Thursday, 28 November 2019

PNA Technology

PNA Technology

Peptide nucleic acids (PNA) were originally conceived and designed as sequence-specific DNA binding reagents targeting the DNA major groove in analogy to triplex-forming oligonucleotides. However, instead of the sugar-phosphate backbone of oligonucleotides, PNA was designed with a pseudo peptide backbone. It also quickly became clear that triplexes formed between one Homopurine DNA (or RNA) strand and two sequences complementary PNA strands are extraordinarily stable.


 Chemical structures of PNA.
Fig. 1. Chemical structures of PNA.

Structural modes for binding of PNA oligomers.

Fig. 2. Structural modes for binding of PNA oligomers.


Triplex invasion by Pomopyrimidine PNA oligomers

Fig. 3. Triplex invasion by Pomopyrimidine PNA oligomers. 

1. PNA Chemistry

PNA oligomers are easily synthesized by standard solid-phase manual or automated peptide synthesis using either Boc or Fmoc protected PNA monomers, of which the four natural nucleobases are commercially available. Typically the PNA oligomers are deprotected and cleaved off the resin using TFMSA/TFA (tBoc) or and purified by reversed-phase high-performance liquid chromatography (HPLC). While sequencing is not yet a routine option, the oligomers are conveniently characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. PNA oligomers can routinely be labeled with fluorophores (fluorescein, rhodamine) or biotin while labeling with radioisotopes requires the incorporation of tyrosine for  125 I-iodination or conjugation to a peptide motif that can be  32 P-phosphorylated. Furthermore, PNA-peptide conjugates can be obtained by continuous synthesis or using standard peptide-conjugation techniques, such as maleimide cysteine coupling or thioester condensation. Finally, the attractive chemistry of PNA has inspired the synthesis of a large number of PNA analog, including the introduction of a variety of non-natural nucleobases.

Double-duplex invasion PNA Chemistry

Fig. 4. Double-duplex invasion PNA Chemistry

2. Cellular Uptake

PNA oligomers used for biological (antisense or antigene) experiments are typically 12–18-Mers having a molecular weight of 3–4000. Because 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 the employment of cell-penetrating peptides, such as penetration Transportation, 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.

 Chemical structures of non-natural PNA oligomers.

Fig. 5. Chemical structures of non-natural PNA oligomers.

3. Antisense Applications

As mentioned earlier, several examples of PNA-directed (antisense) downregulation 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 unless a triplex-forming PNA is used although exceptions are reported. In cells in culture, the picture is less clear, and in one very recent study, it was even reported that among 20 PNA oligomers targeted to the luciferase gene (in HeLa cells) only one at the far 5'end of the mRNA showed good activity.

Because PNA-RNA duplexes are not substrates for RNAseH, antisense inhibition of translation by PNA is mechanistically different from that of phosphorothioates. Consequently, sensitive targets identified for phosphorothioate oligonucleotides are not necessarily expected to be good targets for PNA.

4. Antigene Properties

PNA triplex-invasion complexes have sufficient stability to arrest elongating RNA polymerase, especially when positioned on the template DNA strand. Naturally, DNA recognition by proteins, such as transcription factor and RNA polymerase is also totally blocked by PNA binding (both triplex- and double-duplex invasion) and the concomitant complete distortion of the DNA helix. Therefore, the PNA gene targeting at the DNA level (antigene) should be very efficient. The main obstacle appears to be the access of the PNA to the DNA under physiological conditions that include the presence of cations (K +, Mg 2+, spermine, etc.) that stabilize the DNA double helix and therefore dramatically reduces the rate of helix invasion by the PNA. Furthermore, the effect of chromatin structure on PNA binding is not known but would be expected to decrease the access to the DNA binding sites.

Nonetheless, it has been reported that triplex invading PNAs induced mutations in mouse cells, thereby inferring target binding in the cell nucleus. Binding in vivo may be greatly facilitated by negative DNA supercoiling, e.g., induced by active transcription, or by the transcription process.

5. Gene Delivery

Gene therapy requires efficient delivery of DNA vectors to the nucleus of cells in desired tissues. The specific and strong binding of PNA to double-stranded DNA has been exploited to tag such vectors noncovalently with fluorophores in order to be able to track the vector in the cells, and more recently with targeting ligands conjugated to the PNA. These were either the nuclear localization signal (NLS) peptide improving nuclear entry of the vector (or ligands (such as ferritin) for cell-specific receptors, that target the vector to cells expressing this receptor.

6. Antimicrobial PNAs

Microbes have also been targets for PNA antisense. Many antibiotics interfere with protein-synthesis by specifically binding to prokaryotic ribosomes. The binding sites of such antibiotics often map to the ribosomal RNA. In an effort to mimic the action of such antibiotics, PNA oligomers were targeted to functionally essential regions of the 23S Escherichia coli ribosomal RNA. In particular, a triplex-forming bis-PNA targeting a 7-Mer Homopurine stretch in the α-sarcin loop effectively inhibited translation in a cell-free system and was also able to inhibit the growth of E. coli, albeit with low potency, which was ascribed to poor uptake of the PNA by the bacteria. Conjugate- ing a simple transporter peptide to the PNA increased the potency significantly, and an even more potent antibacterial PNA was developed by targeting an essential gene involved in the fatty acid synthesis, acpP. This PNA was shown to inhibit the growth of bacteria E. coli in the presence of human (HeLa) cells. Analogous PNA conjugates showed antiinfective efficacy in a mouse model. Unmodified antisense PNA oligomers were also recently shown to downregulate targeted genes in an amoeba (Entamoeba Histolytica).

Antimicrobial PNAs

Fig. 6. The sequence of part of the 23S ribosomal RNA from E. coli. 

7. Antiviral PNAs

Reverse transcriptase, one of the key enzymes in the life cycle of retroviruses (such as HIV), is very sensitive to PNA antisense inhibition. Reverse transcription of the RNA template is effectively arrested by PNA oligomers bound to the template. This finding has raised hope that PNA antiviral drugs could be developed, and one report has even shown that HIV replication in cell culture can be inhibited by PNAs targeting the gag-pol gene.

Structure of Antiviral PNAs

Fig. 7. Structure of Antiviral PNAs

8. Genetic Information Carrier

PNA oligomers are potential carriers of genetic information through their nucleobase sequence. As PNAs are also peptides, these molecules formally bridge the chemistry and function of proteins (peptides) and nucleic acids (DNA) and in this respect may be of relevance to the discussion of the prebiotic evolution of life. It is well-established that the formation of amino acids and nucleobases could have occurred in a prebiotic soup on the young Earth, whereas it is very difficult to imagine and mimic conditions that would create sugars (ribose) and nucleosides. Thus one may consider the possibility that a PNA-like prebiotic genetic material may have been a predecessor of RNA and the RNA world.

9. PNA in Diagnostics

The excellent hybridization properties of PNA oligomers combined with its unique chemistry has been exploited in a variety of genetic diagnostic techniques. Thus PNA-fluorescence in situ hybridization (FISH) techniques has been developed for quantitative telomere analyses, chromosome painting, and viral and bacterial diagnostics both in medical as well as environmental samples.

In another very powerful application, PNA oligomers can be used to silent polymerase chain reaction (PCR) amplification in single mutation analyses. This technique is so powerful that it is possible to obtain a specific signal from a single mutation oncogene in the presence of a 1,000–10,000-fold excess of the nonmutated wild-type normal gene.

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