Abstract
A fusion protein comprising an antibody and a cell-penetrating peptide is a candidate molecule capable of efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and arginine-9 (R9) with the Q-tag sequence LLQGS at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 exhibited a maximum reaction efficiency of 85.1%, without changing the properties of the nanobody or R9. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced RNAi of target mRNA with approximately 52% efficiency compared to lipofectamine.
A Myristoylated Cell-Penetrating Peptide Bearing a Transferrin Receptor-Targeting Sequence for Neuro-Targeted siRNA Delivery