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Peptide Nucleic Acid (PNA) Synthesis

​​​​​​​​​​​​​​Peptide nucleic acids mimic oligonucleotides in that the ability to base pair remains while the sugar phosphate backbone has been replaced by a charge neutral polyamide. The neutral charge facilitates a more stable duplex when hybridised to DNA or RNA. As a result, PNA lends itself well to antisense or diagnostic technologies.

Guidelines for synthesis of PNA using an automated DNA synthesiser are provided here, however these could be easily applied to other automated instruments capable of synthetic chemistry, in fact many users have successfully transferred this synthesis to microwave assisted peptide synthesisers. Information on obtaining protocols for manual PNA synthesis or synthesis by alternative automated means is provided below.

Physical & Dilution Data

Dilution volumes (in ml) are for 0.2M solutions in dry NMP. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/0.5ml to achieve 0.2M, regardless of molecular weight.

Item Mol. Formula Mol. Wt. Unit Wt. 250mg 500mg 1g
LK5001 C40H35N7O7 725.76 275.27 1.72 3.44 6.89
LK5002 C39H35N5O8 701.74 251.24 1.78 3.56 7.13
LK5003 C40H35N7O8 741.76 291.27 1.69 3.37 6.74
LK5004 C26H26N4O7 506.52 266.26 2.47 4.94 9.87
LK5005 C21H23NO6 385.42 145.16 3.24 6.49 12.97
LK5010 C13H24O7S 324.39 265.11 3.85 7.71 15.41
LK5011 C19H30N2O7S2 462.58 336.15 2.70 5.40 10.81

General Guidelines for PNA Design

PNA has a higher binding affinity to a DNA strand in a duplex than DNA itself. Therefore, for most applications, a short oligomer length of 12-15 units is sufficient, and in many cases even shorter probes will work well. Longer oligomers tend to aggregate and are difficult to purify and characterise.

Purine-rich PNA tends to aggregate, particularly with G-rich oligomers. As a rule, limit the number of purines to 6 in any 10 units and do not include more than 3 G units in a row. The shorter the oligomer, the less susceptible to aggregation it will be. Avoid, if possible, self-complementary sequences as PNA/PNA interactions are even stronger than those of PNA/DNA.

Incorporating lysine at one or both ends of the PNA oligomer can greatly assist solubility without any adverse effect on the hybridsation properties, as is the incorporation of multiple AEAA (LK5005) linkers. In reality, these guidelines cannot always be adhered to, but there are ways to improve the synthesis and downstream handling of more complex PNA oligos.

Labelling of PNA

PNA can be labelled with e.g. fluorescein and biotin, however this can decrease the solubility of the oligomer, especially with purine-rich sequences. When labelling PNA, adhere to the following:

  • Incorporate two or more spacer monomers (LK5005) between the label and the PNA oligomer;
  • If you are labelling with biotin, fluorescein, rhodamine, or other acid-resistant reporter groups, PNA oligomer labelling is best carried out on the resin before cleavage & deprotection;
  • If you are labelling with acid-sensitive compounds, labels must be attached in solution after cleavage & deprotection.
    C-terminal labels may be similarly prepared by the incorporation of e.g. Lysine.

Direction of Synthesis

In PNA synthesis the amino-terminal is analogous with the 5’-end of a DNA sequence. Using methods conventional to DNA synthesis, therefore, the PNA will be synthesised starting with the C-terminus towards the N-terminus (pseudo 3’-5’).

General PNA Oligomer Synthesis Protocols1

Fmoc-based PNA synthesis consists of a repetitive cycle of deblocking, activation/coupling, and capping. Typically this is done on a 2μmol scale.

Choice of Solid Support

In general, an appropriate solid support must be cleavable in acid, leaving a C-terminal amide, and be compatible with Fmoc chemistry. Both PAL-PEG-PS and XAL-PEG-PS have been found to be suitable in this regard.

Despite the advantages of XAL-handled supports outlined later, it should be remembered that either the PAL or XAL handles may be placed on a wide range of resin types. Best results, however, have been reported with polyethylene glycol derivatised polystyrene (PEG-PS). Note also that PNA synthesis is optimally performed on resins loaded below 0.2mmol/g. Higher loadings can lead to aggregation and poor synthesis quality.

Finally, unlike the most common solid supports used in DNA synthesis, the PNA supports are universal – the first monomer is not contained within the support - and therefore only a single support is needed rather than a range of four. Note that, due to the universal support, the C terminal is an amide rather than a free acid.


PNA monomers are prepared for coupling as 0.2M solutions in peptide-grade N-methylpyrrolidone (NMP). Gentle heating on e.g. a dri-block may be required to fully dissolve the monomers (especially C). Our PNA monomers are provided in 500mg and 1g bulk sizes, and in a convenient 700μmol size ready-packaged in a 30ml vial for use on an Expedite DNA synthesiser (enough for around 30 additions). The AEEA spacer (LK5005) is provided in Expedite-ready 500μmol vials (also 30ml).

Fmoc-Deprotection (Deblocking)

Removal of the Fmoc from the primary aliphatic amine, required for subsequent monomer coupling, is achieved by treatment with 20% piperidine in DMF for 30s (flow rate 0.4ml/min). Transacylation is possible at this step (A>G>T, but not with C), however this is not a significant issue if brief basic treatments are employed. A deblock step is required to remove the final Fmoc group prior to cleavage, unless Fmoc-ON purification is desired.


The coupling conditions were initially based on methods employed for Boc synthesis.2 Activation of the monomer carboxylic acid function is achieved with HATU (or PyBOP®) and a base mixture of 0.2M DIPEA and 0.3M lutidine for 2.5min. This mixture requires to “pre-activate” for this time before introduction to the support for coupling. A slight excess of monomer is used in the coupling (prevents the undesired tetramethylguanidine capping arising from direct reaction of HATU with the primary amine on the growing PNA chain). A minimum activation-coupling time of 7.5min (or 8.25min with washing steps) is suggested.

Capping (Acetylation)

Capping of unreacted product is carried out with a solution of 5% acetic anhydride and 6% lutidine in DMF. This mixture has been found to have a longer shelf life (months) than acetic anhydride and bases such as DIPEA or pyridine. Note also that the piperidine wash step, found to be useful in Boc protocols for elimination of products stemming from acylation of the exocyclic and cyclic amino groups of the nucleobases, is effectively accomplished during Fmoc synthesis during each subsequent deprotection.


PNA oligomers can be labelled at the N-terminus by attachment via the free amine generated on removal of the final Fmoc group. This is best accomplished whilst the PNA is still attached to the solid support (as outlined above, two or more spacer (LK5005) molecules incorporated into the sequence, prior to addition of the label increases the conjugation efficiency). Labelling is then carried out using e.g. an activated NHS ester (5 to 10-fold excess) of biotin, fluorescein, etc using the protocols applicable to those materials. If the PNA is lysine terminated, then the lysine residue can be considered a substitute for one spacer residue. After labelling, oligos are cleaved and deprotected as below. Where multiple Lys incorporations are required, it is important to use orthogonal protection to obtain selective labelling.

Acid-sensitive labels cannot be incorporated on-column as they will not survive the cleavage conditions. Such labels are attached post-synthetically in solution. Again, follow the protocol appropriate to the label being used.

Labelling can also be accomplished in solution via the ε-amino group of an incorporated lysine. This is particularly useful for introducing a C-terminal label or second label to the oligomer. Also, since there are several orthogonal protection strategies applicable to Lys, it is possible to use this as a means of adding multiple labels. In a similar way, it is also possible to use suitably protected Cys residues.

Cleavage and Bhoc Deprotection

PNA is both deprotected and cleaved from the solid support by treatment with TFA containing 5% m-cresol, the latter a scavenger for the resultant benzhydryl cations. However, depending on the amino acids used in PNA-petide synthesis, other scavengers such as 10% phenol and/or TIS (triisopropylsilane) may be required. Deprotection is complete within 1min, however the cleavage takes longer and is dependent on the support used. Approximately 400μl of cleavage solution is required for a 2μmol synthesis.

Generally, PAL-PEG-PS is used for Fmoc-based PNA assembly; this requires treatment with 5% m-cresol in TFA (or similar) for 90min to complete cleavage. The XAL synthesis handle can be employed for more rapid cleavage, complete in 5min. The XAL synthesis handle, like PAL, produces PNA with a carboxy terminal amide. The PNA product is isolated from the cleavage mixture by precipitation with diethyl ether.

Typical Cleavage & Deprotection Protocol (0.2μmol Scale):

  1. After drying the resin, transfer to a microfilter tube.
  2. Add 200μl of the 5% m-cresol in TFA cleavage mixture (or similar) to the top of the microfilter tube. Allow to sit at room temperature for:

    PAL resin — 90min.
    XAL resin — 5min.
  3. Centrifuge the resin for 5min.
  4. Repeat steps 2 and 3.
  5. Remove the filter insert from the microcentrifuge tube.
  6. Add 1 ml of diethyl ether.
  7. Shake or vortex for about 1min.
  8. Centrifuge for 5min. The crude PNA forms a pellet at the bottom of the microcentrifuge tube.
  9. Decant the supernatant. Retain this until cleavage and deprotection is complete, and you have successfully recovered the PNA.
  10. Repeat steps 6-9 twice more.
  11. Dry the PNA by removing the cap from the tube and leaving open for about an hour to allow excess diethyl ether to evaporate.

Analysis and Purification

PNA, like DNA, can be analysed for purity by RP-HPLC and MALDI-TOF mass spectroscopy. Reconstitution of the PNA for analysis is best done in a solution of 0.1% TFA in water. The quantity of PNA produced is measured as a total optical density (OD) at 260nm. This ranges between 80 and 180OD for a 2μmol synthesis depending on the length of sequence.

Suggested HPLC conditions: C8 or C18, A = 0.1% TFA in water, B = 0.1% TFA in acetonitrile. Gradient of 19:1 to 13:7 over 35min at 1ml/min. HPLC analysis is performed at 55°C to avoid aggregation of the PNA.

Purification is best carried out by RP-HPLC using conditions similar to the analytical method. Fmoc-ON purification may be performed. If the PNA has an N-terminal O-spacer unit or an α-amino acid, Fmoc-ON purification is very useful to separate the modified PNA from the unmodified PNA.

Handling, Storage & Stability

The monomers are stored dry in a freezer at –20°C. The dry monomers are prone to static. Typically, 0.2M PNA monomer solutions are stable for 1-2 weeks in peptide-grade NMP.

PNA oligomers have a high affinity for glass surfaces and polystyrene. Where possible use polypropylene or polyethylene materials when working with low (sub-micromolar) concentrations.

Crude PNA oligomers can be aliquoted from 0.1% TFA/water solutions, however if they are not to be used immediately then drying the aliquots prevents degradation. PNA purified by RP-HPLC with TFA present will be protonated at all the basic amino groups (A, C and the amino terminus) and hence carry trifluoroacetate counter-ions when dissolved in water. This aids solubility at high concentrations, however users should be aware of possible toxic effects of TFA in cells. Buffered solutions can be used to regulate pH.

In cases where the oligomer is difficult to dissolve, addition of 10-20% acetonitrile to the aqueous solution and heating to 50°C for 10min aids solubility.

Further Information

Alternative Synthesis Methods

If you are familiar with DNA synthesis then synthesis of PNA on an Expedite instrument may be your simplest “entry-point” for this chemistry. However, other methods are available.

A recent example Fmoc-based protocol of PNA synthesis on a peptide synthesiser (Advanced ChemTech Omega 396) has been reported.5 The Liberty range of microwave-assisted peptide synthesisers is also capable of PNA synthesis (see

A detailed protocol for manual PNA Fmoc synthesis is available from the Schneider research group at Carnegie Mellon University. Please contact Prof. James Schneider by e-mail at Goodwin et al have also described a method applicable to general PNA synthesis.6 Hüsken et al have also recently described a manual method utilised when labelling PNA with ferrocene.7


LINK would like to thank Donna Williams of the MRC in Cambridge, UK, and Professor James Schneider of Carnegie Mellon University in Pittsburgh, US, for their helpful discussions on the use of these products.


  1. Protocols are based on those provided in Synthesis of PNA Oligomers by Fmoc Chemistry, R. Casale, I.S. Jensen and M. Egholm, in Peptide Nucleic Acids: Protocols and Applications, P.E. Nielsen (Ed.), Second Edition, Garland Science, 2003.
  2. (a) Improved synthesis, purification and characterisation of PNA oligomers, L. Christensen, R. Fitzpatrick, B. Gildea, B. Warren and J. Coull, In Solid Phase Synthesis. Peptides, Proteins and Nucleic Acid, R. Epton, Ed., pp149-156, Mayflower Worldwide Ltd, Birmingham, 1994; (b) Solid-phase synthesis of peptide nucleic acids, L. Christensen, R. Fitzpatrick, B. Gildea, K.H. Petersen, H.F. Hansen, T. Koch, M. Egholm, O.Buchardt, P.E. Neilsen, J. Coull and R.H. Berg, J. Peptide Sci., 3, 175-183, 1995.
  3. Effect of Tertiary Bases on O-Benzotriazolyluronium Salt-Induced Peptide Segment Coupling, L.A.Carpino and A. El-Faham, J. Org. Chem., 59, 695-698, 1994.
  4. Preparation and Applications of Xanthenylamide (XAL) Handles for Solid-Phase Synthesis of C-Terminal Peptide Amides under Particularly Mild Conditions, Y. Han, S.L. Bontems, P. Hegyes, M.C. Munson, C.A. Minor, S.A. Kates, F. Albericio and G. Barany, J. Org. Chem., 61, 6326-6339, 1996.
  5. Effect of terminal amino acids on the stability and specificity of PNA-DNA hybridisation, N.C. Silvester, G.R. Bushell, D.J. Searles and C.L. Brown, Org. & Biomol. Chem., 5, 917-923, 2007.
  6. A simple procedure for the solid-phase synthesis of peptide nucleic acids with N-terminal cysteine, T.E. Goodwin, R.D. Holland, J.O. Lay and K.D. Raney, Bioorganic and Medicinal Chem. Lett., 8, 2231-2234, 1998.
  7. “Four-Potential” ferrocene labeling of PNA oligomers via click chemistry, N. Hüsken, G. Gasser, S.D. Köster and N. Metzler-Nolte, Bioconjuagte Chem., 20, 1578-1586, 2009.

PNA monomers are manufactured and sold pursuant to licence under one or more of US Patents Nos. 5,773,571, 6,133,444, 6,172,226, 6,395,474, 6,414,112, 6,613,873, 6,710,163 and 6,713,602, or corresponding patent claims outside the US. PNA Monomers are sold to be used for internal research use only, and are not to be resold unless by separate licence.​​​​​​​

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