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).