What is Peptide Nucleic Acid (PNA)?

The second company to leverage PNA technology is Pittsburgh-based Neubase Therapeutics.

Peptide Nucleic Acid (PNA) is a synthetic polymer similar to DNA or RNA. PNA can be said to have been born in Denmark, where it was originally developed by Peter E. Nielsen (University of Copenhagen), Michael Egholm (University of Copenhagen), Rolf H. Berg (Risø National Laboratory), Technical University of Denmark) and Ole Buchardt (University of Copenhagen) invented it in 1991.

Chemical Structure of PNA

The molecular backbones of DNA and RNA are made up of alternating ribose (deoxyribose) and phosphate sugars held together by phosphodiester bonds. Unlike nucleic acids, the backbone of PNA is made up of repeated N-(2-aminoethyl)-glycine units (Figure 1) through peptide bonds (amide bonds) (blue part in Figure 2). The same thing is that they all contain nucleobases with side chains. In the PNA molecule, various purine and pyrimidine bases (red part in Figure 2) are fused to the N of the glycine moiety on the main chain through an acetyl structure (green part in Figure 2).

It can be said that PNA combines the characteristics of two distinct macromolecules: peptides and nucleic acids. Viewed as a whole, PNA looks like a poly-N-(2-aminoethyl)-glycine polypeptide containing base side chains.

If the base sequences are complementary, PNA can bind to the DNA strand. The bases in the side chain of PNA can form hydrogen bonds with the bases in the main groove of the double helix of double-stranded RNA or DNA, thereby binding to the outside of double-stranded RNA or DNA. This so-called Hoogsteen pairing creates a triple helix. Since the backbone of PNA does not contain negatively charged phosphodiester groups, the binding between PNA and DNA strands is stronger than the binding between DNA and DNA strands due to the lack of electrostatic repulsion. PNA can even disrupt the bond between DNA double strands and insert into the middle of the DNA double strands. But the lack of charge also makes PNA quite hydrophobic, which makes it difficult to deliver it in solution to body cells.

Chemical Modification of PNA

The properties of PNA make this class of compounds ideal for use as antisense drugs, as PNA inhibits protein production by binding to mRNA. In theory, PNAs could bind more strongly than most antisense drugs developed today, which are typically DNA or RNA fragments.

First-generation PNAs are insoluble in water, so these molecules can easily aggregate in solution and bind nonspecifically to other biopolymers in cells, leading to toxicity. This problem was solved through the molecular design of γ-PNA. By introducing a hydroxymethyl group into Cγ on the N-(2-aminoethyl)-glycine unit in the PNA backbone (Figure 3). Modified γ-PNA binds to DNA and RNA more efficiently. Compared with unmodified PNA, γ-PNA binds to DNA more stably. In addition, PNA is not degraded by nucleases and proteases like synthetic DNA or RNA, a property that gives them greater stability. In addition to hydroxymethyl groups, the researchers are also adding diethylene glycol side chains to the peptide backbone to increase the binding strength of the PNA and increase the solubility of the modified PNA. These chemically modified PNAs can solve the problem of delivering PNA drug candidates to target cells.

Applications of PNA

Applications of PNA include altering gene expression, serving as inhibitors and promoters, antigene and antisense therapeutics, anticancer agents, antiviral agents, antibacterial and antiparasitic agents, molecular tools and probes for biosensors, DNA Sequence detection and nanotechnology.

Genome editing

In October 2022, Neubase shifted the focus of its PNA business to gene editing. Neubase, in partnership with a global healthcare company, plans to create PNAs designed to edit genetic mutations in three undisclosed diseases. Aberrant nucleic acid structures are key to endogenous repair, which may occur under sequence-specific conditions. PNA enables non-enzymatic gene editing. The method exploits the ability of PNA, discovered by Nielson in 1991, to invade and pry open double-stranded DNA molecules. By forming high-affinity heteroduplex or triplex structures within the genome, PNAs have been used to correct mutations associated with multiple human diseases with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled the systemic in vivo application of PNAs to enable gene editing in preclinical mouse models. In a β-thalassemia model, treated animals exhibited clinically relevant protein recovery and disease phenotype improvements, suggesting the potential for therapeutic application of PNAs in the treatment of monogenic diseases.

Nucleic acid sensing

The detection of specific nucleic acid sequences is crucial in biomedical research and diagnostics. The unique hybridization properties and metabolic stability of PNAs make them well suited for sensing in complex biological environments and even in whole cells. One of the main strategies for live cell imaging is to use fluorescent probes and increase the fluorescence when duplex complexes are formed. This technology has been applied to detect KRAS oncogenic mutations (SNPs). The use of fluorescent PNA probes has also been extended to the detection of triplexes.

Supramolecular drugs

Over the past decade, many studies have used PNA-ligand conjugates to self-assemble into larger structures. Assemblies of PNA-ligand conjugates have been found to have active functions in vivo. For example, PNA-ligand conjugates targeting αvβ3 integrin, a trimeric receptor overexpressed in many cancers, showed 100-fold enhanced binding upon oligomerization, leading to tumorigenesis in mouse models. Colonies reduced by 50%. PNA-labeled macromolecules have been used to program antibody fragments (Fabs) to rapidly explore bispecific antibodies.

Antisense Therapeutics

The metabolic stability and strong binding affinity of PNA make it an exploitable tool for reverse gene therapy. PNAs are steric blockers that inhibit splicing or translation of target mRNAs by binding to the start site. A PNA molecule modified with four lysines at the C terminus was shown to be effective in correcting abnormal splicing in transgenic mice, demonstrating its potential as a therapeutic agent. In another study, GPNA (α-guanidine-modified PNAs) was successfully used to inhibit the expression of EGFR, an important driver of non-small cell lung cancer, in a mouse model.

PNA Therapeutic Agent

The leading drug candidates for PNA use a direct antisense approach. South Korean pharmaceutical company OliPass has a head start here, with its most advanced drug candidate, the painkiller OLP-1002, undergoing clinical trials. Olipass’s PNAs are modified with bases containing cationic lipid groups, which improves stability and facilitates entry into cells. This makes them active in animal models at doses as low as 10 ng/kg, many orders of magnitude lower than existing antisense oligonucleotides. PNA can enter the nucleus and interact with pre-mRNA. Pre-mRNA is the first version of mRNA produced by transcription before splicing enzymes cut out the unwanted parts. By binding to these pre-mRNA molecules, PNA essentially prevents splicing enzymes from cleaning up the RNA code, causing the mRNA to not be translated correctly, thus preventing protein production.

The company currently has PNA drug candidates targeting three diseases. Their most advanced drug candidate, NT-0200, targets myotonic dystrophy type 1, a progressive muscle disease caused by faulty RNA that traps critical splicing proteins and causes cellular translation errors. According to Neubase’s mouse studies, NT-0200 restores downstream protein production in a wide range of tissues. The company also has a PNA drug candidate that targets the repetitive trinucleotide sequence of the huntingtin gene after it is transcribed into mRNA.

Several PNA drugs are in clinical trials and others are in development. The road to developing viable PNA therapies has been difficult. Scientists in academia and startups have chemically tweaked the PNA backbone to make it easier for the molecule to sneak into cells and latch onto RNA more firmly to silence genes. While developing these therapies, chemists are now able to exploit the ability of PNA to embed into DNA helices, making it an ideal tool for gene editing and a potential alternative to the groundbreaking CRISPR-Cas9 system. Despite CRISPR’s decade-long head start, several gene-editing methods using PNAs are now in development. Although it can be said that PNA’s technology has not yet been fully developed, it should not be too far away before they officially enter the medical stage.


Alex Brown

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