In 1991, Nielsen, Egholm, Berg, and Buchardt reported the synthesis of peptide nucleic acids (PNA’s), a new, completely artificial DNA/RNA analog in which the backbone is a pseudopeptide rather than a sugar.
The most impressive property of PNA’s is their ability to form extremely stable complexes with complementary DNA oligomers. A 10-mer PNA:DNA complex denatures at a melting temperature of 73 °C. This stability suggests PNA’s are superior reagents in antisense and antigene applications, and creates other uses for which sequence specific but thermally stable complexes are required.
Applications for PNA oligomers- Genome Rare Cutting
- Duplex DNA Capture
- Triple helix formation
- PCR Clamping
- Antigene Technology
Structure of PNA
The PNA backbone is made of N-(2-aminoethyl)-glycine units linked by peptides bonds. The different bases (purines and pyrimidines) are linked to the backbone by methylene carbonyl linkages. Unlike DNA or other DNA analogs, PNAs do not contain any pentose sugar moieties or phosphate groups. Like amino acids, PNA monomers have amino and carboxyl termini. PNA monomers are linked by peptide bonds into a single chain oligomer. By convention, the N-terminus of a PNA oligomer is considered equivalent to the 5’ end of the DNA. PNAs can form duplexes in either orientation, but the anti-parallel orientation is strongly preferred and forms the most regular duplex. Anti-parallel is also the preferred configuration for antisense and DNA probe type applications (i.e.: the N-terminus of a PNA hybridizes preferentially to the 3’-end of complementary single-stranded DNA).
DNA triplexes
The PNA backbone is not charged, this confers to these polymers a much stronger binding between PNA/DNA strands than between PNA strands and DNA strands. This is due to the lack of charge repulsion between PNA and DNA strand.
In published reports, the Tm of a 6-mer PNA T:DNA dA mixture, measured by the midpoint in the hypochromic shift, is 31 °C. In comparison, the Tm of the corresponding DNA: DNA duplex is 15 °C. Longer PNA’s exhibit higher Tm’s. A 10-mer PNA I: DNA A mixture melted at 73 °C. The high thermal stability results from the formation of a triple-stranded complex having a 2 PNA: 1 DNA stoichiometry. The complex is hypothesized to be a PNA:DNA duplex maintained by standard Watson-Crick base pair interactions. A second PNA strand lies in the major groove of the duplex where it makes Hoogstein hydrogen bonds to bases in the PNA:DNA duplex.
The same trends have now been replicated with PNA’s of mixed sequences, which are also presumed to form triplexes in solution. A complicating factor, however, is that the Tm of any PNA:DNA complex is influenced by the sequence. Unlike DNA or RNA duplexes, it does not appear to be possible to predict the Tm of a PNA:DNA mixture. Single base mismatches lower the Tm by approximately 15 °C but the magnitude of the decrease depends on the position and the base.
Resistance to nucleases and proteases
Although PNA oligomers share similar structures with peptides and oligonucleotides, the non-standard backbone confers resistance to degradation by proteases and nucleases.
The high thermal stability and resistance to proteases and nuclease make PNA’s ideal reagents in an antigene or antisense study. Resistance to degradation should increase the half-life of the reagent in the cell or in cell culture media, and simultaneously decrease the dose required for inhibition. In addition to that, the fact that they are not charged should facilitate their crossing through the cell membranes and their stronger binding properties should decrease the amount of antisense needed for the inhibition of gene expression.
PNA’s offer the additional advantage that PNA-peptide chimeras can be synthesized. Such combinations may offer additional mechanisms to target PNA’s to specific locations.
Cellular uptake
PNA oligomers used for biological (antisense or antigene) experiments are typically 12-18 mers having a molecular weight of 3-4000. As PNA oligomers are hydrophilic rather than hydrophobic, these are in analogy to hydrophilic peptides (or oligonucleotides) not readily taken up by pro- or eukaryotic cells in general. Consequently, it has been necessary to devise PNA delivery systems. These include employment of cell penetrating peptides, such as penetratin transportan, Tat peptide and nuclear localization signal (NLS) peptide in PNA-peptide conjugates. Alternatively, cationic liposome carriers, which are routinely and effectively used for cellular delivery of oligonucleotides, can be used to deliver PNAs. However, as PNA oligomers do not inherently carry negative charges, loading of the liposomes with PNA is extremely inefficient. However, efficient loading and hence cell delivery can be attained by using a partly complementary oligonucleotide to “piggy-back” the PNA or by conjugating a lipophilic tail (a fatty acid) to the PNA. Finally, techniques that physically disrupt the cell membrane, such as electroporation or streptolysin treatment can be used for cell delivery.
Antisense applications
Several examples of PNA directed (antisense) down regulation of gene expression have been described. Cell free In vitro translation experiments indicate that regions around or upstream the translation initiation (AUG) start site of the mRNA are most sensitive to inhibition by PNA. It has to noted that there are exceptions to that rule. (e.g. in cell culture model).
As PNA-RNA duplexes are not substrates for RNaseH, antisense inhibition of translation by PNA is mechanistically different from that of phosphorothiotes. Consequently, sensitive targets identified for phosphorothioate oligonucleotides are not necessarily expected to be good targets for PNA. Indeed, sensitive RNA targets for PNA oligomers are presumably targets at which the PNA can physically interfere with mRNA function, such as ribosome recognition, scanning or assembly, whereas ribosomes involved in translation elongation appear much more robust. Interestingly, but not too surprisingly, it was recently demonstrated that intro-exon splice junctions are very sensitive targets for PNA antisense inhibition as correct mRNA splicing is prevented. Thus in antisense experiments with PNA as with other DNA analogues and mimics, it is advisable to perform a mRNA scanning (gene-walk) by testing a series of PNAs targeting different regions of the mRNA.
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