PNA for knock-down of miR-9 microRNA in ascidian embyos

MicroRNAs are non-coding transcripts of approximately 21 nucleotides, highly conserved in different metazoans and acting as post- transcriptional regulators of gene expression. Peptide Nucleic Acids (PNA) are pseudopeptide chains bearing the four nucleobases mimicking the antisense oligonucleotide base-matching properties. PNAs exhibit stronger and more selective binding affinity for complementary nucleic acid strands than natural nucleic acids and can be used in efficient antisense approaches. We tested the efficiency of these molecules to block miRNAs by microinjecting in ascidian eggs different concentrations of anti-miR 9 and anti-miR 124 PNAs. These miRNAs are known to be expressed preferentially in the nervous system of vertebrates. Ascidians are sessile invertebrate chordates, which develop through a swimming larva that shows the basic chordate features, comprising a notochord, which runs the length of the tail, and a dorsal tubular central nervous system (CNS). Moreover, ascidian larvae possess a peripheral nervous system (PNS), mainly composed by multiple series of epidermal sensory neurons. In the ascidian Ciona intestinalis, miR-124 is expressed in all the nervous system; mir-9 expression can be detected in few cells of the CNS directly connected to the otolith, a gravity sensory organ. Embryos developed by eggs injected with anti miR-9 PNA showed a negative regulation of peripheral epidermal sensory neuron differentiation, as evidenced by the suppression of gene marker expression. Injection of anti- miR-124 PNAs had no detectable effects at the tested concentrations. These results demonstrated the possibility to use PNAs in antisense experiments in a chordate model organism.