Delivery of cell-penetrating peptide-peptide nucleic acid conjugates by assembly on an oligonucleotide scaffold.

Xing-Liang Zhao,Lai Wei,Xiao-Ben Pan,Bi-Cheng Chen,Jin-Chao Han

doi:10.1038/srep17640

Abstract

Delivery to intracellular target sites is still one of the main obstacles in the development of peptide nucleic acids (PNAs) as antisense-antigene therapeutics. Here, we designed a self-assembled oligonucleotide scaffold that included a central complementary region for self-assembly and lateral regions complementing the PNAs. Assembly of cell-penetrating peptide (CPP)-PNAs on the scaffold significantly promoted endocytosis of PNAs by at least 10-fold in cell cultures, particularly for scaffolds in which the central complementary region was assembled by poly(guanine) and poly(cytosine). The antisense activity of CPP-PNAs increased by assembly on the scaffold and was further enhanced after co-assembly with endosomolytic peptide (EP)-PNA. This synergistic effect was also observed following the assembly of antigene CPP-PNAs\\EP-PNAs on the scaffold. However, antigene activity was only observed by targeting episomal viral DNA or transfected plasmids, but not the chromosome in the cell cultures. In conclusion, assembly on oligonucleotide scaffolds significantly enhanced the antisense-antigene activity of PNAs by promoting endocytosis and endosomal escape. This oligonucleotide scaffold provided a simple strategy for assembly of multiple functional peptide-PNA conjugates, expanding the applications of PNAs and demonstrating the potential of PNAs as antiviral therapeutics.

Highlights

Is most efficient within AT-rich regions and at inverted repeats capable of forming cruciforms[12,13,14,15,16,17,18,19]
Different lengths of flanking oligonucleotides were tested to identify the appropriate balance of the association and disassociation between the Peptide nucleic acids (PNAs) and oligonucleotides; this was expected to affect the assembly of the PNA-oligonucleotide scaffold in the test tube and disassociation of the PNAs from the oligo scaffold following endocytosis
We developed a self-assembled oligonucleotide scaffold that was capable of assembling with multiple peptide-PNAs and greatly improved the efficiency of cell-penetrating peptides (CPPs)-PNA uptake and endosomal escape

Summary

Introduction

Is most efficient within AT-rich regions and at inverted repeats capable of forming cruciforms[12,13,14,15,16,17,18,19] This invasion of duplex DNA by the neutral PNA backbone suggests that antigene PNAs (agPNAs) may be important agents for inhibiting the transcription of genes within cells. A variety of cellular delivery systems have been developed during the last few years These include microinjection, electroporation, cotransfection with DNA, or conjugation to lipophilic moieties, nanoparticles, cell-penetrating peptides (CPPs), oligo-aspartic acid, or nuclear localization signal (NLS) peptides to enhance cellular internalization. In order to overcome the cell membrane barrier and endosomal entrapment of intracellular CPP-PNAs, we designed a self-assembled oligonucleotide scaffold that was capable of assembling with multiple PNA conjugates modified by various functional moieties. We used the hepatitis B virus (HBV) genome as a target for evaluating the activity of the PNA-oligonucleotide scaffold complex in various cell lines

Methods

Results

Conclusion