Monday, December 1, 2008
Peptide nucleic acids (PNA) are synthetic DNA analogues or mimics with a polyamide backbone instead of a sugar phosphate bone. The significant of this post is the importance to biosensing, PNAs exhibit superior hybridization characteristics and improved chemical and enzymatic stability compared to nucleic acids. Both double and triple stranded complexes are capable of being formed by PNA in association with nucleotides the negatively charged ribosephosphate backbone of nucleic acids is replaced by an uncharged N-(2-aminoethyl)-glycine scaffold to which the nucleobases are attached via a methylene carbonyl linker.
The neutral amide backbone also enables PNA to hybridize to DNA molecules in lowsalt conditions because no positive ions are necessary for counteracting the interstrand repulsion that hampers duplex formation between two negatively charged nucleic acids. Consequently, the abundance and stability of intramolecular folding structures in the DNA or RNA analytes are significantly reduced, making the molecules more accessible to complementary PNA oligomers. Because the intramolecular distances and configuration of the nucleobases are similar to those of natural DNA molecules, specific hybridization occurs between PNAs and cDNA or RNA sequences.
The uncharged nature of PNAs is responsible for a better thermal stability of PNA–DNA duplexes compared with DNA–DNA equivalents and, as a result, single-base mismatches have a considerably more destabilizing effect As with DNA, the decrease in duplex stability depends on the position of the mismatch within the sequence. Thus, the use of PNAs will contribute significantly to establishment of faster and more reliable biosensing applications. PNAs have now been used to replace DNA to functionalize gold nanoparticles and, upon hybridization to complementary DNA strands and formation of nanoparticle aggregates, resulted in (1) a red-to-blue color transition and (2) high discrimination of DNA single-base mismatches. PNAs are stable across a wide range of temperatures and pHs. Of significant importance in clinical samples, PNAs are resistant to nucleases and proteases. In contrast to DNA molecular beacons, stemless PNA beacons are less sensitive to ionic strength and the quenched fluorescence of PNA is not affected by DNAbinding proteins. This enables the use of PNA beacons under conditions that are not feasible for DNA beacons.
The very different nature of PNA molecular structure enables new modes of detection, especially procedures that avoid the introduction of a label. The change of color results from the shift of surface plasmon band upon aggregation and this property is now the basis of colorimetric biosensors for selective detection of DNA. PNA functionized gold nanoparticles have been shown to be simple, highly sensitive and selective. Xi and coworkers used DNA and PNA molecular beacons to detect and quantify rRNA in solution and in whole cells. Of clinical relevance, PNA molecular beacons are ideal tools for detection of whole bacteria in solution and in real time. Xi and coworkers use real-time confocal microscopy to detect the fluorescence emitted from DNA and PNA molecular beacons in microfluidic systems.