Enhanced Binding Affinity of siRNA Overhang-Binding Fluorescent Probes by Conjugation with Cationic Oligopeptides for Improved Analysis of the siRNA Delivery Process

Carrier-mediated delivery of small interfering RNAs (siRNAs) into the living cells is important for the realization of siRNA therapeutics that can silence target genes through RNA interference. We recently proposed a new strategy for analyzing the siRNA delivery process based on affinity labeling with a peptide nucleic acid (PNA)-based fluorescent probe (PyAATO; Py: pyrene, A: adenine; TO: thiazole orange) capable of selectively binding to the overhanging structures of siRNAs. We have prepared new probes with improved binding affinity by conjugation with a cationic oligopeptide. The probe, carrying six lysine residues (PyAATO-Lys ₆ (Lys6)), displayed a 39-fold increase in affinity, compared with that of the parent probe containing no oligopeptides. Thermodynamic characterization revealed that enhanced affinity resulted from the favorable polyelectrolyte effect, due to the electrostatic interaction between lysine residues and phosphate anions of the RNA duplexes near the overhanging structure. Lys6 showed the improved imaging ability of the carrier-mediated siRNA delivery process in living cells, in which 20 nm siRNA could be analyzed and was considered to show the minimal off-target effects.