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Peptide nucleic acid (PNA) is a synthetic analog of DNA with a peptide-like backbone, possessing strong specificity for nucleic acid binding, good stability in biological tissues and cells, and a long half-life. It is utilized for gene regulation by targeting DNA/RNA to inhibit their replication, transcription, and translation processes. Introducing chiral functional groups at the γ-position in the PNA backbone allows for the formation of a right-handed helical structure, significantly enhancing its hybridization properties with target DNA/RNA. These derivatives of PNA are referred to as γPNA. γPNA exhibits improved chemical and biological properties such as solubility, thermal stability, and specificity, presenting promising applications in gene editing and probe-based detection.
In the structure of γPNA, the neutral N-(2-aminoethyl) glycine backbone substitutes the negatively charged ribose-phosphate backbone of DNA, eliminating the electrostatic interactions when two single-stranded nucleic acids undergo complementary base pairing. This enhances its affinity for the target gene and increases binding stability. Introducing an appropriate stereo center at the γ-position of PNA can result in either left-handed or right-handed helical structures. Adding an S-type stereo center yields a classical right-handed helical structure, while adding an R-type stereo center forms a left-handed helical structure. Incorporating L-amino acids leads to a right-handed helical structure, whereas D-amino acids result in a left-handed helical structure. However, only the right-handed helical γPNA exhibits high affinity and specificity for DNA, readily forming complementary nucleic acids in DNA double-stranded structures, thus enhancing binding to the target gene.
Fig 1. Structure of γPNA and DNA
Adding diethylene glycol (miniPEG) to the γPNA structure enhances thermal stability, while incorporating amino and guanidino groups increases cell permeability. Adding lysine, aspartic acid, or polyethylene glycol enhances specificity. These modifications significantly improve the physicochemical and biological properties of γPNA, allowing for enhanced hybridization with target genes. Additionally, γPNA inherits some inherent properties of PNA, such as high biological stability, lack of recognition sites for nucleases and proteases, and resistance to degradation by nucleases and proteases. Under normal physiological conditions, the negatively charged aspartic acid in the γPNA structure exhibits high specificity for RNA, while the positively charged lysine demonstrates higher specificity for DNA.
Adding diethylene glycol to γPNA increases water solubility and enhances affinity for hybridizing with target DNA, facilitating cellular uptake. The cell-penetrating abilities conferred by amino and guanidino groups have been demonstrated through live-cell imaging techniques. Studies have shown that γPNA can effectively penetrate MCF-7 cells, accumulating near the nuclear membrane, indicating that optimized γPNA structures do not necessarily require the addition of cell-penetrating peptides or other methods to enter cells for exerting antisense effects.
γPNA exhibits selective secondary structures and forms γPNA-DNA hybrid complexes with DNA through Hoogsteen and Watson-Crick hydrogen bonding, which can stabilize in the presence of sodium or potassium cations. γPNA binds to single-stranded or double-stranded DNA molecules, forming stable complexes with different chemical structures and stoichiometries. γPNA can form stable double-stranded structures with single-stranded DNA through complementary base pairing principles. Furthermore, γPNA can form several hybrid complexes with double-stranded DNA: γPNA-DNA2 complex structure; γPNA2-DNA complex; and the formed γPNA2-DNA complex can also undergo strand invasion into double-stranded DNA, replacing one DNA strand, while the other strand protrudes from the complex, forming a D-loop structure; the two strands of γPNA can also independently bind to DNA strands, forming γPNA2-DNA2 complexes. Studies have shown that a three-molecule PNA can form a tetramer complex with one molecule of DNA, indicating the possibility of designing γPNA with functional group modifications in an asymmetric structure.
γPNA exhibits bidirectional control over transcription, capable of both inhibiting and activating transcription. When inhibiting transcription, γPNA binds to the template strand of double-stranded DNA, forming stable complex structures that terminate the extension of RNA polymerase, disrupting the DNA double helix structure, and completely blocking recognition by proteins (such as transcription factors and RNA polymerase). When activating transcription, γPNA binds to the non-template strand of double-stranded DNA, forming a (PNA)2·DNA-DNA complex, allowing the single-stranded DNA loop produced on the template strand to be recognized by RNA polymerase, thus activating transcription. PNA can also effectively bind to the RNA of retroviruses (such as HIV), forming steric hindrance, and blocking the extension of reverse transcriptase on the RNA chain. Regulation of translation by γPNA occurs through binding to RNA, interfering spatially with RNA processing, transport to the cytoplasm, and translation processes, preventing the binding of ribosomes to mRNA, thereby regulating protein expression. Reversible translation regulation has been achieved through the use of two complementary γPNA monomers: one γPNA targeting mRNA can inhibit translation, while another γPNA, through PNA-PNA displacement, can de-repress translation. During the de-repression process, mRNA transcription releases a complete functional original mRNA from each site through strand displacement, eliminating antisense inhibition, and translation begins.
There have been numerous studies on gene editing using PNA, including transcriptional regulation, pre-mRNA splicing, mRNA translation, miRNA function, etc. As a new generation derivative of PNA, γPNA not only inherits some inherent advantages of PNA but also significantly improves upon its shortcomings due to various functional group modifications at the γ-position. It can be used as molecular probes to detect nucleic acid sequence information, regulate gene expression levels, perform gene editing, and achieve therapeutic and diagnostic purposes.
The length of γPNA typically ranges from 12 to 18 base pairs, targeting conserved regions within microbial genomes or species, such as bacterial 16S rRNA or fungal 18S rRNA. In studies of Staphylococcus aureus and Staphylococcus epidermidis, γPNA probes can rapidly and selectively capture 16S rRNA fragments amplified by 5' hapten-modified PCR primers, with homogeneity greater than 99.5% reaching saturation within 5 minutes during capture; whereas under stringent hybridization conditions, DNA probes typically require several hours for capture.
PNA can inhibit the expression of essential bacterial genes (such as gyrA, rpoD, FtsZ, acpP genes) by binding to cell-penetrating peptides in vitro, significantly downregulating the expression levels of target genes and effectively inhibiting bacterial growth, especially in the case of multidrug-resistant strains, with no reports of PNA resistance. As a modified derivative of conventional PNA, γPNA exhibits superior structure and properties, suggesting its potential in antibacterial therapy is inestimable.
