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Synthesis and Purification of Peptide Nucleic Acids
Publication Name:
Current Protocols in Nucleic Acid Chemistry
Unit Number:
Unit 4.11
DOI:
10.1002/0471142700.nc0411s09
Online Posting Date:
August, 2002 Abstract
Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2-aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson-Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high-affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.
Table of Contents
- Unit Introduction
- Basic Protocol 1: Automated Synthesis of Peptide Nucleic Acids
- Support Protocol 1: Adding Peptides to PNAs
- Support Protocol 2: Addition of Biotin
- Basic Protocol 2: Manual Synthesis of Peptide Nucleic Acids
- Support Protocol 3: Preparation of Carrier Resin for Manual PNA Synthesis
- Basic Protocol 3: Purification and Analysis of Peptide Nucleic Acids
- Commentary
- Bibliography
- Figures
- Tables
Materials
Basic Protocol 1: Automated Synthesis of Peptide Nucleic Acids
Materials
- PNA Expedite reagents (Applied Biosystems)
- FMOC-PNA monomers (Fig.
- Diluent: N-methylpyrrolidone (NMP)
- Activator: 7-aza-1-hydroxybenzotriazole (HOAt) or O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
- Linker: 2-aminoethoxy-2-ethoxy acetic acid (AEEA)
- Base solution: 0.2 M diisopropylethylamine (DIPEA)/0.3 M 2,6-lutidine
- Deblocking solution: 20% (v/v) piperidine in N,N-dimethylformamide (DMF)
- Capping solution: 5% (v/v) acetic anhydride/6% (v/v) 2,6-lutidine in DMF
- N,N-Dimethylformamide (DMF), anhydrous (Burdick Jackson; wash A is Opti-Dry DMF from Fisher; wash B is anhydrous DMF from Applied Biosystems)
- Amino acids (Novabiochem, Advanced Chemtech)
- Isopropyl alcohol (optional)
- Cleavage cocktail: 20% (v/v) m-cresol (Sigma-Aldrich) in trifluoroacetic acid (TFA; Burdick Jackson)
- Diethyl ether, –20°C
Expedite 8909 synthesizer (Applied Biosystems)
FMOC-XAL-PEG-PS synthesis column (0.2 µmol prepacked; Applied Biosystems)
10-mL syringe
1.5-mL, 0.2-µm polytetrafluoroethylene (PTFE) or regenerated cellulose spin column (Millipore)
Additional reagents and equipment for automated synthesis (see manufacturer's instructions) and for purification and analysis of PNAs (see Basic Protocol 3 )
NOTE: Powdered reagents such as monomers, activator, and linker (AEEA) should be unpacked on arrival and stored at –20°C in a sealed container containing Drierite desiccant. Monomers should be inspected upon arrival. Clumps of reagent may indicate that water has been introduced during shipping.
NOTE: The authors use three sources of DMF because the bottles from Fisher (Opti-Dry) and Applied Biosystems fit onto the input fitting and reagent port on the synthesizer, and the third can be used following synthesis. All DMF must be anhydrous and should have a low amine content to reduce the likelihood of side reactions. DMF should be purchased in 100-mL volumes to ensure that it is used quickly, minimizing the likelihood that contaminating water will interfere with synthesis.
Support Protocol 2: Addition of Biotin
Additional Materials (also see Basic Protocol 1 )
- Biotin (Sigma)
- 42°C water bath
- 1-mL syringe
Basic Protocol 2: Manual Synthesis of Peptide Nucleic Acids
Materials
- Nitrogen source
- N,N-Dimethylformamide (DMF; OptiDry; Fisher)
- 4-Hydroxymethylphenylamidomethyl (PAM) resin protected with tert-butyloxycarbonyl (BOC; Applied Biosystems)
- Carrier resin: PAM resin capped with an acetyl group (see
- BOC-PNA monomers (Fig.
- tert-butyloxycarbonyl-protected peptide nucleic acid monomers (A, C, G, and T), base protected with benzyloxycarbonyl
- 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) activators (Applied Biosystems)
- Fresh dichloromethane (DCM; Fisher)
- m-Cresol
- Trifluoroacetic acid (TFA; Burdick Jackson)
- Pyridine
- Diisopropylethylamine (DIPEA)
- Methanol
- Thioanisole
- Trifluoromethanesulfonic acid (TFMSA; Aldrich)
- Diethyl ether, ice cold
250°C oven
125-mL vacuum filtration side-arm flasks
24/40 rubber septa
15-mL medium (C) fritted Pyrex funnel
Vacuum tubing
3-way valves
250-mL Wheaton bottles with caps
Lyophilizer
10-mL flask with a ground glass joint
Desiccator
Additional reagents and equipment for purification and analysis of PNAs (see Basic Protocol 3 )
NOTE: All powdered reagents such as monomers and activator should be unpacked on arrival and stored at –20°C in a sealed container containing Drierite desiccant. Monomers should be inspected upon arrival. Clumps of reagent may indicate that water has been introduced during shipping. DMF should have a low amine content to reduce the likelihood of side reactions.
Support Protocol 3: Preparation of Carrier Resin for Manual PNA Synthesis
Additional Materials (also see Basic Protocol 2 )
- tert-Butyloxycarbonyl-protected 4-hydroxymethylphenylamidomethyl resin (e.g., BOC-Ala-PAM, BOC-Val-PAM, BOC-Ile-PAM; Applied Biosystems)
- Acetic anhydride
- HPLC-grade dichloromethane
- HPLC-grade methanol
Basic Protocol 3: Purification and Analysis of Peptide Nucleic Acids
Materials
- PNA sample solution (see
- RP-HPLC buffer A: 0.1% (v/v) trifluoroacetic acid (TFA; Burdick Jackson) in water, passed through a 47-mm, 0.4-µm nylon membrane (Whatman)
- RP-HPLC buffer B: 0.1% (v/v) TFA in acetonitrile (Optima grade; Fisher), filtered through an Anodisc 47 filter (0.22-µm; Whatman)
- -Cyano-4-hydroxycinnamic acid (Sigma)
- Isopropanol
High-performance liquid chromatograph (HPLC) with C18 reversed-phase column (300-Å Microsorb-MV column; Varian Analytical Instruments)
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Voyager-DE workstation; Applied Biosystems)
Lyophilizer
UV spectrophotometer
Additional reagents and equipment for HPLC and MALDI-TOF-MS
Looking for materials? Suppliers Guide
Figures
-
Figure 4.11.1Structure of a peptide nucleic acid. -
Figure 4.11.2Automated PNA synthesis as described in2 O, acetic anhydride; base*, N-protected nucleobase (see PNA monomers); BHOC, benzhydryloxycarbonyl; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; FMOC, 9-fluorenylmethoxycarbonyl; HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; NMP, N-methylpyrrolidone; P, FMOC-XAL-PEG-polystyrene; TFA, trifluoroacetic acid. -
Figure 4.11.3Manual PNA synthesis as described in -
Figure 4.11.4Apparatus for manual synthesis. Reprinted from Norton et al. ( -
Figure 4.11.5Example of a spreadsheet for the manual synthesis of a 14-base PNA including lysine at the C terminus. The resin used produces a theoretical yield of 24.6 mg of PNA upon cleavage. -
Figure 4.11.6Typical (A) HPLC and (B) MALDI-TOF mass spectrometry data.
Literature Cited
| Literature Cited | |
| Braasch, D.A. and Corey, D.R. 2001. Synthesis, analysis, purification, and intracellular delivery of peptide nucleic acids. Methods 23:97-107. | |
| Doyle, D.F., Braasch, D.A., Simmons, C.G., Janowski, B.A., and Corey, D.R. 2001. Intracellular delivery and inhibition of gene expression by peptide nucleic acids. Biochemistry 40:53-64. | |
| Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S.M., Driver, D.A., Berg, R.H., Kim, S.K., Norden, B., and Nielsen, P.E. 1993. PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365:566-568. | |
| Goodwin, T.E., Holland, R.D., Lay, J.O., and Raney, K.D. 1998. A simple procedure for solid-phase synthesis of peptide nucleic acids with N-terminal cysteine. Bioorg. Med. Chem. Lett. 8:2231-2234. | |
| Hamilton, S.E., Iyer., M., Norton, J.C., and Corey, D.R. 1996. Specific and nonspecific inhibition of RNA synthesis by DNA, PNA and phosphorothioate promoter analog duplexes. Bioorg. Med. Chem. Lett. 6:2897-2900. | |
| Hamilton, S.E., Simmons, C.G., Kathriya, I., and Corey, D.R. 1999. Cellular delivery of peptide nucleic acids and inhibition of human telomerase. Chem. Biol. 6:343-351. | |
| Herbert, B.-S., Pitts, A.E., Baker, S.I., Hamilton, S.E., Wright, W.E., Shay, J.W., and Corey, D.R. 1999. Inhibition of telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc. Natl. Acad. Sci. U.S.A. 96:14726-14281. | |
| Lohse, J., Dahl, O., and Nielsen, P.E. 1999. Double-duplex invasion by peptide nucleic acid: A general principle for sequence-specific targeting of double-stranded DNA. Proc. Natl. Acad. Sci. U.S.A. 96:11804-11808. | |
| Mayfield, L.D. and Corey, D.R. 1999. Automated synthesis of peptide nucleic acids (PNAs) and peptide nucleic acid-peptide conjugates. Anal. Biochem. 268:401-404. | |
| Nielsen, P.E. 2001. Targeting double-stranded DNA with PNA. Curr. Med. Chem. 8:545-550. | |
| Nielsen, P.E., Egholm, M., Berg, R.H., and Buchardt, O. 1991. Sequence-selective recognition of double stranded DNA by a thymine-substituted polyamide. Science 254:1497-1500. | |
| Norton, J.C., Waggenspack, J.J., Varnum, E., and Corey, D.R. 1995. Targeting peptide nucleic acid protein conjugates to structural features within duplex DNA. Bioorg. Med. Chem. 3:437-445. | |
| Simmons, C.G., Pitts, A.E., Mayfield, L.D., Shay, J.W., and Corey, D.R. 1997. Synthesis and membrane permeability of PNA-peptide conjugates. Bioorg. Med. Chem. Lett. 7:3001-3007. | |
| Smulevitch, S.V., Simmons, C.G., Norton, J.C., Wise, T.W., and Corey, D.R. 1996. Enhanced strand invasion by oligonucleotides through manipulation of backbone charge. Nature Biotech. 14:1700-1704. | |
| Zhang, X., Ishihara, T., and Corey, D.R. 2000. Strand invasion by mixed base PNAs and PNA-peptide chimera. Nucl. Acids Res. 28:3332-3338. | |
| Internet Resources | |
| http://www.appliedbiosystems.com/ds/pna.taf | |
| Ordering information and bibliography. | |
| http://www.horizonpress.com/gateway/pna.html | |
| Links to PNA-related sites. | |
| http://www.isogen.nl/pna.html | |
| PNA synthesis provider in The Netherlands. | |
| http://www.bostonprobes.com | |
| Supplier of PNA diagnostic probes. | |
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