PNA Clamps and Triplexes for Diagnostics, Gene Editing, and Therapeutics

Triplex-based PNA clamping utilizes the synthetic DNA analog "Peptide Nucleic Acid" (PNA) to form a triple helix structure with double-stranded DNA (dsDNA). This triple helix, or "triplex," formation serves as the basis for several applications in diagnostics, gene editing, and therapeutics. The neutral backbone of PNAs eliminates electrostatic repulsion with the charged DNA backbone, resulting in strong and specific interactions. PNAs interact with their complementary target in double-stranded DNA by strand displacement. The introduction of modified PNA bases, such as pseudoisocytosine, ensures stable triplex formation at physiological pH, crucial for in vivo applications. The helical distortion caused by the PNA clamp is a key feature that can initiate cellular repair pathways, utilized in triplex-based gene editing. PNA clamps can target both DNA and RNA for various diagnostic and therapeutic applications.

PNAs are synthetic polymers similar to DNA and RNA polymers but with different molecular building blocks. PNAs mimic the structure of DNA and RNA but have a different backbone. PNAs have a peptide-like backbone composed of N-(2-aminoethyl)glycine units linked by peptide bonds, which gives PNAs unique properties and makes them useful in a variety of applications. Their neutral backbone and hybridization properties enable the design of PNA-based biosensors and their application in various biomedical fields.

The unique properties of PNA, with its neutral, protein-like backbone, allow it to bind to complementary nucleic acid sequences with very high affinity and specificity. A PNA "clamp" can interact with a dsDNA target in a specific way: In the strand invasion mode, a "bis-PNA" consisting of two PNA strands connected by a linker, recognizes a particular sequence on the dsDNA. When the bis-PNA invades the DNA duplex, it displaces one of the DNA strands.

During triplex formation, a PNA binds to a DNA strand, typically a purine-rich sequence, to form a stable triple-stranded structure known as a PNA/DNA/PNA complex. The third strand of the triplex is formed by the PNA binding to the DNA strand via Hoogsteen or reverse Hoogsteen hydrogen bonding, in addition to the standard Watson-Crick base pairing.

PNA "clamps" create a stable, localized disruption in the DNA's helical structure, resulting in a highly stable and difficult-to-dissociate complex. The formation of a triplex-based PNA clamp can block or modulate cellular processes.

Binding modes of PNA Clamps

TC-PNAs form a Triplex to target specific DNA sequences!

A tail-clamp Peptide Nucleic Acid (TC-PNA) is a modified PNA molecule designed to bind to DNA or RNA through both Watson-Crick and Hoogsteen base pairing. TC-PNA forms a stable triplex structure, allowing TC-PNAs to target specific sequences and potentially modulate gene expression or be used in diagnostic assays. Due to their unique binding mechanism, high stability, and specificity, TC-PNAs have a wide range of applications in research, diagnostics, and therapeutics. {WC Base_pair, Hoogsteen_base_pair, Triple-stranded_DNA}

Triplex-forming PNAs are a chemical tool for mediating the recombination of 50 to 60 bp donor DNA fragments with genomic DNA to introduce gene modifications. These PNA molecules form a PNA-DNA-PNA triplex that provokes the cell’s own DNA repair machinery and stimulates recombination with donor DNAs to cause heritable changes in targeted genes.

Correctly designed TC-PNAs target specific DNA or RNA sequences with high accuracy enabling gene regulation via modulating gene expression by targeting specific DNA or RNA sequences. Further, TC-PNAs used as guides for genome editing allow directing the delivery of other molecules to specific DNA sequences. In diagnostics, TC-PNAs allows the design of diagnostic assays for the specific detection of DNA or RNA sequences. TC-PNAs target and bind to both DNA and RNA, potentially modulating their function or allowing for detection.

A brief history of PNA

Nielsen et al. (1991) reported the design of a PNA recognizing its complementary target in dsDNA by strand displacement. The PNA contained an N-2-aminoethylglycine (aeg) system obtained by replacing a deoxyribose phosphate backbone with an achiral polyamide backbone, resulting in an oligomer without a charged backbone.

Betts et al. (1995) solved the crystal structure of a nucleic acid triplex revealing a helix, designated P-form, that differs from previously reported nucleic acid structures. The triplex consists of one polypurine DNA strand complexed to a polypyrimidine hairpin peptide nucleic acid (PNA) designed to promote Watson-Crick and Hoogsteen base pairing. The P-form helix is underwound, with a base tilt similar to B-form DNA. The bases are displaced from the helix axis even more than in A-form DNA. Hydrogen bonds between the DNA backbone and the Hoogsteen PNA backbone explain the observation that polypyrimidine PNA sequences form highly stable 2:1 PNA-DNA complexes.

Rogers et al. (2002) showed that a bis-PNA coupled to a short donor DNA fragment mediated specific sequence changes within the supFG1 reporter gene in vitro in human cell-free extracts, and that the PNA–DNA conjugate was more active than its TFO–DNA counterpart. The bis-PNA even stimulated recombination without covalently linked to the donor DNA. Rogers et al. designed the PNA to bind as a clamp by including two distinct segments connected by two 8-amino-3,6-dioxaoctanoic acid (O-linker) units to allow flexibility in binding. The PNA clamp has the sequence JJJ-JJT-TJJ-T-O-Lys-(SMCC)-O-TCC-TCC-CCC-C, containing a heterofunctional crosslinker succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) for covalently attachment to a lysine residue. Pseudoisocytosine (J) mimicking N-3-protonated cytosine is routinely used in PNA clamps for recognition of double-stranded DNA. This modified base is important for pH-independent binding to G in the Hoogsteen mode of triplex formation in a PNA:DNA:PNA triplex.

Guha et al. (2013) developed a PNA clamp method for the detection of low-abundance T790M EGFR mutations from target DNA.

To enhance cellular delivery, McNeer et al. (2013) engineered poly(lactic-co-glycolic acid) (PLGA) nanoparticles for the encapsulation of PNAs/DNAs to introduce modifications in the CCR5 gene by utilizing a double emulsion solvent evaporation technique, to produce particles in size of approximately 150 nm in diameter. The tail-clamp PNA (tcPNA-697) used forms a PNA/DNA/PNA triple helix “clamp” at positions 679–688 of CCR5 and includes a 5-bp “tail” forming a PNA/DNA duplex at positions 674–678. tcPNA-697 induces the recombination of donor DNA sequences that contain a 6 bp modification which includes an in-frame stop codon.

TC-PNA 654-1: N terminus KKK-JTTTJTTTJTJT-OOO-TCTCTTTCTTTCAGGGCA-KKK C terminus.

[Legend: “O” = 8-amino-2,6-dioxaoctanoic acid linker; “J” = pseudoisocytosine, a replacement for cytosine that allows pH-independent triplex formation.]

Ricciardi et al. (2014) described the use of PNA TFOs for targeted genome modification and discuss the method, its applications, and protocols for TFO design, delivery, and evaluation of activity in vitro and in vivo.

Gupta et al. (2017) reviewed hybridization properties along with potential applications of PNAs and modified PNAs for diagnostics and pharmaceuticals. Also, the review investigated the impact of chemical modifications on the backbone of PNAs and their hybridization properties.

Sawada et al. (2017) showed that homopyrimidine TC-PNA consisting of a C4-AZO linker, nine homopyrimidine bases in the N-terminal strand, and five homopyrimidine bases in the C-terminal strand enable the detection of homopurine DNA target sequences with good binding affinity and sequence specificity.

Piotrowski-Daspit et al. (2018) showed that PNAs can be utilized as non-viral vectors for the delivery of genome editing agents in vivo. PNAs can serve as a non-nuclease-based technology for gene editing to enhance the limitations of current transfection methods.

Brodyagine et al. (2021) reviewed chemical approaches, including the use of cell-penetrating peptides, for the discovery of biomedical applications of PNAs covering selected examples of diagnostic and therapeutic PNAs.

Oyaghire et al. (2023) showed that hydroxymethyl-γ-modified PNAs (serγPNA) retain the helical pre-organization present in diethylene glycol-γ-modified PNAs but are more efficient at strand invasion and gene modification. These custom PNAs are considered to be useful for biochemical studies related to DNA recognition. Diethylene glycol or minipeg [mp]γ-substituted PNAs enhance editing frequencies over unmodified PNAs. mp-modified γPNAs (mpγPNAs) have all the biophysical features and improved binding properties of γPNA oligomers but possess enhanced aqueous solubility due to the hydrophilic mp moiety.

Chemical structures of PNAs

El-Fateh et al. (2024) reported that PNAs have antibacterial efficiency at a micromolar dose, and that the efficiency is influenced by several design factors. However, for efficient function, optimization of the delivery system and infection testing models is required. The research group found that PNAs have potential as target-based antibacterial agents, potentially by down regulating or blocking bacterial mRNAs.

References

Betts L, Josey JA, Veal JM, Jordan SR. A nucleic acid triple helix formed by a peptide nucleic acid-DNA complex. Science. 1995 Dec 15;270(5243):1838-41. [1PNN]

BNA PCR Clamping

BNA PCR Protocol

Brodyagin N, Katkevics M, Kotikam V, Ryan CA, Rozners E. Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications. Beilstein J Org Chem. 2021 Jul 19;17:1641-1688. [PMC]

Brown SC, Thomson SA, Veal JM, Davis DG. NMR solution structure of a peptide nucleic acid complexed with RNA. Science. 1994 Aug 5;265(5173):777-80. [176D]

Clamp Oligonucleotide

El-Fateh M, Chatterjee A, Zhao X. A systematic review of peptide nucleic acids (PNAs) with antibacterial activities: Efficacy, potential and challenges. Int J Antimicrob Agents. 2024 Mar;63(3):107083. [sciencedirect]

EGFR single mutation T790M BNA-NC Clamping RT-PCR

Guha M, Castellanos-Rizaldos E, Makrigiorgos GM. DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation. PLoS One. 2013 Jun 21;8(6):e67782. [PMC]

Gupta A, Mishra A, Puri N. Peptide nucleic acids: Advanced tools for biomedical applications. J Biotechnol. 2017 Oct 10;259:148-159. [PMC]

McNeer NA, Schleifman EB, Cuthbert A, Brehm M, Jackson A, Cheng C, Anandalingam K, Kumar P, Shultz LD, Greiner DL, Mark Saltzman W, Glazer PM. Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo. Gene Ther. 2013 Jun;20(6):658-69. [PMC]

Nielsen PE, Egholm M, Berg RH, Buchardt O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science. 1991 Dec 6;254(5037):1497-500. [PubMed]

Oyaghire SN, Quijano E, Perera JDR, Mandl HK, Saltzman WM, Bahal R, Glazer PM. DNA recognition and induced genome modification by a hydroxymethyl-γ tail-clamp peptide nucleic acid. Cell Rep Phys Sci. 2023 Oct 18;4(10):101635. [PMC]

Piotrowski-Daspit AS, Glaze PM, Saltzman WM. Debugging the genetic code: non-viral in vivo delivery of therapeutic genome editing technologies. Curr Opin Biomed Eng 2018, 7, 24–32. [PMC] [PubMed]

PNA Clamp PCR for K-ras mutation detection

Ricciardi AS, McNeer NA, Anandalingam KK, Saltzman WM, Glazer PM. Targeted genome modification via triple helix formation. Methods Mol Biol. 2014;1176:89-106. [PMC]

Rogers FA, Vasquez KM, Egholm M, Glazer PM. Site-directed recombination via bifunctional PNA-DNA conjugates. Proc Natl Acad Sci U S A. 2002;99(26):16695–700. doi: 10.1073/pnas.262556899. [PMC] [PubMed]

Triplex formation for the detection of microRNA

Sawada S, Takao T, Kato N, Kaihatsu K. Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA. Molecules. 2017 Oct 27;22(11):1840. [PMC]

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