title: Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds.
abstract: Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and 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 HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine-lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively (15)N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1-H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5-1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.
authors.affiliation: Department of Pathology, University of Maryland Baltimore, MSTF Building, 10 South Pine Street, Baltimore, MD 21201, United States; Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States. Electronic address: stchou@umd.edu.
authors.forename: Szu-Ting
authors.initials: ST
authors.lastname: Chou
authors.forename: Kellie
authors.initials: K
authors.lastname: Hom
authors.forename: Daoning
authors.initials: D
authors.lastname: Zhang
authors.forename: Qixin
authors.initials: Q
authors.lastname: Leng
authors.forename: Lucas J
authors.initials: LJ
authors.lastname: Tricoli
authors.forename: Jason M
authors.initials: JM
authors.lastname: Hustedt
authors.forename: Amy
authors.initials: A
authors.lastname: Lee
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shapiro
authors.forename: Joonil
authors.initials: J
authors.lastname: Seog
authors.forename: Jason D
authors.initials: JD
authors.lastname: Kahn
authors.forename: A James
authors.initials: AJ
authors.lastname: Mixson
mesh_terms: D001216:Asparagine / Q000737:chemistry; D045744:Cell Line, Tumor; D020868:Gene Silencing; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D034741:RNA, Small Interfering / Q000737:chemistry*; D014162:Transfection
chemical_list: D010455:Peptides; D011108:Polymers; D034741:RNA, Small Interfering; D011107:Polylysine; D006639:Histidine; D001216:Asparagine; D008239:Lysine
keywords: HK peptide; Histidine; Hydrogen bond; Isothermal titration calorimetry; Nuclear magnetic resonance; siRNA