Standard peptide nucleic acids (PNAs) contain a pseudopeptide backbone made up of N-(2-aminoethyl) glycine (aeg) motifs. This motif replaces the sugar-phosphate backbone of natural nucleic acids.

Gamma-PNA (γPNA) is a backbone-modified Peptide Nucleic Acid (PNA). Nucleobases (A, C, T, G) are attached to this backbone via tertiary amide linkages, allowing PNAs to base-pair with DNA and RNA. PNAs can form Watson–Crick hydrogen bonds, exhibiting stronger binding affinity to complementary DNA/RNA oligomers with increased specificity, and excellent single-nucleotide mismatch discrimination. Gamma peptide nucleic acids (γPNAs) are a promising nucleic acid mimic that adopt either a right- or left-handed helical motif as individual strands and hybridize to DNA or RNA with high affinity and sequence specificity. However, depending on the helical sense of the strand, they may not at all hybridize to it.

PNAs can target structured DNA and RNA in a sequence-specific manner, which is a key aspect of nucleic acid targeting, since DNA and RNA adopt various structures under physiological conditions, making them less accessible. PNA sequences are resistant to nuclease- and protease-mediated degradation in serum and cell extracts, extending their lifetime both in vitro and in vivo. Unlike DNA, PNAs remain stable across a wide range of temperatures and pH levels. PNAs are ideal candidates for therapeutic and diagnostic applications.

γPNA structures can also form bundles of nanofibers. Kumar et al. (2020) showed that γPNAs form a tight distribution of nanofiber diameters in the presence of the surfactant SDS during self-assembly. Also, the nanostructure morphology of γPNAs can be tuned by correct solvent selection, by strand substitution with DNA and unmodified PNAs. This work introduced the science of γPNA nanotechnology.

Structures of PNAs

Chemical structure of PNA and γ-PNAs. The resulting oligomer contains regular nucleobases and the modified nucleobase, or even in some cases, functionalized at the γ-position.

Bahal et al. (2011) showed that chiral γPNAs containing miniPEG sidechains can invade any sequence of double helical B-form DNA, with the recognition occurring through direct Watson-Crick base-pairing.

Sacui et al. (2015) suggested that these modified nucleic acids are an attractive platform for the design of molecular self-assembly devices because of their specific nucleobase interactions and defined lengths.

Legend: Alpha (α) and Gamma (γ) Modifications: The Greek letters denote the position of modifications on the aeg backbone. α-PNA modification occurs at the alpha position. γ-PNA modification occurs at the gamma (γ) position. A common modification involves creating a chiral center at the gamma position, which leads to the formation of a pre-organized, right-handed helix in the PNA strand.

PNA and γPNA differ by a functional group at the gamma (γ) position of the PNA backbone. This modification adds a chiral center, allowing the molecule to exist in different stereoisomeric forms (e.g., D- and L-isomers). The chirality influences the molecule's properties. The γ-modification "preorganizes" the PNA into a more rigid, helical structure before it binds to a target, reducing the entropic penalty of binding, resulting in a higher binding affinity and increased thermal stability of the resulting PNA-DNA or PNA-RNA duplexes.

The higher affinity and structural pre-organization of γ-PNA lead to better discrimination between a perfectly matched target sequence and one with a single mismatch. The addition of specific functional groups, for example, a lysine, glutamic acid, or serine, at the γ-position can improve the solubility of the PNA, often preventing self-aggregation. γ-PNAs are promising tools for the development of therapeutic oligonucleotide mimics.

γ-PNA can be used as an antigen or antisense agent to silence specific genes by binding to DNA or RNA, thereby blocking transcription or translation, which makes it useful for antiviral therapy and gene editing.
The high affinity and specificity of PNAs make them ideal candidates for detecting gene mutations and as a component of molecular probes. The ability to create different helical conformers, right- and left-handed, and their selective recognition properties allow γ-PNA to be used as a building block for organizing complex molecular structures.

Typically, γ-PNA oligomers are synthesized using solid-phase peptide synthesis (SPPS) methods. Typical synthesis steps involve the sequential coupling of protected monomers onto a solid support. However, the synthesis of γPNAs is more challenging than that of peptides due to issues with monomer preparation, solubility, and potential side reactions. 

A brief history of PNA

Nielsen et al. (1991) designed an achiral polyamide backbone consisting of thymine-linked aminoethylglycyl units. These oligomers recognized their complementary target in double-stranded DNA by strand displacement. Their results showed that the backbone of DNA can be replaced by a polyamide, resulting in an oligomer with base-specific hybridization.

Egholm et al. (1993) showed that PNA oligomers containing thymine and cytosine can hybridize to complementary oligonucleotides by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes. The resulting triplexes are much more stable than the corresponding DNA-DNA duplexes. Further, PNAs containing all four natural nucleobases hybridize to complementary oligonucleotides as well by obeying the Watson-Crick base-pairing rules.

Demidov et al. (1994) investigated the stability of PNAs in human blood serum, Escherichia coli extracts, Micrococcus luteus extracts, and nuclear and cytoplasmic extracts from mouse Ehrlich ascites tumor using HPLC analysis. This study did not detect any significant degradation of PNAs, showing that PNAs have sufficient biostability, allowing them to be candidates for therapeutic drugs.

Uhlman et al. (1998) reported that PNAs are better nucleic acid mimetics than many other oligonucleotides. This paper also summarizes the synthesis, physical properties, and biological interactions of PNAs as well as their chimeras with DNA and RNA.

Schwarz et al. (1999) investigated the thermodynamics of 13 hybridization reactions between 10 base DNA sequences of design 5'-ATGCXYATGC-3' with X, Y = A, C, G, T, and their complementary PNA and DNA sequences using isothermal titration calorimetry (ITC) measurements at ambient temperature. The study found that most of the PNA sequences exhibited tighter binding affinities than their corresponding DNA sequences as a result of minor entropy changes in the PNA/DNA hybridization reactions.

Ratilainen et al. (2000) characterized the hybridization thermodynamics of mixed sequence PNA-DNA duplexes by characterizing the binding affinity to DNA of perfectly matched duplexes and sequence specificity of binding forming singly mismatched duplexes using absorption hypochromicity melting curves and isothermal titration calorimetry.

Ray & Nordan (2000) reported that in vitro studies indicate that PNA can inhibit both transcription and translation of targeted genes, suggesting their use for antigene and antisense therapy. However, the delivery of PNAs through the cell membrane is a general problem.

γ-PNA

Kuhn et al. (2010) reported γ-PNA as a new class of peptide nucleic acids, promising to overcome previous sequence limitations of double-stranded DNA (dsDNA) targeting with PNAs. This study found that strand invasion reactions of the γ-PNA oligomer to its complementary target within dsDNA occur with significantly higher binding rates than to targets containing single mismatches. The research demonstrated that a linear DNA target fragment with the correct target sequence can be affinity-purified from DNA mixtures containing mismatched target or unrelated genomic DNA by affinity capture with streptavidin-coated magnetic beads.

Sahu et al. (2012) showed that adding a relatively small, hydrophilic (R)-diethylene glycol (`miniPEG') unit at the γ-backbone transforms a randomly-folded PNA into a right-handed helix. This paper describes the synthesis of optically pure R-MPγPNA monomers, accomplished in a few steps from a commercially available and relatively cheap Boc-L-serine. Once synthesized, R-MPγPNA oligomers are preorganized into a right-handed helix and hybridize to DNA and RNA with greater affinity and sequence selectivity. They are also more water-soluble and aggregate less than the parental PNA oligomers.

Seo et al. (2011) reported that an aegPNA-DNA hybrid is a much more stable duplex and less dynamic compared to a DNA duplex. In this duplex, the base pairs are opened and reclosed much more slowly. The site-specific incorporation of a γ3T monomer in the aegPNA-DNA hybrid can destabilize a specific base pair and its neighbors. A hydrogen exchange study revealed the unique kinetic features of base pairs in the aegPNA-DNA and chiPNA-DNA hybrids, providing insights into method development for specific DNA recognition by PNA fragments.

Bahal et al. (2011) showed that chiral γPNAs containing miniPEG side-chains can invade any sequence of double helical B-form DNA through direct Watson-Crick base-pairing.

Sugiyama et al. (2012) reviewed chiral PNAs with a substituent in the N-(2-aminoethyl)glycine backbone. This review describes the syntheses, properties, and applications of chiral PNAs.

Manicardi et al. (2014) showed that modifying the PNA backbone by introducing new functional groups will broaden its utility. For example, the introduction of one side chain results in a chiral backbone in which the stereochemistry determines the binding properties of the modified PNA, opening the way to the exploitation of stereochemical features in diagnostic assays and in nanofabrication.

Sacuie et al. (2015) observed that because of the stereogenic center, a γ-PNA oligonucleotide forms an α-helical structure. This conformation reduces self-aggregation, improves solubility, and forms a more stable duplex with the target DNA, providing higher binding affinity to the target. In addition, various modifications, such as internal multi-labeling, are possible at any γ-position. These features make γPNA an attractive material for diagnostics and drug development.

Gupta et al. (2016) added a guanidinium group to the γ-position of γPNAs. The research group found that PNA-peptide conjugates are more toxic than Guanidinium PNAs (GPNAs) because of the amphipathic nature of PNA peptide conjugates. Guanidinium-containing PNAs are less harmful because they are less amphipathic than PNA peptide conjugates. Gupta et al. utilized poly(lactic-co-glycolic acid) (PLGA) poly(β-amino ester) (PBAE) based nanoparticles to deliver small molecules like paclitaxel to MCF7 tumor-bearing mice in a sustained manner because PLGA/PBAE nanoparticles increase the retention time of drugs at tumor sites by reducing their clearance rate. PLGA/PBAE blends also allow the efficient administration of paclitaxel and C6 ceramide.

Gupta et al. (2017) developed a miR-210 inhibition strategy based on conformationally preorganized antisense γPNAs with superior RNA-binding affinity, improved solubility, and favorable biocompatibility. For cellular delivery, the research group encapsulated the γPNAs in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). γPNAs targeting miR-210 resulted in a significant delay in the growth of a human tumor xenograft in mice compared to conventional PNAs. Further, histopathological analyses showed considerable necrosis, fibrosis, and reduced cell proliferation in γPNA-treated tumors compared to controls. This work provides a chemical framework for a novel anti-miR therapeutic approach using γPNAs.

Quijano et al. (2017) reviewed developments of PNAs by focusing on advances in PNA therapeutic applications, in which chemical modifications improve PNA function and the use of nanoparticles enhances PNA delivery.

More recently, Muangkaew et al. (2020) reviewed recent progress in new targeting modes of structured DNA and RNA by PNA and PNA-mediated gene editing.

Kumar et al. (2020) showed that γ-modified peptide nucleic acids (γPNA) enable the formation of complex, self-assembling nanostructures in select polar aprotic organic solvent mixtures. However, unlike the diameter-monodisperse populations of nanofibers formed using analogous DNA approaches, γPNA structures appear to form bundles of nanofibers with a tight distribution of the nanofiber diameters in the presence of the surfactant SDS during self-assembly.

Dhami et al. (2022) introduced γPNAs as promising nucleic acid mimics that adopt either a right- or left-handed helical motif as individual strands and hybridize to DNA or RNA with high affinity and sequence specificity, or not at all, depending on the helical sense, suggesting that γPNAs are attractive as antisense and antigen reagents, and as building blocks for molecular self-assembly.

Malik et al. (2022) engineered PNA amphiphiles using chemically modified γ PNAs, 8mer in length, consisting of hydrophilic diethylene glycol units at the gamma position and a hydrophobic moiety in the form of lauric acid (C12) covalently linked to it. The authors selected a γ PNA sequence complementary to the seed region of oncomiR-155, enabling it to self-assemble into spherical vesicles. Further, the researchers also formulate nano-assemblies using the amphiphilic γPNA as a polymer via ethanol injection-based protocols.

Specifically, Malik et al. designed and synthesized lauric acid (C12) conjugated regular PNA-155 and γPNA-155 amphiphiles to target the seed region of oncomiR-155, upregulated in B-cell lymphomas. The researchers compared the self- and nano-assembly characteristics of designed γPNA-155 amphiphile conjugates for their ability to deliver these γPNA into cells.

Recently, Brazil R. (2023) reviewed PNAs as drug prospects and as candidates for gene editing.

Dhuri et al. (2023) evaluated the in vitro and in vivo efficacy of pH-low insertion peptide (pHLIP)-conjugated serine and diethylene-based γPNAs. pHLIP targets only the acidic tumor microenvironment and not the normal cells. Dhuri et al. synthesized and parallelly tested pHLIP-serine γPNAs and pHLIP-diethylene glycol γPNAs that target the seed region of microRNA-155, a microRNA upregulated in various cancers. The research group performed an all-atom molecular dynamics simulation-based computational study to elucidate the interaction of pHLIP-γPNA constructs with the lipid bilayer. It determined the biodistribution and efficacy of the pHLIP constructs in the U2932-derived xenograft model. The study established that the pHLIP-serine γPNAs showed superior results in vivo compared with the pHLIP-diethylene glycol-based γPNA. Dhuri et al. suggested that this comparative study established the advantages of pHLIP-serine γPNA as a more effective delivery system for anti-miRNA therapy compared to the diethylene glycol-modified version, highlighting the potential of this approach for various therapeutic applications.

Aritablie et al. (2025) reviewed the delivery of PNAs into cells and showed that PNAs can target structured DNA and RNA in a sequence-specific manner. Because DNA and RNA adopt various structured forms under physiological conditions, making them less accessible, PNAs are ideal candidates for their specific targeting. PNA sequences are resistant to nuclease- and protease-mediated degradation in serum and cell extracts, extending their lifetime both in vitro and in vivo, making them an ideal potential drug for therapeutic and diagnostic applications. Unlike DNA, PNAs remain stable across a wide range of temperatures and pH levels.

References

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Bio-Synthesis provides custom synthesis of peptide nucleic acids (PNAs), modified and unmodified, including conjugates such as PNA-peptide conjugates.

Also, Bio-Synthesis offers a full spectrum of oligonucleotide and peptide synthesis including bio-conjugation services as well as high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.
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