**1. Introduction**

Peptide Nucleic Acids (PNAs) are artificial nucleic acids mimics [1], extensively used for the regulation of gene expression in cellular and molecular systems [2]. In PNAs, the neutral pseudo-peptide backbone, based on *N*-(2-aminoethyl)glycine units (Figure 1A), replaces the negatively charged sugar-phosphate chain of nucleic acids. PNAs can recognize and bind to DNA or RNA sequences according to regular Watson–Crick base pairing rules [3]. Unlike DNA or RNA, PNAs are chemically stable across a wide range of temperatures and pHs, and they are resistant to enzymatic degradation since they are not easily recognized by nucleases or proteases [4]. Moreover, one of the most remarkable properties of PNA is the excellent thermal stability of PNA/DNA and PNA/RNA duplexes, in comparison with DNA/DNA or DNA/RNA duplexes [5]. Indeed, the lack of charge repulsion between the neutral PNA strand and the DNA or RNA strand provides extremely stable complexes. For example, Shakeel et al. reported that the melting temperature (*Tm*) values for a 15-mer PNA/DNA or PNA/RNA duplex are generally 20 ◦C higher than the natural nucleic acid duplexes [6]. All these properties make PNAs excellent candidates for in vivo antisense and antigene therapies, targeting oncogenes, viruses, and bacteria [7]. Indeed, studies on the use of PNAs in knockdown technologies are accumulating, demonstrating PNAs potential for future therapeutic purposes, as well as for basic

research. However, in vivo PNAs applications are still rare [8,9] due to some drawbacks, such as poor cellular uptake and low solubility in aqueous media [10], which can be improved either by conjugation with carrier molecules or by chemical modifications [11].

PNAs poor cellular uptake could be also overcome by using model organisms, such as ascidians, in which molecules can be directly injected in the target tissue/cell. Ascidians are marine invertebrates, closely related to vertebrates [12]. They develop through a swimming larva that shows the basic chordate features, comprising a notochord, which runs along the tail, and a dorsal tubular central nervous system (CNS) [13,14]. Particularly, the ascidian *Ciona intestinalis* is amenable to embryological manipulations. A variety of molecular tools were developed to perturb gene activity during its development, including microinjections of antisense molecules directly into unfertilized eggs [15,16].

To verify PNAs efficiency in gene downregulation during ascidian development, we chose one of the most evolutionarily conserved microRNAs (miRNAs), miR-7 [17], as PNAs target. miRNAs are a class of non-coding RNAs that regulate gene expression at post-transcriptional level. They are found in all animal lineages, where they modulate multiple biological processes [18,19]. A single miRNA has the potential to target a broad spectrum of mRNAs, possessing great regulatory potential [20]. However, in many cases, knockout of individual miRNA does not lead to critical effects, as the same pathway is often controlled by many of these molecules that collectively affect the pathway by exerting fine-tune functions and ensuring the correct progression of cellular and developmental programs [21].

In mammals, miR-7 is expressed predominantly in the pancreas, neural tissues and pituitary [22,23]. miR-7 is highly expressed in neurons with sensory or neurosecretory functions in fish and animals distantly related from vertebrates, such as annelids [24]. miR-7 expression in photoreceptors is similarly conserved, being reported in rodents [25], amphioxus [26], and even *Drosophila* [27]. A gene encoding for miR-7 is also present in the genome of the ascidian *C. intestinalis* (www.mirbase.org), but its expression has not been described yet.

Thus, in this communication, we aim to characterize miR-7 expression profile in the ascidian *C. intestinalis*, and then test PNAs in vivo knockdown efficiency in this species, comparing it with the commercial antisense molecule, Antagomirs (Dharmacon, USA) [28].

**Figure 1.** (**A**) DNA and Peptide Nucleic Acids (PNA) backbone; (**B**) multi-alignment of mature miR-7 sequences in different species: *Phallusia mammillata*, *Ciona intestinalis*, *Ciona savignyi*, *Oikopleura dioica*, *Branchiostoma floridae*, *Danio rerio*, *Homo sapiens*, *Drosophila melanogaster*; (**C**) miR-7 and PNAs sequences used in this study; (**D**) schematic representation of miR-7 genomic position inside the last Ci-hnRNP K intron (blue rectangles and interconnecting lines represent exons and introns, respectively; the red graph corresponds to the miR-7 sequence).
