Peptide nucleic acids (PNAs) were first proposed in the 1990s by Peter E. Nielsen of the University of Copenhagen.1 Based on computer modeling data, he proposed that replacement of the deoxyribose (or sugar) phosphate backbone of DNA with a neutral, achiral peptide-like backbone comprising N-(2-aminoethyl) glycine units attached to nucleobases via methylene carbonyl linkers would afford greater nuclease stability (resistance to enzyme degradation) and cell membrane permeability. 2

When actual PNAs were synthesized, their properties were found to be as predicted by the computer model. They are typically produced using established automated solid-phase peptide synthesis methods (SPPS), which has the advantage of making modification of the backbone to mediate physicochemical properties and incorporation of fluorescent probes more facile.

PNAs today are widely used synthetic oligonucleotide analogues, serving as artificial oligonucleotide mimetics with higher metabolic stability and greater specificity for target DNA and RNA. The neutral backbones of PNA play a crucial role in its superior stability compared to DNA. Unlike DNA and RNA with their charged phosphoribosyl backbones, PNA lacks this charge, preventing repulsive electrostatic interactions when binding to DNA or RNA.As a result, hybridization with complementary DNA and RNA oligonucleotides via Watson-Crick base pairing proceeds with strong H-bonding and base stacking and greater affinity than can be achieved with DNA. In addition, this greater stability is retained regardless of the salt concentration used for hybridization.

The increased stability and specificity of PNA binding with DNA and RNA makes PNA useful in molecular biology and biochemical applications. 3 Short, fluorescently labeled PNA probes are valuable in the identification of point mutations and single nucleotide polymorphisms due to their enhanced specificity. Additionally, they are employed in fluorescent in situ hybridization studies (FISH). The stability of PNA:RNA duplexes in low salt concentrations, meanwhile, enables PNA probes to differentiate between regions of RNA not accessible to DNA probes that must be used in higher salt concentrations in which the RNA would be highly structured. The ability of PNA probes to tolerate wider salt ranges also creates opportunities for conducing analyses not possible with DNA and RNA probes.

Due to their different backbone structures, PNAs are not susceptible to nucleic acid- and protein modifying enzymes. Analysis of PNAs using common analytical techniques such as polymerase chain reaction, restriction enzyme degradation, and proteolysis is therefore not possible. They can, however, be used in other ways, such as to enhance amplification of target DNA in PCR studies.

The main therapeutic application for PNAs is antisense gene silencing; their ability to bind DNA and RNA makes it possible for PNAs to block replication, transcription, and protein synthesis. 3

[1] Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, Science 1991, 254, 1497.
[2] Egholm, M.; Buchardt, 0.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895.
[3] J. Saarbach, P. M. Sabale, N. Winssinger, Curr. Opin. Chem. Biol. 2019, 52, 112–124.