Peptide nucleic acid

Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA.^[1]

Synthetic peptide nucleic acid oligomers have been used in recent years in molecular biology procedures, diagnostic assays, and antisense therapies.^[2] Due to their higher binding strength, it is not necessary to design long PNA oligomers for use in these roles, which usually require oligonucleotide probes of 20–25 bases. The main concern of the length of the PNA-oligomers is to guarantee the specificity. PNA oligomers also show greater specificity in binding to complementary DNAs, with a PNA/DNA base mismatch being more destabilizing than a similar mismatch in a DNA/DNA duplex. This binding strength and specificity also applies to PNA/RNA duplexes. PNAs are not easily recognized by either nucleases or proteases, making them resistant to degradation by enzymes. PNAs are also stable over a wide pH range. Though an unmodified PNA cannot readily cross the cell membrane to enter the cytosol, covalent coupling of a cell penetrating peptide to a PNA can improve cytosolic delivery.^[3]

PNA is not known to occur naturally but N-(2-aminoethyl)-glycine (AEG), the backbone of PNA, has been hypothesized to be an early form of genetic molecule for life on Earth and is produced by cyanobacteria and is a neurotoxin.^[4]

PNA was invented by Peter E. Nielsen (Univ. Copenhagen), Michael Egholm (Univ. Copenhagen), Rolf H. Berg (Risø National Lab), and Ole Buchardt (Univ. Copenhagen) in 1991.^[1]

Structure

DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge (-CH
₂-) and a carbonyl group (-(C=O)-). PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the last (right) position.^[5]

Binding

Since the backbone of PNA contains no charged phosphate groups, the binding between PNA/DNA strands is stronger than between DNA/DNA strands due to the lack of electrostatic repulsion. Unfortunately, this also causes it to be rather hydrophobic, which makes it difficult to deliver to body cells in solution without being flushed out of the body first. Early experiments with homopyrimidine strands (strands consisting of only one repeated pyrimidine base) have shown that the T_m ("melting" temperature) of a 6-base thymine PNA/adenine DNA double helix was 31 °C in comparison to an equivalent 6-base DNA/DNA duplex that denatures at a temperature less than 10 °C. Mixed base PNA molecules are true mimics of DNA molecules in terms of base-pair recognition. PNA/PNA binding is stronger than PNA/DNA binding.

PNA translation from other nucleic acids

Several labs have reported sequence-specific polymerization of peptide nucleic acids from DNA or RNA templates.^[6]^[7]^[8] Liu and coworkers used these polymerization methods to evolve functional PNAs with the ability to fold into three-dimensional structures, similar to proteins, aptamers and ribozymes.^[6]

Delivery

In 2015, Jain et al. described a trans-acting DNA-based amphiphatic delivery system for convenient delivery of poly A tailed uncharged nucleic acids (UNA) such as PNAs and morpholinos, so that several UNA's can be easily screened ex vivo.^[9]^{[non-primary source needed]}

PNA world hypothesis

It has been hypothesized that the earliest life on Earth may have used PNA as a genetic material due to its extreme robustness, simpler formation, and possible spontaneous polymerization at 100 °C^[10] (while water at standard pressure boils at this temperature, water at high pressure—as in deep ocean—boils at higher temperatures). If this is so, life evolved to a DNA/RNA-based system only at a later stage.^[11]^[12] Evidence for this PNA world hypothesis is, however, far from conclusive.^[13] If it existed though, it must have preceded the widely accepted RNA world.

Applications

Applications include alteration of gene expression - both as inhibitor and promoter in different cases, antigene and antisense therapeutic agent, anticancer agent, antiviral, antibacterial and antiparasitic agent, molecular tools and probes of biosensor, detection of DNA sequences, and nanotechnology.^[14]^[15]

PNAs can be used to improve high-throughput 16S ribosomal RNA gene sequencing of plant and soil samples by blocking amplification of contaminant plastid and mitochondrial sequences.^[16]

Cellular – Functional Antagonism/Inhibition. In 2001, Strauss and colleagues reported the design of an application for PNA oligomers in living mammalian cells. The Xist chromatin binding region was first elucidated in female mouse fibroblastic cells, and embryonic stem cells though the use of a PNA molecular antagonist. The novel PNA approach directly demonstrated function of a lncRNA. The long non-coding (lncRNA) RNA, Xist directly binds to the inactive X-chromosome. Functional PNA inhibition experiments revealed that specific repeat regions of the Xist RNA were responsible for chromatin binding, and hence could be considered domain regions of the RNA transcript. The PNA molecular antagonist was administered to living cells and functionally inhibited the association of Xist with inactive X-chromosome using the approach for studying noncoding RNA function in living cells called peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of Xist RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.^[17]

References

^ ^a ^b Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science. 254 (5037): 1497–500. Bibcode:1991Sci...254.1497N. doi:10.1126/science.1962210. PMID 1962210.
^ Gupta A, Mishra A, Puri N (October 2017). "Peptide nucleic acids: Advanced tools for biomedical applications". Journal of Biotechnology. 259: 148–159. doi:10.1016/j.jbiotec.2017.07.026. PMC 7114329. PMID 28764969.
^ Zhao XL, Chen BC, Han JC, Wei L, Pan XB (November 2015). "Delivery of cell-penetrating peptide-peptide nucleic acid conjugates by assembly on an oligonucleotide scaffold". Scientific Reports. 5: 17640. Bibcode:2015NatSR...517640Z. doi:10.1038/srep17640. PMC 4661726. PMID 26612536.
^ Banack SA, Metcalf JS, Jiang L, Craighead D, Ilag LL, Cox PA (7 November 2012). "Cyanobacteria Produce N-(2-Aminoethyl)Glycine, a Backbone for Peptide Nucleic Acids Which May Have Been the First Genetic Molecules for Life on Earth". PLOS ONE. 7 (11): e49043. doi:10.1371/journal.pone.0049043. PMC 3492184. PMID 23145061.
^ Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE (October 1993). "PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules". Nature. 365 (6446): 566–8. Bibcode:1993Natur.365..566E. doi:10.1038/365566a0. PMID 7692304. S2CID 4318153.
^ ^a ^b Brudno Y, Birnbaum ME, Kleiner RE, Liu DR (February 2010). "An in vitro translation, selection and amplification system for peptide nucleic acids". Nature Chemical Biology. 6 (2): 148–55. doi:10.1038/nchembio.280. PMC 2808706. PMID 20081830.
^ Kleiner RE, Brudno Y, Birnbaum ME, Liu DR (April 2008). "DNA-templated polymerization of side-chain-functionalized peptide nucleic acid aldehydes". Journal of the American Chemical Society. 130 (14): 4646–59. doi:10.1021/ja0753997. PMC 2748799. PMID 18341334.
^ Ura Y, Beierle JM, Leman LJ, Orgel LE, Ghadiri MR (July 2009). "Self-assembling sequence-adaptive peptide nucleic acids". Science. 325 (5936): 73–7. Bibcode:2009Sci...325...73U. doi:10.1126/science.1174577. PMID 19520909. S2CID 13327028.
^ Jain HV, Verthelyi D, Beaucage SL (2015). "Amphipathic trans-acting phosphorothioate DNA elements mediate the delivery of uncharged nucleic acid sequences in mammalian cells". RSC Advances. 5 (80): 65245–65254. Bibcode:2015RSCAd...565245J. doi:10.1039/C5RA12038A.
^ Wittung P, Nielsen PE, Buchardt O, Egholm M, Nordén B (April 1994). "DNA-like double helix formed by peptide nucleic acid". Nature. 368 (6471): 561–3. Bibcode:1994Natur.368..561W. doi:10.1038/368561a0. PMID 8139692. S2CID 551986.
^ Nelson KE, Levy M, Miller SL (April 2000). "Peptide nucleic acids rather than RNA may have been the first genetic molecule". Proceedings of the National Academy of Sciences of the United States of America. 97 (8): 3868–71. Bibcode:2000PNAS...97.3868N. doi:10.1073/pnas.97.8.3868. PMC 18108. PMID 10760258.
^ Alberts B, Johnson A, Lewis J (March 2002). Molecular Biology of the Cell (4th ed.). Routledge. ISBN 978-0-8153-3218-3.
^ Zimmer C (January 2009). "Evolutionary roots. On the origin of life on Earth". Science. 323 (5911): 198–9. doi:10.1126/science.323.5911.198. PMID 19131603. S2CID 206583796.
^ Anstaett P, Gasser G (2014). "Peptide nucleic acid - an opportunity for bio-nanotechnology" (PDF). CHIMIA. 68 (4): 264–8. doi:10.2533/chimia.2014.264. PMID 24983612.
^ D'Souza AD, Belotserkovskii BP, Hanawalt PC (February 2018). "A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system". Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1861 (2): 158–166. doi:10.1016/j.bbagrm.2017.12.008. PMC 5820110. PMID 29357316.
^ Lundberg DS, Yourstone S, Mieczkowski P, Jones CD, Dangl JL (October 2013). "Practical innovations for high-throughput amplicon sequencing". Nature Methods. 10 (10): 999–1002. doi:10.1038/nmeth.2634. PMID 23995388. S2CID 1751531.
^ "Beletskii et al 2001"/Beletskii, Anton; Strauss, William (2001). "PNA interference mapping demonstrates functional domains in the noncoding RNA Xist". Proceedings of the National Academy of Sciences of the United States of America. 98 (16): 9215–20. Bibcode:2001PNAS...98.9215B. doi:10.1073/pnas.161173098. PMC 55400. PMID 11481485.