Phenylboronic acid ligands enhance peptide-based messenger RNA delivery into root tissues in diverse vascular plants

Abstract Efficient nucleic acid delivery across diverse plants remains challenging because carrier performance often depends on plant species and tissue type. Herein, we developed peptide-based messenger RNA (mRNA)-loaded polyplex micelles decorated with phenylboronic acid (PBA) ligands designed based on the conserved boric acid uptake system in the roots of vascular plants. Among the tested PBA ligands, installation of 3-methyl-substituted PBA possessing a relatively high acid dissociation constant significantly increased cellular mRNA uptake and reporter gene expression in the root tissues of Arabidopsis thaliana , Oryza sativa and Nicotiana benthamiana , revealing the applicability of the same ligand design to both monocot and eudicot species. The PBA ligand effect was pH dependent. All the tested PBA derivatives enhanced mRNA uptake under acidic conditions, where PBA derivatives predominantly adopt uncharged trigonal structures, whereas no enhancement was observed under alkaline conditions, where negatively charged tetrahedral forms predominate. Furthermore, reduced boric acid availability in the culture medium increased the expression of the boric acid channel (NOD26-like intrinsic protein 5;1, NIP5;1), accompanying increased cellular uptake. These findings reveal the substituent structure and ionization state of PBA ligands as important parameters for peptide-based nucleic acid carriers and provide a strategy for broadening transient mRNA-based expression technologies across vascular plants.