riDOM, a Cell-Penetrating Peptide. Interaction with DNA and Heparan Sulfate

Abstract

DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 10(7) M(-1) and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 10(6) M(-1) and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 10(6) M(-1). Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin.

A retro-inverso cell-penetrating peptide for siRNA delivery

“…Our results revealed that this retro - inverso peptide RICK retained the main unique properties of CADY-K such as conformational versatility, formation of RICK:siRNA nanoparticles and remarkable efficacy in siRNA cellular delivery. Interestingly, in contrast to other retro - inverso peptides [51, 62], we observed a structural difference concerning the tryptophan cluster between CADY-K ( l -isomer), d -cady-k ( d -(isomer) and RICK, highlighting the important role of this amino acid. Moreover, because tryptophan has emerged as an important amino acid for membrane translocation [63, 64], a tryptophan clustering is a potential feature that should be taken into account for future PBN designs.…”

“…In sum, we demonstrated through three approaches that RICK is more stable against proteolysis phenomena (trypsin or serum) compared to CADY-K. To our knowledge, only Seelig and Coll are working on retro - inverso CPP in a non-covalent strategy (riDOM:plasmid) [51, 52]. However, they did not look at trypsin or serum resistance of their peptide.…”

Peptide and protein mimetics by retro and retroinverso analogs

“…Retroinverso melittin has fused to dioleoyl phosphoethanolamine for making a gene therapy vehicle riDOM, which has DNA binding as well as cell penetrating properties. Milletin has cell penetration properties whereas dioleoyl phosphoethanolamine binds DNA (Ngoei et al, 2013;Québatte, Kitas, & Seelig, 2013).…”

The Formation of Nanoparticles between Small Interfering RNA and Amphipathic Cell-Penetrating Peptides

“…The same could apply of PF/NFs, as they are also quite hydrophobic. A similar layer theory was proposed by Québatte et al., 44 where the authors speculated that a portion of riDOM, their developed CPP, is not involved in DNA binding and will instead bind to a sulfated GAG, thereby starting an endocytotic translocation process. Nanoparticles formed with CPPs have been reported to use scavenger receptors for internalization and also interact with heparin sulfate proteoglycans,13, 14, 15, 24, 45 indicating the presence of CPP able to interact with cell membrane structures.…”