Development of a Peptide Nucleic Acid Based siRNA Delivery System

Abstract

Small interfering RNA (siRNA) plays a major role in gene silencing. The ability to harness siRNA for therapeutic benefit can have a widespread impact on a variety of diseases. However, the efficacy of such treatments is limited due to the many barriers associated with siRNA delivery. In this work, we lay the groundwork for the development of a peptide nucleic acid (PNA)-based surface-mediated siRNA delivery system. PNAs are nucleic acid analogs that hybridize with complementary DNA or RNA sequences, enabling the direct attachment of various macromolecules such as peptides. Conjugation of targeting and protective moieties can potentially enhance the delivery of siRNA. We first established a cell transfection model utilizing stably transfected B16FO mouse melanoma cells producing GFP. Anti-GFP siRNA was designed and its efficacy was evaluated via flow cytometry and fluorescence microscopy. Optimization of cell seeding density, siRNA concentration, use of antibiotics, and time of transfections was accomplished. Our second task was to prepare molecular conjugates for PNA-based siRNA modifications. PNA-peptide conjugates were assembled and purified through Reverse Phase HPLC. Future development of this delivery system will include linkage of anti-GFP siRNA to surfaces via the PNA-peptide tethers, and exploration of the delivery system in B16FO cells.