(8f) Histidine-Mediated Hydrogen Bonding Enhances Stability and Silencing Activity of Peptide siRNA Polyplexes

Branched peptides containing histidines and lysines (HK) have been determined to be effective carriers for DNA or siRNA.  We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the polyplex.  In siRNA silencing experiments with mammalian cells, a four-branched HK peptide siRNA polyplex maintained silencing activity even with prolonged pre-incubation with serum.  In contrast, siRNA in complex with four-branched N3K4b peptide, in which histidines were substituted with asparagines, showed a marked decreased in silencing activity with pre-exposure to serum.  Consequently, we explored the hypothesis that histidines might form non-covalent bonds with nucleic acids to enhance the stability of siRNA polyplexes.  To accomplish this, we initially compared the biophysical properties of H3K(+H)4b with N3K4b and polylysine.  Consistent with siRNA silencing experiments, gel electrophoresis analysis demonstrated that the HK siRNA polyplex maintained its integrity for more than 24 h incubation in 50% serum, whereas siRNA in complex to N3K4b or polylysine was degraded in a time-dependent manner.  We next studied the thermodynamic profiles of various peptides binding to siRNA at pH 7.3 with isothermal titration calorimetry.  While four-branched polylysine and linear lysine-alanine peptide (K4b and A2K) interacted with siRNA resulting in an endothermic reaction, branched and linear lysine-histidine peptides (H3K(+H)4b and H2K) both exhibited an exothermic reaction at the initial binding step.  This indicates that an important part of the exothermic interaction was based on non-ionic bond formation of histidines with siRNA.  To investigate the type of non-ionic bond, we studied the protonation state of the imidazole ring of a selectively N15 labeled H3K(+H)4b upon siRNA binding with heteronuclear single quantum coherence (HSQC) NMR.  The peak of N^δ1-protonated tautomers of imidazole (type α, neutral form) shifted downfield (in the direction of deprotonation) by 0.5 to 1.0 ppm with addition of siRNA at pH 7.3, providing direct evidence that uncharged histidines formed hydrogen bonds with siRNA at physiological pH.  Moreover, unshifted N^ε1 tautomers described the directionality of hydrogen bonds.  Taking together, these results indicate that histidine-rich peptides form non-ionic bonds, and in particular hydrogen bonds, with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.