胞壁对植物中的生物分子传递构成了主要的物理屏障。我们已经证明DNA纳米结构可以用作成熟植物中直接siRNA递送和基因沉默的货物载体。 DNA 纳米结构的大小、形状、紧密度和硬度影响植物细胞的内化以及随后的基因沉默效率。在此，我们提供了一个详细的协议，可以很容易地采用标准生物台式设备来生成几何优化的 DNA 纳米结构，用于成熟植物中的无转基因和力独立的 siRNA 传递和基因沉默。我们进一步讨论如何合理设计此类DNA纳米结构以有效进入植物细胞并将货物输送至成熟植物，并为DNA纳米结构表征、储存和使用提供指导。本文描述的协议可以在 4 d 内完成。