Synthesis, purification and characterization of the peptide dendrimer KK-46
The design of the cationic dendrimeric peptide KK-46 for intracellular delivery of siRNA was based on in silico calculation of the molecular properties (Chemsketch software, Dock Prep software, PyMOL software) to achieve an optimal range of positive charge and amphiphilicity for cell penetration and RNA-binding. The positive charge was attributable to the use of arginine and histidine residues for the N-terminal ends of the peptide branches (the basic sequences are described previously(29) ). The peptide for this study was de novo synthesized using automatic solid-phase peptide synthesis with Fmoc-protection strategy of α-amino groups (PS3 Peptide Synthesizer, Gyros Protein Technologies, Tucson, USA). The purification of the peptide was performed by means of preparative reverse-phase high performance liquid chromatography with ODS column (octadecylsilyl groups chemically bonded to a silica gel carrier) and 0.1% TFA and acetonitrile as the mobile phase. The peptide molecular mass was confirmed by matrix assisted laser desorption/ ionization time-of-flight (Bruker Daltonics Microflex LT MALDI-TOF, Bruker Corporation, Billerica, USA). The peptide was tested for its ability to inhibit the binding of RBD to ACE2 using a molecular interaction assay(30).