Cell-Penetrating Peptide-Mediated Delivery of Peptide Nucleic Acid (PNA) Oligomers
This protocol was adapted from “Cell-Penetrating Peptide-Mediated Delivery of Peptide Nucleic Acid Oligomers,” Chapter 59, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.INTRODUCTION
Techniques for knockdown of specific genes and nonviral DNA delivery are important for studying protein functions, and are a means for future therapeutics. The problems with many techniques are low bioavailability and toxicity, especially in vivo. Some cationic peptides are able to translocate across the plasma membrane, and more importantly, to carry a cargo many times their own size, which may improve the bioavailability of hydrophilic macromolecules. These cationic peptides, often referred to as cell-penetrating peptides, have been used to transport a wide variety of cargos, including gene-regulating oligonucleotides and analogs, both in vitro and in vivo. One oligonucleotide analog that shows great potential in protein knockdown is peptide nucleic acid (PNA). PNA is a DNA-mimicking molecule that binds more strongly to DNA and RNA than DNA itself does and is more stable than its DNA counterpart. Furthermore, it is also easily synthesized and modified using standard peptide synthesis protocols. This protocol presents a synthesis strategy for covalently linking a cell-penetrating peptide (CPP) to PNA using a disulfide bridge. This can improve existing protocols for protein knockdown as well as DNA delivery, due to its relatively high efficacy and low toxicity.
