Key featuresShow Hide
- Milder chemistry of the Fmoc/Bhoc protection allows the synthesis of PNA with e.g. sensitive reporter groups.
- The benzhydryloxycarbonyl (Bhoc) group protection of the exocyclic amino groups of the nucleobases provides sufficient protection during synthesis, is readily removed under the cleavage conditions, and renders solubility to the monomers.
- Cleavage and deprotection can also be achieved in minutes, provided a suitable resin is used.
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Peptide Nucleic Acid (PNA) was originally conceived as a ligand for the recognition of double- stranded DNA. (1) The concept was to mimic an oligonucleotide binding to double stranded DNA via Hoogsteen base pairing. However it is the favourable properties of PNA when mimicing and/or binding to single strands of DNA that have seen PNA gather interest in many areas of modern chemical biology.
The structure of PNA is quite simple, consisting of repeating N-(2-aminoethyl)- glycine units linked by amide bonds. The purine (A, G) and pyrimidine (C, T) bases are attached to the backbone by methylene carbonyl linkages. Unlike DNA or its analogues, PNAs do not contain any sugar moieties or phosphate groups. Again, unlike DNA, the backbone is acyclic, achiral and neutral.
It is tempting to regard PNA as a DNA analogue, however its chemical structure shows that it is in fact more similar to a protein or peptide molecule. Nevertheless, for applications using PNA the basis of analysis is using sequence information just like with DNA etc. By convention, PNAs are represented like peptides, with the N-terminus (or pseudo 5’) at the left hand side position and the C-terminus (pseudo 3’) at the right.
PNA oligomers are less soluble in water than DNA, and in some aqueous buffers (especially phosphate) poor solubility can be an issue. This is particularly true with increasing length (>12 units) and purine content (especially G above 60%). Often the inclusion of one or two lysine residues can alleviate this problem, as can use of the AEEA spacer.
The neutrality of the PNA backbone is a significant feature that has several consequences. One of the most important is the stronger binding between complementary PNA/DNA strands than between DNA/DNA strands at low to medium ionic strength. This can be attributed to the lack of charge repulsion between PNA and DNA. This is also thought to be the reason that the sequence specificity of PNA to DNA is also higher than in native DNA/DNA strands.(2)
In general, homopyrimidine PNAs form extremely stable triplexes that have sufficient stability to invade intact double stranded DNA. Studies have also shown that 2PNA/DNA triplex formation follows the rules of homopyrimidine DNA triplex formation, i.e. with an antiparallel Watson-Crick duplex and a parallel bound Hoogsteen strand. Even more stable triplexes can be formed when the Watson-Crick PNA strand is connected by continuous synthesis via ethylene glycol type linkers (e.g. AEAA Spacer) to the Hoogsteen strand. Such constructs are called bis- PNAs.(3)
Although PNA was first synthesised using tBoc/Z chemistry, the milder chemistry of the Fmoc/Bhoc protection allows the synthesis of PNA with e.g. sensitive reporter groups. The simplified final cleavage and deprotection can also be achieved in minutes, provided a suitable resin is used.
After extensive screening, the benzhydryloxycarbonyl (Bhoc) group was selected as the best choice for protecting the exocyclic amino groups of the nucleobases. This group provides sufficient protection during synthesis, is readily removed under the cleavage conditions, and renders solubility to the monomers. For PNA synthesis, therefore, we provide the four Fmoc/ Bhoc monomers and a hydrophilic spacer molecule, AEEA.
The latter is used in bis PNA and can be added to PNA to aid solubility. It is also useful to add to the N-terminus (pseudo 5’) when labelling PNA with e.g. biotin, ROX, TAMRA etc.
- (a) Sequence selective recognition of DNA by strand displacement with a thymine-substituted polyamide, P.E. Nielsen, M. Egholm, R.H. Berg and O. Buchardt, Science, 254, 1497-1500, 1991; (b) Peptide nucleic acids (PNA). Oligonucleotide analogues with an achiral peptide backbone, M. Egholm, O. Buchardt, P.E. Nielsen and R.H. Berg, J. Amer. Chem. Soc., 114, 1895-1897, 1992; (c) Peptide nucleic acids (PNA). DNA analogues with a polyamide backbone, P.E. Nielsen, M. Egholm, R.H. Berg and O. Buchardt, In “Antisense Research and Application”, S. Crook and B. Lebleu (eds.), CRC Press, Boca Raton, pp. 363-373.
- PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules, M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S.M. Freier, D.A. Driver, R.H. Berg, S.K. Kim, B. NordJn and P.E. Nielsen, Nature, 365, 556-568, 1993.
- Single and bis peptide nucleic acids as triplexing agents: binding and stoichiometry, M.C. Griffith, L.M. Risen, M.J. Greig, E.A. Lesnik, K.G. Sprangle, R.H. Griffey, J.S. Kiely and S.M. Freier, J. Amer. Chem. Soc., 117, 831-832, 1995.