Side chain modified peptide nucleic acids (PNA) for knock-down of six3 in medaka embryos

Abstract

Background: Synthetic 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.

Results: We 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.

Conclusions: PNAs 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.

Figure 1

Chemical structure of PNAs. Schematic structure of aegPNAs, pePNAs, mixed PNAs (containing both aeg- and pePNA monomers) and HypNA-pPNAs is shown. pePNAs contain C2-phosphonoester residues, mixed PNAs contain C3-residues.

Figure 2

Sequences of the antisense molecules and their targets. The mRNA targets are shown in the 5’-3’ orientation, capital letters indicate the coding region. The AUG start codon is marked by red overlay. A morpholino oligo is indicated by MO (Six3-MO). All other sequences represent PNAs, pePNA-C2 components are shown in black, pePNA-C3 components in red and aegPNAs components are marked by gray overlay. Underlined bases (black) represent mismatches. All PNA and morpholino oligos are shown in the 3’-5’ (NH2-H for PNAs) orientation. L means trimethyl-lysine and LLLL a combination of 4 such residues. SuRho indicates sulforhodamine B.

Figure 3

Translational blocking of pePNAs after injection of gfp mRNA into medaka embryos. The pePNAs were coinjected with gfp mRNA into medaka embryos at the one cell stage. 24 hours later the intensity of the gfp fluorescence was qualified as weak (B), moderate (C; mod) or strong (D). (A) Shows an uninjected embryo (no signal, corresponding to a complete knock down of gfp). The embryos are shown in a dorsal view, anterior to the top. A graph summarizing the experiments for optimisation of the PNA length is shown in (E). The names of the PNAs are explained in Figure 2. The embryos were injected with a mixture of 10 ng/μl gfp mRNA and 200 μM PNAs preincubated for 30 minutes on ice. After 24 hours the embryos were divided into groups according to their gfp signal intensity. Above the columns the calculated average gfp intensity in percent is indicated (see text for calculation). Similarly the results for mixed PNAs are shown in (F). Injections and evaluations were performed as in (E), except that 20 ng/μl mRNA were used. Note that the increased amount of mRNA results in higher numbers of average gfp intensity for comparable antisense function.

Figure 4

Injection of medaka embryos with Six3-PNAs. A wild-type embryo (no PNA injection) at 3 days is shown in (A). Embryos injected with 400 μM Ref16mix-PNA are indistinguishable from wild-type embryos; at 900 μM unspecific phenotypes were detected (both in A). The Six3mix-PNA injected embryos were evaluated 3 days after injection at stage 29. The phenotypes were divided into weak, moderate and strong (for criteria see text). Arrows indicate the anterior parts of the eyes pointing to the midline in weak phenotypes. The black arrowhead indicates remnants of the eye structures. All embryos in (A) are at stage 29 (34 somites). In (B) the expression of the bf1 gene in wild-type and strongly affected embryos injected with Six3mix-PNAs is shown (both at stage 22; 9 somites). Note the presence of bf1 expression in the otic vesicles, both in wild-type as well as in Six3mix-PNA injected embryos (marked by red arrowheads). Dorsal views of the anterior part of the embryos are shown. The quantitative evaluation of the six3 knock down experiments is shown in (C) and (D) for the indicated PNA molecules. The percentages of phenotypes for the diagrams were calculated from the surviving embryos. PNAs shown in (D) were injected at 400 μM. (E) Shows the mortality rate of embryos injected at the indicated concentration (survival was examined 24 hrs after injection, see also Additional file 5: Table S3).