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

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.

The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

“…At concentrations with high mortality rates, some embryos with unspecific phenotypes appeared which looked similar for both pePNAs and morpholino oligonucleotides. We previously described such unspecifically affected medaka embryos, which have smaller eyes but show severe defects over the entire brain [ 9 ]. Such unspecific phenotypes were not detected at non-toxic concentrations.…”

“…In particular combined versions containing both aeg non-modified- and pe-modified PNA blocks were most effective. In a direct comparison morpholino oligonucleotides were active at lower concentrations, but also showed higher toxicity [ 9 ].…”

The Genomic and Genetic Toolbox of the Teleost Medaka (Oryzias latipes)

“…The nonribose-based backbone renders morpholinos insensitive to enzymatic degradation. Recently, gene knockdown using peptide nucleic acids (PNAs) has been applied in medaka ( Dorn et al 2012 ). Both, morpholinos and PNAs are powerful antisense tools.…”

“…PNAs have a higher affinity for RNA, yet they are less soluble and therefore the in vivo use is limited. Dorn et al (2012) changed the chemical composition of the PNA backbone to increase solubility and showed efficient knockdown of the six3 gene in medaka. In most cases these antisense morpholinos are injected into fertilized eggs at early cleavage stages to ensure a ubiquitous distribution to all cells of the developing embryo.…”

Applied Molecular Cloning: Present and Future for Aquaculture

“…PNAs have a higher affinity for RNA, yet they are less soluble and therefore the in vivo use is limited. Dorn et al [51] changed the chemical composition of the PNA backbone to increase solubility and showed efficient knockdown of the six3 gene in medaka. In most cases, the chemical/RNA is micro-injected or electroporated into fertilized eggs at early cleavage stages to ensure a ubiquitous distribution to all cells of the developing embryo.…”