The DNA–carbon nanotube binding mode determines the efficiency of carbon nanotube-mediated DNA delivery to intact plants

Efficient delivery of DNA, RNA, and genome engineering machinery to plant cells will enable efforts to genetically modify plants for global food security, sustainable energy production, synthetic biology applications, and climate change resilience. For the delivery of functional genetic units into plant cells, charged nanoparticles, particularly carbon nanotubes (CNT), have attracted considerable interest. Although some success has been achieved using CNT-based approaches, the efficiency, batch reproducibility, and the limits of their applicability remain to be assessed. Here, we provide a mechanistic understanding of plasmid DNA-loaded CNTs based transfection of plant cells, and factors affecting the expression of the transformed plasmid. We show that transfection is inherently limited by the presence of the cell wall, Coulomb interactions between DNA and polymer coated CNT, and DNA size, whereas expression of the transformed plasmid is limited by relative gene-to-plasmid size and the intracellular accessibility of DNA. We further show that the formation of partially condensed DNA on the CNT surface is a prerequisite for successful transfection and expression. Furthermore, DNA does not detach completely from the CNT, so the accessibility of the transcription machinery to DNA is the key for transformation efficiency. This irreversible DNA plasmid binding and partial condensation limit the length of DNA that can be expressed, thus negatively affecting efficiency and reproducibility. Understanding the underlying mechanisms and limitations of CNT-mediated delivery of DNA through the plant cell wall is of considerable importance in guiding efforts to design nanomaterials for efficient transformation, trait engineering, and synthetic biology applications.