PNA Monomers
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PNA Monomers

Peptide Nucleic Acid

Peptide nucleic acid (PNA) is a structural analog of deoxyribonucleic acid (DNA) with a neutral amide bond as the backbone. As a new molecule formed by replacing the ribose phosphate backbone in nucleic acids, it has many advantages that DNA/RNA does not have, so it has a very wide range of molecular biological effects, especially in the diagnosis and gene therapy of diseases. PNAs can alter gene expression, including inhibition of transcription and translation, gene activation and mutagenesis, and gene delivery. In recent years, PNA has been used in gene therapy, biosensors and diagnostics. Furthermore, due to its neutral charge, PNA can bind to complementary DNA or RNA with high affinity and specificity, resulting in a more stable hybrid than naturally occurring RNA or DNA.

Peptide Nucleic Acid Structure

Phosphodiester linkages hold the alternating ribose (deoxyribose) and phosphate sugars that make up the molecular backbone of DNA and RNA together. In contrast to nucleic acids, PNA is mostly composed of repeated N-(2-aminoethyl)-glycine units, which are connected by amide bonds (peptide bonds; see the blue portion of Fig. 2). The same is that they all contain nucleobases with side chains. The N of the glycine moiety on the main chain of the PNA molecule is fused to a variety of purine and pyrimidine bases (red portion in Fig. 2) via an acetyl structure (green part in Fig. 2). It can be said that PNA combines the characteristics of two distinct macromolecules, peptide and nucleic acid. PNA appears as a poly-N-(2-aminoethyl)-glycine polypeptide with base side chains when viewed as a whole.

Structure of peptide nucleic acid

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-stranded RNA or DNA double helix, thereby binding to the outside of the 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.

Peptide Nucleic Acid Synthesis

Currently, similar to solid-phase peptide synthesis, solid-phase peptide nucleic acid synthesis is used to synthesize PNA. This method uses the synthesis technology of 9-fluoroalkenylmethoxycarbonyl (Fmoc) peptide. The amino skeleton in the monomer is protected by Fmoc. This method combines PNA with peptides and labels such as biotin or fluorescent dyes. Each synthesis cycle takes approximately 30 minutes and is mainly divided into the following steps:

1) Column load: Unlike conventional peptide synthesis where a synthetic C-terminal amino acid is attached to a solid support, the PNA synthesis column contains only a polystyrene solid support.

2) Washing: Prior to synthesis, the PNA synthesis column was washed with dimethylformamide (DMF).

3) Unblock: Use hexahydropyridine/DNE to remove the Fmoc protecting group.

4) Washing: Wash with DMF to remove excess reagent.

5) Activation and coupling: In the presence of a mixture of diisopropylethylene rubber and hexafluorophosphate, the newly synthesized PNA monomer is activated and coupled to the solid phase fat (or coupled to the extended PVA chain).

6) Washing: Wash with DMF to remove excess reagent.

7) Add a cap: Unreacted genes were capped with acetic anhydride dissolved in lutidine/DMF.

8) Washing: Wash with DNF to remove excess reagent.

Modified PNA Monomers

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. 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.

Peptide Nucleic Acid Probes

Peptide nucleic acids have unique advantages in the field of probes due to their excellent chemical and biological stability and flexibility in chemical synthesis. Peptide nucleic acid probes generally refer to short peptide nucleic acids of 13-18 bases with a specific sequence, and their length is shorter than DNA probes. The electrically neutral backbone gives it a higher hybridization affinity than DNA. At the same time, the specificity of base pairing is also significantly improved, and different nucleic acid sequences can be distinguished through single base mismatches. Hybridization conditions are also milder than DNA probes and do not affect hybridization at low salt concentrations. Moreover, the non-natural peptide skeleton can resist degradation by nucleases and proteases and has high biological stability.

What are the Advantages of PNA?

Although PNA is very similar to DNA/RNA, PNA monomers are connected to each other through an unnatural peptide bond. Therefore, PNA has the properties of both peptides and nucleic acids, and has its unique advantages:

  • Compared with the negatively charged ribose phosphate backbone of DNA and RNA, the backbone of peptide nucleic acid is uncharged, which reduces the repulsion between its double strands and thus has higher affinity and stability than DNA or RNA.
  • Since peptide nucleic acids are artificially synthesized nucleic acid-like substances and do not contain amino acid residues or pentose phosphate units in their structure, most nucleases and proteases cannot effectively degrade them.
  • The achiral conformation of peptide nucleic acids facilitates monomer synthesis and purification. The backbone contains repeated amide bond structures, so solid-phase synthesis methods can be used to synthesize oligomers.
  • Compared with other oligonucleotide analogs such as locked nucleic acids, peptide nucleic acids have better specific recognition ability of target sequences.

What are the Uses of Peptide Nucleic Acid?

Based on the above advantages, peptide nucleic acids have shown great potential in the fields of antisense nucleic acid therapy, gene editing, biosensing, and drug development. It can serve as inhibitors and promoters, antigene and antisense therapeutics, anticancer, antiviral, antibacterial and antiparasitic agents, molecular tools and probes for biosensors, DNA sequence detection and nanotechnology.

  • PNA for Genome Editing

Aberrant nucleic acid structures are key to endogenous repair, which may occur under sequence-specific conditions. PNA enables non-enzymatic gene editing. 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 improvement, suggesting the potential of PNAs for therapeutic application in the treatment of monogenic diseases.

  • PNA for 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.

  • PNA for 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, a PNA-ligand conjugate targeting αvβ3 integrin showed 100-fold enhanced binding upon oligomerization, resulting in a 50% reduction in tumor colonies in a mouse model. PNA-labeled macromolecules have been used to program antibody fragments (Fabs) to rapidly explore bispecific antibodies.

  • PNA for 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.

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