miR-7 Knockdown by Peptide Nucleic Acids in the Ascidian Ciona intestinalis

Abstract

Peptide Nucleic Acids (PNAs) are synthetic mimics of natural oligonucleotides, which bind complementary DNA/RNA strands with high sequence specificity. They display numerous advantages, but in vivo applications are still rare. One of the main drawbacks of PNAs application is the poor cellular uptake that could be overcome by using experimental models, in which microinjection techniques allow direct delivery of molecules into eggs. Thus, in this communication, we investigated PNAs efficiency in miR-7 downregulation and compared its effects with those obtained with the commercially available antisense molecule, Antagomir (Dharmacon) in the ascidian Ciona intestinalis. Ascidians are marine invertebrates closely related to vertebrates, in which PNA techniques have not been applied yet. Our results suggested that anti-miR-7 PNAs were able to reach their specific targets in the developing ascidian embryos with high efficiency, as the same effects were obtained with both PNA and Antagomir. To the best of our knowledge, this is the first evidence that unmodified PNAs can be applied in in vivo knockdown strategies when directly injected into eggs.

Exploring miR-9 Involvement in Ciona intestinalis Neural Development Using Peptide Nucleic Acids

“…In ascidians, recent comparisons between PNAs and the commercial AntagomiRs (Dharmacon, USA) antisense molecules have demonstrated the ability of PNAs to efficiently downregulate miRNAs. The effects induced by injections of these molecules (PNAs and AntagomiRs) into C. intestinalis eggs were comparable, confirming the reliability of PNA-based knockdown [22].…”

“…In the experimental animals, treatment with anti-miR-155 PNAs did not affect levels of unrelated miRNAs, and the effects of the PNA-based inhibition mainly recapitulated the effects of miR-155 genetic deletion [21]. More recently, our research group successfully downregulated miR-7 by injections of anti-miR-7 PNAs directly into the eggs of a basal chordate, the ascidian Ciona intestinalis [22].…”

“…To describe Cin-miR-9 (MIMAT0016396) expression during C. intestinalis development, in situ hybridization with a DIG-labeled locked nucleic acid (LNA; Exiqon, Vedbaek, Denmark) probe (cin-miR-9: 5 -TCAAACTGGATAACCAAAGA/3Dig_N; DNA Tm = 74 • C) was carried out as described before [22].…”

“…Thus, considering the therapeutic potential of miR-9 and the advantages of PNA-based approaches, in this study we aim to investigate the ability of a PNA specifically designed to target miR-9 to inhibit its function in vivo, using C. intestinalis as a model organism [22]. Moreover, we evaluated the effects of this inhibition on the development of the C. intestinalis nervous system and proposed a possible mechanism of function by identifying its putative targets.…”

Embryotoxicity characterization of the flame retardant tris(1‐chloro‐2‐propyl)phosphate (TCPP) in the invertebrate chordate Ciona intestinalis

“…A DIG‐labelled riboprobe were synthetized as previously described (Mercurio et al, 2019) and the following neural markers genes were used: Ci‐Pans , to investigate larvae neural architecture (Alfano et al, 2007); Ci‐Syn , encoding for specific synaptic molecules associated with synapsis (Candiani et al, 2010); and Ci‐TH , to explore differentiation of dopaminergic neurons (Moret et al, 2005).…”