Peptide Nucleic Acids: Synthetic DNA Analogues with Many Applications

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 DNA analogues, serving as artificial oligonucleotide
mimetics with higher metabolic stability and greater specificity for target DNA and RNA. The key
to the enhanced stability of PNA compared to DNA is their neutral backbones. Because they do
not have the same charge as the phosphoribosyl backbones of DNA and RNA, repulsive
electrostatic interactions do not occur when PNA bind 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

The increased stability and specificity of PNA binding with DNA and RNA makes PNA useful in
molecular biology and biochemical applications. 3 Because of their greater specificity, short,
fluorescently labelled PNA probes are useful for identifying point mutations and single
nucleotide polymorphisms and for 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

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.

PNA Monomers: Building Blocks for Peptide Nucleic Acids

As with DNA and RNA oligonucleotides, peptide nucleic acids (PNAs) are typically produced via
automated, continuous solid-phase synthesis on peptide synthesizers, but from PNA monomers
with appropriate protecting groups.

For SPPS fluorenylmethyloxycarbonyl (Fmoc)- and benzhydryloxycarbonyl (Bhoc)-protected
PNA monomers are preferred because they allow for mild deprotection of all protecting groups
as well as easy cleavage of the resulting PNA oligomer from the resin. The Bhoc protecting
group is particular to PNA synthesis (vs. RNA and DNA synthesis), as it is used to protect the
primary amines of the heterocyclic bases in PNA monomers which are linked to Fmoc-protected
back bone. The benzyloxycarbonyl (Cbz) protecting group on the other hand is combined with
Boc-protected back bone. It, too, can be removed under acidic conditions, although they tend to
be harsh.

PNA monomers include Cbz- and Bhoc-protected natural nucleobases (cytosine C, guanine G,
adenine A, thymine T) attached to N-(2-aminoethyl) glycine units. Newer PNA monomers are
developed on a regular basis that include protected synthetic nucleobases such as J, U, thioU,
IG, M and D modified to afford certain physicochemical attributes. All PNA monomers are
designed to bind to the natural nucleobases present in DNA and RNA.

The modifications to PNA monomers that have been developed help overcome a range of
issues with the monomers themselves and those of the highly stable DNA and RNA duplexes
formed with PNAs. For instance, some Fmoc/Bhoc-protected PNA monomers exhibit low
solubility and tend to precipitate upon storage in solutions. PNA:DNA duplexes, meanwhile, also
tend to have lower aqueous solubility and a greater propensity to aggregate than DNA:DNA

PNA monomers with modified chemical structures, particularly at the gamma position, have
been shown to improve these properties. The synthesis of gamma-modified PNA monomers is
based on amino acids bearing exclusively heteroatoms. Modifications that cause changes in the
conformations of PNA monomers have also been explored.