Peptide Nucleic Acid in Antiviral Therapy

Peptide nucleic acid (PNA) is a class of oligonucleotide analogs in which the pentose-phosphate backbone of natural nucleic acids is replaced by a peptide backbone. The neutral peptide backbone structure of PNA retains high target affinity similar to oligonucleotides with sugar-phosphate chains, while possessing stronger enzyme stability and thermal stability than sugar-phosphate backbones. PNA has become a hot topic in current research on oligonucleotide analogs. On one hand, PNA exhibits rapid, efficient, and accurate detection capabilities for virus replication and mutation levels. On the other hand, based on PNA's sequence specificity and dose dependence, it can selectively regulate the virus lifecycle at the genetic level, thereby effectively inhibiting the virus's survival and replication in host cells.

Utilizing the antisense DNA or antisense RNA mechanism, PNA specifically binds to viral DNA and RNA at various stages of the virus replication process, influencing virus gene expression through a spatial blocking mechanism. This imparts PNA with significant antisense and antigenic potential. PNA, as a novel antisense nucleic acid drug, finds broad applications in areas such as antibacterial, antitumor, polymerase chain reaction (PCR), fluorescence in situ hybridization, biosensors, and other biomedical fields. In recent years, there have been numerous reports on the role of PNA and modified PNA in antiviral therapy.

Fig. 1 PNA and modified PNAs with pendant side chains. (Volpi, 2021)

Structure and Properties of PNA

A PNA monomer consists of a N-(2-aminoethyl) glycine unit and the base is linked to the backbone through a methylenecarbonyl group. The spatial structure and distances of PNA are similar to nucleic acids, allowing PNA bases to specifically bind to complementary DNA/RNA sequences through Watson-Crick base pairing.

Table 1. List of PNA monomers used for PNA synthesis.

Compared to DNA, PNA lacks the charged phosphate groups, and its neutral backbone structure prevents electrostatic repulsion, resulting in higher hybridization stability and thermal stability. Additionally, PNA exhibits good chemical stability under weakly basic, acidic, and appropriate pH and temperature conditions. The PNA chain, composed of peptide bonds of polyamide, is not recognized by intracellular nucleases or proteases, providing excellent biological stability. However, PNA faces challenges in low cellular uptake due to poor penetration through lipid cell membranes. Research indicates that modifying the PNA structure, conjugating with cell-penetrating peptides, and constructing new drug delivery systems can significantly enhance cellular uptake of PNA.

Applications of PNA in Antiviral Therapy

PNA is primarily used in the treatment of viruses such as hepatitis viruses, influenza viruses, human immunodeficiency virus (HIV), and Japanese encephalitis virus.

Table 2. PNA base sequences used in antiviral therapy

• Hepatitis DNA Viruses

Hepatitis DNA viruses include duck hepatitis B virus (DHBV) and HBV. Currently, drugs used for treatment are mostly nucleoside analogs and interferons, which have strong resistance, significant side effects, and limited therapeutic efficacy. PNA, as an antisense agent, can regulate target gene expression at the genetic level, reducing side effects.

1. Avian Hepatitis DNA Viruses

In the replication process of HBV, the epsilon signal, a stem-loop structure of the pre-genomic RNA (pgRNA), plays a crucial role in reverse transcription. PNA targeting the ε signal of DHBV inhibits reverse transcription (RT) and has been studied. The PNA/DR sequence interaction disrupts the secondary structure of εRNA, preventing reverse transcription and initiating sequence competition with the HBV polymerase. To enhance cellular uptake, cell-penetrating peptides (CPP) were conjugated with PNA. Modifying CPPs with fatty acids can increase the biological activity of PNA.

Fig. 2 Linkers used for PNA conjugation. (Tsylents, 2023)

CPP serves as a PNA carrier and can destabilize or translocate through bacterial membranes. Furthermore, CPPs typically have a positive net charge that attracts PNA conjugates to their negatively charged DNA or RNA targets. The common linkers for conjugation of CPPs to PNAs or other oligonucleotides include nondegradable ethylene glycol (eg1), also known as mini-PEG, and degradable disulfide linkers.

2. Positive Hepatitis DNA Viruses

HBV has a unique lifecycle where the pre-genomic RNA (pgRNA) not only participates in the translation of viral core and polymerase proteins but also serves as a template for genome DNA replication. PNA targeting the 98 nt sequence (X-RNA) at the 3' end of the HBV genome was designed and effectively inhibited HBV replication. Tat-PNA-DR, a compound with multiple functions and a dual-action mechanism, can interfere with various aspects of the HBV lifecycle.

• Hepatitis RNA Viruses

Hepatitis C virus (HCV) is a single-stranded positive RNA virus, and the internal ribosomal entry site (IRES) plays a crucial role in viral translation. PNA sequences of 15-21 bases targeting the IRES were found to dose-dependently inhibit HCV translation. PNA targeting the 98 nt sequence (X-RNA) at the 3' end of the HCV genome was designed to interfere with RNA polymerase binding, blocking RNA synthesis initiation.

• Influenza Virus

Influenza A viruses, with various subtypes, have a conserved elongated double-stranded RNA structure in their genomes crucial for virus RNA replication, transcription, and packaging. PNA fragments complementary to the post-infection genome were designed to interfere with normal virus replication. Constructing suitable PNA delivery systems, such as TiO2·PL·DNA/PNA nanocomposites, improved cellular uptake and significantly inhibited virus particle replication and assembly.

1. HIV

HIV, attacking the immune system, poses challenges in treatment due to its long latency period. PNA targeting specific sequences on the virus genome has shown potential as an anti-HIV drug. In vivo evaluation of anti-HIV-1 TAR RNA PNA-penetratin conjugates demonstrated tolerability and immunoinert properties. Studies showed that Tat-PNA-DR effectively inhibits HIV long terminal repeat sequence (LTR) transcription, demonstrating high antiviral activity and low cytotoxicity.

2. Japanese Encephalitis Virus (JEV)

Specific elements in the 5' untranslated region (UTR), 3' UTR, and 5' and 3' cyclization motifs (CYC) of JEV are critical for host and virus protein binding, RNA-RNA interactions, and RNA synthesis during virus genome expression. PNA targeting the top loop region of the 3' UTR showed inhibitory activity against the virus. PNA forming a closed loop within the conserved SL structure of the 3' UTR blocked RNA synthesis initiation or host factor binding required for virus genome translation.

In Conclusion

PNAs possess unique structural features, specific sequence selectivity, and special biological stability, enabling them to inhibit the transcription or replication of target genes. In antiviral therapy, PNAs have demonstrated enormous potential. Purposeful chemical modifications of PNAs can fine-tune their properties, potentially increasing stability and specificity towards target sequences. Specific antisense PNA sequences can be designed to bind to individual or multiple targets in the viral genome, and targeting carriers such as cell-penetrating peptides can be used to increase their targeting capabilities to achieve inhibition of transcription, reverse transcription, translation, and RNA replication, thus showing significant antiviral activity.

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