Side chain modified peptide nucleic acids (PNA) for knock-down of six3in medaka embryos

Sebastian Dorn,Narges Aghaallaei,Thomas Czerny,Birgit Werner,Gerlinde Jung,Thomas Lindhorst,Holger Bock,Baubak Bajoghli

doi:10.1186/1472-6750-12-50

Abstract

BackgroundSynthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications.ResultsWe generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes.ConclusionsPNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.

Highlights

Synthetic antisense molecules have an enormous potential for therapeutic applications in humans
Synthesis of novel peptide nucleic acids aminoethyl glycine PNAs (aegPNA) bind corresponding RNA sequences with high affinity and specificity, unfavourable properties like their low solubility hamper their application in vivo
By employing the building blocks shown in Additional file 1: Figure S1, we introduced phosphonic ester side chains into the otherwise non-modified backbone of aegPNAs

Summary

Introduction

Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. Antisense oligonucleotides are designed for sequence specific binding of complementary regions on their target mRNA. The ribose is replaced by morpholino rings and non-ionic phosphorodiamidate is used instead of phosphodiester linkages [3]. Their binding strength closely resembles that of RNA or DNA molecules, molecules with a length of 25 nucleotides are used for efficient transcriptional blockage. Today morpholino oligos represent the gold standard for gene specific knock down in many species

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