Peptide Nucleic Acid

dnaWhat is a Peptide Nucleic Acid? The name is self suggestive. One can easily deduce that the molecule must be a combination of a peptide and a nucleic acid.Well, for all practical purposes that is exactly what it is. A Peptide Nucleic Acid or a PNA is a synthetic molecule that is a nucleic acid analogue or a structural mimic. A natural nucleic acid (DNA or RNA) has a sugar phosphate backbone linking together the nucleotide bases. In a PNA, the nucleotides are retained, but the charged Sugar phosphate bridges are replaced with a synthetic peptide backbone that is usually composed of N-(2-amin-ethyl)-glycine units. This modification yields an uncharged and a chiral molecule, which follows the rules of the Watson and Crick base pairing as faithfully as its Nucleic acid cousin. In addition, PNA now becomes resistant to enzymatic degradation and exhibits increased thermal and ionic tolerance. Now, the PNA due to its unique structural features can recognise DNA and RNA in a sequence-specific manner. Also what is most interesting is that it recognises duplex DNA, and binds to it by strand invasion forming a triplex PNA-DNA-PNA.This form is extremely stable. Any student of Biotechnology would have by now grasped the immense significance of this molecule with respect to its Pharmacological and Diagnostic abilities. It is the tremendous versatility and the potential of this molecule that brings it into focus in this week’s Cutting Edge.

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PNA was born in the 1980s in the Labs of Prof.Ole Buchardt, an organic chemist based in Copenhagen. Whilst working on synthesis of Nucleic acid-Specific reagents, the first generation of PNAs bearing nucleobases alternately with Acridine moieties was created. Today’s molecules however, are peptide based. PNA binds to DNA and RNA in a sequence dependent manner and this property can be exploited for therapeutic purposes. One application that immediately comes to mind is Antisense technology. Simply put, antisense uses an analogues molecule to either hybridize to specific genes thus inhibiting transcription or to hybridize to mRNA and inhibit translation. These genes will of course be linked to diseased states. Thus prevention of expression would result in alleviation of the symptoms. PNA, being a structural analogue and an extremely stable biologically active molecule, fits the bill as an antisense molecule. Add to this the fact that PNA bind strongly to coiled DNA (Active DNA is negatively supercoiled) and that it binds more effectively to ds DNA being actively transcribed, and one has a potent and effective tool.

Another very interesting application of this molecule is as what is known as a Rare Genome Cutter. The method is known as PNA assisted Rare Cleavage (PARC). Rare cleavage attempts to achieve cleavage of a large genomic molecule into a relatively smaller number of pieces for sequencing purposes. Generally restriction endonucleases used to chew up the DNA into fragments recognise sequences about 4-7 nt long. These sites are frequently encountered and as a natural consequence the DNA yields too many small fragments after restriction digestion. This creates some problems while sequencing large genomes. PNA has been used very elegantly to solve this problem. A PNA is synthesised that overlaps with the Restriction or methylation site in the genome. Thus, when the PNA is bound to the target DNA, it will act as a shield to these sites against methylation. Other sites will be methylated. Now if the PNA is removed and the DNA is treated with endonucleases, the methylated sites will be resistant to cleavage and only the previously shielded and, therefore unmethylated sites will be cleaved. Thus by reducing the target sites for restriction enzymes, one can very easily obtain larger fragments of the Genomic DNA for Sequencing proposes.

The unique applications of this molecule seem to be never-ending. Specially designed PNAs can be used in Affinity Chromatography Columns to purify target nucleic acids by sequence specificity. Flourescein labelled PNAs can be used to hybridize to Telomeric repeats and to determine the length of these regions. Telomeric regions are under study for their possible role in senescence and in the emergence of certain diseases. The high affinity binding of PNAs and their extreme adherence to base-pairing rules enables them to detect mismatched bases in a nucleic acid. This can be an extremely valuable diagnostic tool to detect dangerous mutations. A single stranded PNA can be immobilised as a probe and used to detect target DNA sequences. Hybridisation of the ssPNA will send a signal that can be read off a digital meter.

Although this amazing molecule has certain problems associated with low water solubility and impermeability barriers in the cell, one cannot deny the tremendous potential it has locked within its unique structure. Recent research is aiming to reduce these barriers so that effective delivery of this molecule into the cell can be achieved. The results are promising and the molecule has already found its niche in the field of molecular diagnostics. Biotechnologists will follow this wonder- molecule closely, hoping to use its power to diagnose and treat better.

References:

  1. Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future, ARGHYA RAY and BENGT NORDÉN; The FASEB Journal. 2000; 14:1041-1060.
  2. Kinetics and mechanism of the DNA double helix invasion by  pseudocomplementary peptide nucleic acids; Vadim V. Demidov et al; PNAS.
  3. Image ;   www.dkfz.de/funct_genome/