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Peptide nucleic acid (PNA) is an unnatural informational molecule, containing a neutral peptide-like backbone and the heterocyclic bases of DNA. The normal phosphate linkage found in DNA and RNA is replaced by a 2-aminoethylglycine peptide backbone (Figure 1, Figure 2).
Figure 1 | Chemical structure of a PNA oligonucleotide
Figure 2 | Three-dimensional structure of a PNA duplex
PNA synthesis is normally performed by automated solid-phase methods in a similar manner to peptide and oligonucleotide synthesis (Figure 3). The coupling step in PNA synthesis is a simple amide bond forming reaction, as in solid-phase peptide synthesis.
Figure 3 | PNA synthesis cycle
The monomers used as building blocks for PNA oligomers synthesis are protected at the terminal amine using Fmoc protecting groups, and the heterocyclic bases are protected with Bhoc groups. (Figure 4).
Figure 4 | Structures of monomers used in PNA synthesis
The Fmoc protecting groups are removed by treatment of the PNA oligonucleotide with a solution of 20% piperidine in DMF, on the PNA synthesizer (Figure 5). The Bhoc protecting groups on the heterocyclic bases, and the linker between the PNA and the solid support, are acid-labile, and so can be removed in a single step by treatment with trifluoroacetic acid at the end of PNA assembly (Figure 6).
Figure 5 | Mechanism of Fmoc deprotection
Figure 6 | Mechanism of Bhoc deprotection
PNA oligonucleotides have unique properties that have been utilized in many molecular biology and biochemical applications. PNA oligomers hybridize to complementary RNA or DNA by normal Watson-Crick base pairing with higher affinity and specificity than normal oligonucleotides, and bind strongly under conditions in which normal nucleic acid hybrids are disfavoured. DNA probes often fail to distinguish between certain regions of highly structured RNA, but PNA probes can be used to detect these regions, if the hybridization is carried out at low salt concentration so that the RNA is unstructured. Mismatched base pairs in PNA:DNA hybrids are very unstable, so PNA probes can be used to identify single point mutations and SNPs. The destabilizing effect of the mismatch can be increased by the use of very short PNA probes. Like DNA and RNA, PNA probes can be labelled with fluorescent dyes, biotin or haptens such as dinitrophenol during or after solid-phase synthesis. One limitation of PNA oligomers is that they are unnatural, and are therefore not substrates for DNA, RNA or protein modifying enzymes.
LNA, first described by Wengel and co-workers in 1998, is an unnatural conformationally restricted oligonucleotide analogue bearing a close structural resemblance to DNA, but possessing monomer units with a 2′-O-4′-C-methylene bridge. The bicyclic structure locks the molecule in a C3′-endo sugar (N-type) configuration (Figure 7), ensuring the oligonucleotide adopts the A-form helix (which is associated with high duplex stability).
Figure 7 | Chemical structure of an LNA oligonucleotide (left), and three-dimensional structure of C(3)-endo LNA (right)
Figure 8 | 3D representation of two successive G bases in an LNA molecule. Carbon atoms are shown in white; nitrogen − blue; oxygen − red; phosphorus − orange
The original LNA oligomers were "oxy-LNAs". Subsequently "thio-LNA" (2′-S-CH2-4') and "amino-LNA" (2′-NH-CH2-4') analogues were prepared (Figure 9).
Figure 9 | Structures of thio- and amino-analogues of LNA (or "oxy"-LNA)
LNA oligonucleotides (and mixed LNA/DNA and LNA/RNA oligomers) can be synthesized on standard automated DNA synthesizers using commerically available LNA phosphoramidite monomers (Figure 10). The only alterations to the standard DNA synthesis cycle are increased reaction times for the coupling and oxidation steps. LNA oligonucleotides can be labelled with fluorescent dyes and other chemical modifications in the same way as DNA and RNA oligonucleotides.
Figure 10 | Structures of phosphoramidite monomers used in the synthesis of LNA oligonucleotides
LNA forms stable hybrid duplexes with DNA and RNA. Duplexes formed between two LNA oligonucleotides exhibit unparalleled affinity and specificity. LNA units can be incorporated into normal DNA or RNA oligonucleotides to increase the stability of probe-target hybrids. This is manifested as an increased melting temperature (Tm) of the hybrid duplex. LNA oligonucleotides can be used for mismatch discrimination, their high binding affinity allowing short probes to be used. LNA oligos have been used in triplex forming oligonucleotides, antisense oligonucleotides and microarrays.
UNA is an analogue of RNA in which the C2′-C3' bond has been cleaved. Like LNA, unlocked nucleic acid (UNA) was first described by Wengel and co-workers, in 1995. Whereas LNA is conformationally restricted (relative to DNA and RNA), UNA is very flexible, as a result of the lack of the C2′-C3' bond (Figure 11).
Figure 11 | Chemical structure of a UNA oligonucleotide
UNA can be synthesized using an automated solid-phase approach, using UNA phosphoramidite monomers (Figure 12).
Figure 12 | Structures of phosphoramidite monomers used in the synthesis of UNA oligonucleotides
While the incorporation of LNA units into DNA and RNA oligonucleotides increases the stability of duplexes, UNA has a destabilizing effect. Incorporation of UNA units into an oligonucleotide probe lowers the Tm of the probe-target duplex. With their complementary effect on probe-target stability, LNA and UNA units can therefore be used to fine tune the thermodynamic properties of DNA and RNA probes.
Recently, oligonucleotides have been synthesized in which the normal phosphate backbone is been replaced with triazole linkages, synthesised using click chemistry (in particular, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction; Figure 13).
Figure 13 | Simplified scheme showing the synthesis and structure of triazole DNA
These triazole-linked oligonucleotides form duplexes with complementary strands of natural DNA, and have recently been shown to be active in biological systems.
For a recent review see El-Sagheer AH, Brown T. Click chemistry with DNA. Chem Soc Rev 2010; 39:1388-405.
This article on Nucleic acid analogues is part of the Nucleic Acids Book.
