*4.3. PNAs Synthesis and Characterization*

Two PNA oligomers were designed: PNA-a7 complementary to mature miR-7; PNA-sc7 with a scrambled sequence, but the same base composition of PNA-a7 as control (Figure 1C). They were synthesized by automated solid-phase synthesis using Boc/Z chemistry by means of the automated synthesizer, Applied Biosystems 433A Peptide Synthesizer (Monza, Milan, Italy), equipped with Synthassist 2.0 software. The commercially available Boc/Z-protected PNA monomers were purchased from ASM Research Chemicals GmbH (Hannover, Germany). The MBHA resin was purchased from VWR International, and it was loaded manually to 0.2 mmol/g with Boc/Z-adenine PNA monomer for PNA-a7, and with Boc/Z-cytosine PNA monomer for PNA-sc7 [43]. The PNA purification was performed using reverse phase high pressure liquid chromatography (RP-HPLC) with an Agilent 1200 Series system (Cernusco sul Naviglio, Milan, Italy), equipped with DAD analyzer (UV detection at 260 and 280 nm, Cernusco sul Naviglio). The purity of PNA-a7 and PNA-sc7 was checked by RP-HPLC analyses, and their identity was confirmed by electrospray-ionisation quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) mass analysis (Q-Tof Micro, Waters). PNA-a7, calculated MW: 5875.4; ESI-MS: m/z found (calculated): 1470.1 (1469.9) [MH4 <sup>4</sup><sup>+</sup>], 1176.2 (1176.1) [MH5 <sup>5</sup><sup>+</sup>], 980.4 (980.2) [MH6 <sup>6</sup>+], 840.5 (840.3) [MH7 <sup>7</sup><sup>+</sup>], 735.5 (735.4) [MH8 <sup>8</sup><sup>+</sup>], 653.9 (653.8) [MH9 <sup>9</sup>+]. PNA-sc7, calculated MW: 5875.4; ESI-MS: m/z found (calculated): 1470.0 (1469.9) [MH4 <sup>4</sup><sup>+</sup>], 1176.2 (1176.1) [MH5 <sup>5</sup><sup>+</sup>], 980.3 (980.2) [MH6 <sup>6</sup>+], 840.4 (840.3) [MH7 <sup>7</sup><sup>+</sup>], 735.5 (735.4) [MH8 <sup>8</sup><sup>+</sup>], 653.9 (653.8) [MH9 <sup>9</sup>+].

The melting temperature (*Tm*) of PNA-a7/DNA duplex was calculated according to the linear model for the melting temperature prediction of PNA/DNA duplexes [44]. In particular, taking into account the following formula:

$$T\_{m,\text{pred}} = c\_0 + c\_1 \times T\_{m,\text{rnDNA}} + c\_2 \times f\_{\text{PT}} + c\_3 \times length \tag{1}$$

in which *Tm*, nnDNA is the melting temperature as calculated using the nearest neighbor model for the corresponding DNA/DNA duplex, applying Δ*H*<sup>0</sup> and Δ*S*<sup>0</sup> values as described by SantaLucia et al. [45], *f* pyr denotes the fractional pyrimidine content, *length* is the PNA sequence length in bases, and the constants were determined to be *c*<sup>0</sup> = 20.79, *c*<sup>1</sup> = 0.83, *c*<sup>2</sup> = –26.13, *c*<sup>3</sup> = 0.44. The calculated *Tm*, pred of PNA-a7 was found to be 65.2 ◦C.

#### *4.4. Microinjections*

For microinjections, only batches in which 90% or more of the embryos developed normally were used. Concentrations of injected solutions were determined by preliminary experiments. We tested the following concentrations: 0.3, 0.5, and 0.7 mM of PNAs (PNA-a7 and PNA-sc7); and 0.3 and 0.5 mM of AmiR-7. For each molecule, the maximum non-lethal concentration was chosen. Dechorionated eggs were microinjected with a solution of 0.7 mM PNAs (PNA-a7 or PNA-sc7) in distilled water or 0.3 mM AmiR-7 plus 5 μg/μL Fast Green as vital dye, as previously described [15]. Embryos were reared at 18 ± 1 ◦C until they reached late tailbud stage [46].

#### *4.5. Whole Mount In Situ Hybridization*

To describe gene expression during development and evaluate microinjection effects, a standard protocol for whole mount in situ hybridization (WISH) was employed [29] with some modifications. Dechorionated embryos and larvae were permeabilized with 2 μg/mL proteinase K in PBS + 0.1% Tween20 for 5 min at 37 ◦C. To detect miR-7 mature transcripts (MIMAT0003552), a hybridization step was carried out with a DIG-labeled Locked Nucleic Acid (LNA) probe (cin-miR-7-5p: 5 -UGGAAGACUAGUGAUUUUGUUG; RNA *Tm* = 76 ◦C) for 5 days at 50 ◦C. The specificity of the miR-7 signal was confirmed by results obtained using the LNA probe against *C. intestinalis* miR-124, whose expression pattern is well known [30]. The riboprobe specific for hnRNP K was obtained from a GC27a23 plasmid contained in the *C. intestinalis* gene collection release I [47]. DIG-labelled riboprobes were transcribed with Sp6 (antisense) and T7 (sense) RNA polymerase, using a DIG RNA labelling kit (Roche, Monza, Italy). Microinjection effects were explored employing riboprobes against the pan-neural marker Ci-ETR [31] and the gene Ci-Syn, encoding for synapsin, a protein specifically associated with synaptic vesicles [32]. For each probe, at least 40 injected and control embryos were analyzed.

#### **5. Conclusions**

Overall, our results demonstrate the in vivo biological activity of PNA oligomers directed against miR-7 in *C. intestinalis* embryos. This animal model allowed direct injection of the anti-miR PNA in eggs, overcoming the typical drawbacks associated with the PNAs poor cellular uptake [48]. One still open problem in antisense approaches is the way of delivering antisense molecules to their target cells in a complex organism. Our results suggest that PNA-a7 is able to reach its specific target in the developing ascidian embryos with high efficiency, as underlined by the lack of effects induced by the scrambled sequence PNA-sc7. To the best of our knowledge, this is the first evidence that unmodified PNA can be successfully used in knockdown strategies in a multicellular organism.

Moreover, our results could be the basis for future quantitative analyses investigating in detail the effect of PNAs.

**Author Contributions:** Conceptualization, S.M. and S.C. (Silvia Cauteruccio); ascidian manipulation and investigation, S.M., R.M., and R.P.; methodology, G.S.; PNA synthesis, S.C. (Silvia Cauteruccio); writing—original draft preparation, S.M.; writing—review and editing, S.C. (Silvia Cauteruccio), S.C. (Simona Candiani), and R.P.; supervision, E.L., R.P.; project administration, E.L., R.P.; funding acquisition, E.L., R.P.

**Funding:** This research was funded by FONDAZIONE CARIPLO, grant number 2013-0752.

**Conflicts of Interest:** The authors declare no conflict of interest.
