EMISSIVE PEPTIDE NUCLEIC ACIDS (PNAS): POTENTIAL TOOLS FOR INNOVATIVE DIAGNOSTICS AND THERAPEUTICS

ABSTRACT I. Introduction and aim of the thesis Peptide nucleic acids (PNAs)1 are synthetic mimics of natural nucleic acids, in which the chiral and negatively charged sugar-phosphate backbone is replaced by achiral and neutral N-(2-aminoethyl)-glycine (aeg) units, where the four nucleobases (thymine, adenine, cytosine, guanine) are inserted through a carboxymethylene spacer. PNAs are able to bind complementary DNA and RNA single strands through Watson-Crick base pairing, with high sequence specificity and affinity2. Thanks to their synthetic nature, PNAs also display high chemical and enzymatic stability3. All these unique features make PNAs excellent candidates for diagnostic and therapeutic applications4,5, such as detecting genetic mutations and interfering with protein production in antigene and antisense therapies, for cancer and antibacterial treatments. However, despite their potential, PNAs face challenges in clinical applications due to their poor cell membrane penetration and water solubility. Therefore, the research in the field of PNAs is still ongoing to improve PNA delivery and fully exploit their potential especially for therapeutic purposes. This Ph.D. thesis aimed to broaden the opportunities to employ PNAs in biomedical applications, developing new and attractive tools for diagnostics and therapy. To this purpose, this thesis focused on exploring and validating synthetic methodologies for the creation of modified PNAs. The work of the thesis was divided into four main research activities, with the common feature of creating emissive PNAs. Emissive molecules offer various advantages when paired with PNAs. Their luminescent properties enable cellular localization studies, while their ability to penetrate cells makes them potential carriers for PNAs. Additionally, emissive compounds could introduce new therapeutic capabilities to PNAs. Emissive PNAs could allow to handle the current limitations and propose valid alternatives from which both the diagnostic and the therapeutic fields can benefit. Finally, the study of unmodified PNAs for in vivo application through antisense strategy was also preliminary explored. II. Intrinsically emissive PNAs containing isomorphic thieno[3,4-d]pyrimidine nucleobase The isomorphic emissive nucleobase thieno[3,4-d]pyrimidine6 was inserted in different positions of a model PNA decamer to create intrinsically emissive PNAs. Such PNAs could elicit changes in their emission upon hybridization with complementary DNA or RNA strands, opening up new opportunities for diagnostic purposes. Three novel modified PNAs (PNA1, PNA2 and PNA3) were synthesized manually through standard solid phase protocols and characterized through photophysical studies and melting temperature measurements, which were performed using UV absorption spectroscopy and micro differential scanning calorimetry (micro-DSC), in collaboration with Professor Fin (University of Turin). These studies demonstrated that the nucleobase conferred emissive properties to the PNAs, which maintained their ability to bind complementary antiparallel DNA. Additionally, it was possible to verify the hybridization event between the emissive PNA and the DNA by monitoring the changes in the fluorescent properties of the PNA. Although further research is required to better understand the results and optimize the photophysical and hybridization properties of these PNAs, the results of this study provided valuable insights for the development of emissive PNAs that could be employed in the diagnostic field. III. Development of a luminescent bioorganometallic Ir-PNA conjugate as potential dual-action system against cancer Organometallic cyclometalated iridium (III) complexes, containing two phenylpyridine ligands and a phenathroline as third ligand, display excellent photophysical properties, and can act as photosensitizers (PSs) generating cytotoxic singlet oxygen (1O2) for applications in photodynamic therapy (PDT)7. Moreover, they show good cell permeability, for example towards cancer cells. In this research activity, in collaboration with Professor Maggioni (University of Milan), the Ir-COOH and Ir-NH2 were investigated as carriers for delivering PNAs into cancer cells and create a potential dual-action system, which combines the therapeutic properties of both components of the conjugate. This work introduced the first example of cyclometalated Ir(III) complexes conjugated to a model PNA tetramer. Two Ir(III) complexes with different functional groups (Ir-NH2 and Ir-COOH) were conjugated to the PNA tetramer, using a coupling reaction on solid phase, to investigate the conjugation method. The Ir-PNA conjugate retained the photophysical properties of the iridium complex, including its ability to generate singlet oxygen in solution, a feature that can be employed for PDT. Confocal microscopy studies confirmed the ability of the Ir(III) complexes to effectively deliver the PNA into HeLa cells and demonstrated that the intracellular localization of the system could be tracked using the luminescent features of the iridium component. Cytotoxicity tests in the absence of light showed that the Ir-PNA conjugate is non-toxic for HeLa cells, but its cytotoxicity increased under light irradiation, likely due to the generation of 1O2 by the Ir(III) complex, confirming its ability to act as a photosensitiser (PS) when conjugated to the PNA strand. Therefore, this innovative bioorganometallic Ir-PNA conjugate offers promising features for the future development of dual-action anticancer agents, in which a microRNA target will be selected for the PNA, and its antisense effect will be combined with the PDT related to the iridium complex to synergically target tumors. IV. Luminescent Pt(II) complexes as potential carriers for PNAs to bacteria Neutral and planar N^C^N tridentate Pt(II) complexes have been widely investigated for bioimaging applications and in PDT, thanks to their outstanding photophysical features and ability to act as PSs8. Thus, their conjugation to PNAs could provide a novel approach for therapeutic applications. In particular, given the growing challenge of treating bacterial infections, the combination of Pt(II) complexes as antimicrobial PDT (aPDT) agents and PNAs as antisense oligonucleotides could offer several advantages for the development of new antibacterials. In collaboration with Professor Colombo (University of Milan), two Pt(II) complexes with different spacers (Pt-1 and Pt-2) were conjugated to a model PNA decamer, using a coupling reaction on solid phase, validating the conjugaton method for this class of platinum complexes. The ability of the Pt-PNA conjugates (Pt-1-dPNA and Pt-2-dPNA) to hybridize with complementary single strand DNA (ss-DNA) and single strand RNA (ss-RNA) was assessed through melting temperature measurements and circular dichroism (CD) studies. The results suggested that the conjugation of the Pt(II) complexes to PNAs could affect the hybridization properties of the PNA sequence towards complementary ss-DNA and ss-RNA depending on the length of the spacer. The formation of stable duplexes was observed with the Pt-2-dPNA conjugate, bearing a longer spacer between the metal centre and the PNA sequence. Notably, the photophysical properties of the Pt(II) complex were conserved and the quantum yield of the conjugate Pt-PNA was even higher, suggesting that the conjugation with the PNA could be beneficial for the intrinsic features of the complex. This result also demonstrated that this class of Pt(II) complexes could be suitable for future intracellular localization studies, allowing the tracking of the PNA inside cells. Next, in collaboration with Professor Trylska (Centre of New Technologies, University of Warsaw), the Pt-PNA conjugates targeting the mRNA of the essential acpP gene in E. coli9 were tested for their antibacterial activity. They inhibited the growth of E. coli strains with defective LPS membranes (AS19) but showed no effect on other strains, suggesting that the LPS layer plays a key role for their effectiveness. Current research is focusing on understanding how Pt-PNA conjugates interact with bacterial membranes and optimizing their ability to penetrate cells. In future, these conjugates will be evaluated as dual-action antibacterial agents, combining the antisense activity of the PNA with the cytotoxic singlet oxygen production from the Pt(II) complex for a synergistic antimicrobial effects. V. Cyclic triimidazoles as potential luminescent probes and carriers for PNAs This project, carried out in the final part of this Ph.D. thesis, aimed to study the conjugation of PNAs to three different cyclic triimidazoles as potential luminescent carriers for PNAs to bacterial cells10, in a joint collaboration with Professor Cariati (University of Milan) and Professor Trylska (Centre of New Technologies, University of Warsaw). The conjugation of three different cyclic triimidazoles to PNAs designed to target the acpP mRNA was successfully accomplished via a coupling reaction on solid phase. Melting temperature studies of cyclic triimidazole-PNA conjugates with the complementary antiparallel RNA strand revealed that the recognition properties of the PNA conjugates were affected with varying degrees by the different functionalizations. Absorption and photoluminescence studies on the TT-Th-anti-acpP-PNA in water confirmed that its photophysical properties were in agreement with those typically observed for this class of cyclic triimidazoles. Indeed, the TT-Th-anti-acpP-PNA displayed an intense emission at 464 nm, which could be useful for the future development of new emissive probes. Preliminary studies performed to evaluate the antibacterial activity of these conjugates showed that they were not effective in inhibiting the growth of E. coli AS19, meaning they were not able to penetrate the bacterial membrane. Future work will involve the evaluation of the cyclic triimidazoles on additional bacterial strains, such as Gram-positive bacteria, to further explore their potential as PNA carriers. Moreover, considering the versatility of functionalizing cyclic triimidazoles through synthetic techniques established in Professor Cariati’s group, upcoming research will also focus on developing new derivatives as carriers. In summary, the initial findings highlight the promise of cyclic triimidazole-PNA conjugates, though further studies are necessary to confirm these preliminary results and fully realize the objectives of the project. VI. Anti-miR124 PNAs for in vivo studies in ascidian embryos Besides the work on emissive PNAs, a part of this Ph.D. thesis was also dedicated to the study of anti-miR124 PNAs for in vivo studies using ascidian embryos as animal model. Anti-miR PNAs have been employed for studying miRNA functions and interfere with their activity, potentially treating various diseases11-13. The goal of this research was to investigate the ability of PNA to target miR-124 and study the effects on the neural development of ascidian Ciona intestinalis embryos, in collaboration with Dr. Hitoyoshi (Laboratoire de Biologie du Développement de Villefranche-sur-mer, Sorbonne Université). By using this approach, it would be possible to investigate how miRNA-124 regulation could influence the neural development and gain deeper insights into the role of miRNA-124 in the CNS diseases. Two unmodified PNA sequences, the antiparallel complementary sequence to miRNA-124 (PNA-a124) and the scrambled PNA (PNA-sc124), were synthesized using manual solid phase synthesis. Unfortunately, the unmodified PNA-a124 sequence did not seem suitable for this study, mainly due to its low solubility in the seawater used for the in vivo experiments, so in future the insertion in this sequence of some lysine- or arginine-based residues could be performed to improve its water solubility and potentially prevent their precipitation, which makes the injection in the embryos difficult. Additionally, the toxicity of the PNA-sc124 could arose from off-target binding. Thus, it could be mitigated rearranging its sequence. VII. Conclusions This Ph.D. thesis started and developed several projects within three years, leading to mostly preliminary results that require further optimization to fully unlock the potential of the newly developed PNAs. Despite this, the thesis achieved its goal of designing and creating novel emissive PNAs that combine the advantages of both PNAs and emissive molecules. These emissive PNAs represent a promising starting point for expanding their use in clinical applications, which has been limited by the poor cellular uptake displayed by PNAs. Notably, Ir(III) and Pt(II) complexes emerged as effective carriers for delivering PNAs into cancer and bacterial cells, respectively. VIII. References (1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Sequence-Selective Recognition of DNA by Strand Displacement with a Thymine-Substituted Polyamide. 1991, 254, 1497-1499. https://www.science.org/doi/10.1126/science.1962210. (2) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. H.; Norden, B.; Nielsen, P. E. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules. Nature. 1993, 365, 566–568. https://doi.org/10.1038/365566a0. (3) Demidov, V. V.; Potaman, V. N.; Frank-Kamenetskii, M. D.; Egholm, M.; Buchard, O.; Sönnichsen, S. H.; Nielsen, P. E. Stability of peptide nucleic acids in human serum and cellular extracts. Biochem. Pharmacol. 1994, 48, 1310-1313. https://doi.org/10.1016/0006-2952(94)90171-6. (4) Saarbach, J.; Sabale, P. M.; Winssinger, N. Peptide Nucleic Acid (PNA) and Its Applications in Chemical Biology, Diagnostics, and Therapeutics. Curr. Opin. Chem. Biol. 2019, 52, 112–124. https://doi.org/10.1016/j.cbpa.2019.06.006. (5) MacLelland, V.; Kravitz, M.; Gupta, A. Therapeutic and Diagnostic Applications of Antisense Peptide Nucleic Acids. Molecular Therapy Nucleic Acids. 2024, 35. https://doi.org/10.1016/j.omtn.2023.102086. (6) Srivatsan, S. G.; Weizman, H.; Tor, Y. A Highly Fluorescent Nucleoside Analog Based on Thieno[3,4-d]Pyrimidine Senses Mismatched Pairing. Org. Biomol. Chem. 2008, 6, 1334–1338. https://doi.org/10.1039/b801054d. (7) Maggioni, D.; Galli, M.; D’Alfonso, L.; Inverso, D.; Dozzi, M. V.; Sironi, L.; Iannacone, M.; Collini, M.; Ferruti, P.; Ranucci, E.; D’Alfonso, G. A Luminescent Poly(Amidoamine)-Iridium Complex as a New Singlet-Oxygen Sensitizer for Photodynamic Therapy. Inorg. Chem. 2015, 54, 544–553. https://doi.org/10.1021/ic502378z. (8) De Soricellis, G.; Fagnani, F.; Colombo, A.; Dragonetti, C.; Roberto, D. Exploring the Potential of N^C^N Cyclometalated Pt(II) Complexes Bearing 1,3-Di(2-Pyridyl)Benzene Derivatives for Imaging and Photodynamic Therapy. Inorganica Chim. Acta. 2022, 541, 121082-121095. https://doi.org/10.1016/j.ica.2022.121082. (9) Good, L.; Kumar Awasthi, S.; Dryselius, R.; Larsson, O.; Nielsen, P. E. Bactericidal Antisense Effects of Peptide-PNA Conjugates. Nat. Biotechnol. 2001, 19, 360–364. https://doi.org/10.1038/86753. (10) Previtali, A.; He, W.; Forni, A.; Malpicci, D.; Lucenti, E.; Marinotto, D.; Carlucci, L.; Mercandelli, P.; Ortenzi, M. A.; Terraneo, G.; Botta, C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z.; Cariati, E. Tunable Linear and Nonlinear Optical Properties from Room Temperature Phosphorescent Cyclic Triimidazole-Pyrene Bio-Probe. Chemistry - A European Journal. 2021, 27, 16690–16700. https://doi.org/10.1002/chem.202102839. (11) Gambari, R.; Fabbri, E.; Borgatti, M.; Lampronti, I.; Finotti, A.; Brognara, E.; Bianchi, N.; Manicardi, A.; Marchelli, R.; Corradini, R. Targeting MicroRNAs Involved in Human Diseases: A Novel Approach for Modification of Gene Expression and Drug Development. Biochem. Pharmacol. 2011, 82, 1416–1429. https://doi.org/10.1016/j.bcp.2011.08.007. (12) Mercurio, S.; Cauteruccio, S.; Manenti, R.; Candiani, S.; Scarì, G.; Licandro, E.; Pennati, R. Mir-7 Knockdown by Peptide Nucleic Acids in the Ascidian Ciona Intestinalis. Int. J. Mol. Sci. 2019, 20, 5127-5137. https://doi.org/10.3390/ijms20205127. (13) Mercurio, S.; Cauteruccio, S.; Manenti, R.; Candiani, S.; Scarì, G.; Licandro, E.; Pennati, R. Exploring Mir-9 Involvement in Ciona Intestinalis Neural Development Using Peptide Nucleic Acids. Int. J. Mol. Sci. 2020, 21, 2001-2012. https://doi.org/10.3390/ijms21062001.