In 1991, Nielsen, Egholm, Berg, and Buchardt reported the synthesis of peptide nucleic acids (PNA*), a new, completely artificial DNA/RNA analog in which the backbone is a pseudopeptide (polyamide) rather than a sugar (phosphate ribose).
Despite a radical structural change, PNA is capable of sequence-specific binding in a helix form to its complementary DNA or RNA sequence. Due to its superior binding affinity and chemical/biological stability, PNA has been widely applied in the field of biology.
What is PNA (Peptide Nucleic Acid)?
Origin
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PNA (Peptide Nucleic Acid) were firstly discovered by Dr. Nielsen, and Egholm group.
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PNA chemistry was firstly commercialized in 1993.
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Over one thousand of articles have been published in fields of chemistry, molecular biology, antisense therapy and gene based diagnostics.
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Many products are already on the market in fields of molecular diagnostics and viral or bacterial detection.
Structure of PNA (Peptide Nucleic Acid)
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The backbone is made from repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The different bases (purines and pyrimidines) are linked to the backbone by methylene carbonyl linkage.
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Unlike DNA or other DNA analogs, PNAs do not contain any pentose sugar moieties or phosphate group.
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PNAs are depicted like peptides from the N-terminus to the C-terminus and it corresponds to 5’ à 3’ as in DNA.
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Figure 1. Structural comparison between DNA and PNA |
Characteristics of PNA
Binding properties of PNA with DNA or RNA
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The PNA backbone is not charged, this confers to PNA strands a much more stronger binding between PNA and DNA or RNA than that between DNA and DNA or RNA. This is due to the lack of charge repulsion between PNA and DNA strand.
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The PNA sequences containing all four bases showed that the Tm are higher for PNA hybrids than for either DNA/DNA or DNA/RNA, in general, there is an increase of the Tm of about 1 °C per base pair (Table 1). Generally Tm of 10mer PNA and 15mer PNA shows around 50 °C and 70 °C, respectively.
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The formation of triple-stranded complex by invasion is comprised of a PNA/DNA double helix (formed by Watson-Crick hydrogen bonds) with a second PNA strand lying in the major groove of the duplex (formed by Hoogsteen hydrogen bonds) and is preferred at acidic pH (pH 4.5-6.5). This binding is very strong and the 6mer- and 10mer- homopyrimidine PNA binds to its target with Tm upper than 30 °C and 70 °C, respectively (Table 1, Figure 2).
Table 1. The Tm value of PNA/DNA or PNA/RNA
| Hybrid |
Tm
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| 6-mer PNA T/DNA A |
31 °C
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| 6-mer DNA T/DNA A |
< 10 °C
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| 10-mer PNA T/DNA A |
73 °C
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| 10-mer DNA T/DNA A |
23 °C
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| 15-mer mixed PNA/DNA |
69 °C
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| 15-mer mixed DNA/DNA |
54 °C
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| 15-mer mixed PNA/RNA |
72 °C
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| 15-mer mixed DNA/RNA |
50 °C
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Figure 2. Binding of PNA with Duplex DNA
Advantage of PNA with the stronger binding property
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Shorter PNA can be used as probe due to higher binding property
Typical DNA probe: 20 - 25 mer, Typical PNA probe: 13-17 mer
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PNAs also show greater specificity in binding to complementary DNAs.
A PNA/DNA mismatch is more destabilizing than a mismatch in a DNA/DNA duplex. A single mismatch in mixed PNA/DNA
A single mismatch in 15mer PNA/DNA à 15 °C decreased Tm
A single mismatch in 15mer DNA/DNA à 11 °C decreased Tm
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PNA/DNA(RNA) hybridization reaction is known to be over 100 times faster than DNA/DNA(RNA) hybridization (hybridization time, DNA: 3 hours to overnight, PNA: 30-45 minute)
PNA Hybridization and salt concentration
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Unlike DNA PNA does not need proper salt concentration for hybridization due to neutral charge of PNA backbone. The Tm of a 15-mer duplex decreased by only 5 °C as the NaCl concentration was raised from 10 mM to 1 M (Figure 3).
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PNA can act as a good probe to detect target sequence of secondary structure which is not stable in low slat concentration.
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Figure 3. Stability of PNA and DNA duplex at different salt concentration |
Biological and chemical stability of PNA
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PNA is not a molecular species easily recognized by nucleases or proteases. They are thus resistant to the enzyme degradation, in vivo and in vitro.
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PNA is very stable over a wide range of pH and against various chemicals.
DNA: depurination at acidic pH (pH4.5~6.5)
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PNA is also stable in wide range of temperature.
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Due to this stability against enzymes, stronger binding properties, and easier crossing through cell membranes, PNA is a good candidate material for antisense research.
PNA applications
PNAs can be used in the same applications than traditional synthetic DNA or RNA, but with the added benefits of tighter binding and greater specificity.
PNA probe for diagnosis and detection
Binding properties of PNA give more specific, more sensitive, and more accurate result for the detection of target sequences. Almost commercialized PNA products are probes to detect genetic disease and or to detect viral or bacterial infection.
- FISH probe (telomere detection kit)
- SNP detection
- LightUp probe, Q-PNA probe etc.
Oligonucleotides for antisense and antigene therapy
The stronger binding properties and biologicla stability of PNA implies that a small quantity of PNA can be effective. Triplex invasion of PNA shows a good potential as antigene material. As a third generation of antisense therapy, there have been many experimental data that shows good effect in vitro and in vivo.
Tools for molecular biology and functional genomics
PNA is used as tools of molecular biology abd functional genomics research.
- Northern and Southern blot
- PCR clamping
- Enhanced PCR
- Alternative splicing
- Rare enzyme cutting, etc.
How to use PNA oligonucleotides
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To dissolve PNA
- Resuspend your PNA in 200-1000 µl of 0.1 % aqueous trifluoroacetic acid (TFA) and fractionnate the solution into aliquots. Stock your aliquots frozen.
- Some particular sequences are hard to dissolve, in this case, add 10-20 % of acetonitrile to the TFA solution and heat the sample to 50 °C.
- It has been reported that PNAs have an affinity for glass surfaces, so, if you work with sub-micro molar concentrations of PNA, the majority of the PNA may be bound to the glass. So, if possible, use polypropylene or polyethylene materials during handling and storage of PNA.
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Ionic strength and buffers
- Due to the fact that PNAs are not charged, no salt is necessary to favor and to stabilize the formation of duplex PNA/DNA or PNA/RNA, in comparison of DNA/DNA or DNA/RNA duplex. This means that the Tm of PNA/DNA duplex is almost independent of ionic strength.
- The Tm of a 15-mer duplex decreas by only 5 °C as the NaCl concentration is raised from 10 mM to 1 M.
- PNA, at low ionic strength (10 mM phosphate buffer, without NaCl), effectively binds to its target in the presence of competing DNA strand because the stability of the DNA/DNA duplex is small in these conditions. When using RNA, the low ionic strength permits to destabilize intramolecular hybridization favoring the hybridization with the PNA.
- Hybridization of PNA must always be done in the presence of a buffer, because PNA/DNA duplexes precipitate at low pH or in absence of counterions.
- Self-complementary, hairpin and palindromic sequences should be avoided in designing PNAs since PNA/PNA interactions are stronger than PNA/DNA interactions.
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Strand Displacement
- For experiment of strand displacement, the parallel PNA-oligomer is used at low pH (5 to 5.5) and at low ionic strength. The ionic strength must be as low as possible due to the buffer, only, without any divalent metal ions.
- After strand displacement, the salt concentration can be increased to carry out enzymatic reactions, for instance.
- Buffer should be used to perform the experiment of strand displacement since PNA/DNA complexes precipitate at low pH or when no counter ions are present.
Competitive features of Eurogentec PNA
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High quality and high pure PNA products
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Competitive prices
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Broad range of PNA products including any type of PNA chimeras (i.e. DNA/PNA, PNA/DNA and PNA/peptides)
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All kind of modification is possible
All custom PNA oligomers sold by Eurogentec are manufactured by Panagene under an exclusive license and to be used for internal research only. And such custom PNA oligomers are not to be resold unless properly licensed to do so by separate license. All custom PNA oligomers are made and sold pursuant to license under one or more of US Patents Nos. US 5,773,571, US 6,133,444, US 6,172,226, US 6,395,474, US 6,414,112, US 6,613,873, US 6,710,163 and US 6,713,602 or corresponding patent claims outside the US.
More information on PNA Telomere or Centromere FISH probes.
More info
* The PNA bases are only available in the following countries: Belgium, France, Germany, Italy, Luxembourg, Netherlands, Denmark, Ireland, United Kingdom, Greece, Portugal, Spain, Austria, Finland, Sweden, Cyprus, Czech Republic, Estonia, Hungary, Latvia, Lithuania, Malta, Poland, Slovakia, Slovenia, Bulgaria, Romania, Norway, Iceland, Bosnia, Serbia, Montenegro, Turkey, Switzerland, Israel, Russia and African countries.
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