Synthesis and Purification of Peptide Nucleic Acids

Dwaine A. Braasch1, Christopher J. Nulf1, David R. Corey1

1 University of Texas Southwestern Medical Center at Dallas, Dallas, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.11
DOI:  10.1002/0471142700.nc0411s09
Online Posting Date:  August, 2002
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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.

     
 
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Table of Contents

  • 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
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Automated Synthesis of Peptide Nucleic Acids

  Materials
  • PNA Expedite reagents (Applied Biosystems)
  •  FMOC‐PNA monomers (Fig. ): 9‐fluorenylmethoxycarbonyl‐protected peptide nucleic acid monomers (A, T, C, and G), base protected with benzhydryloxycarbonyl (BHOC)
  •  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 protocol 6)
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 1: Adding Peptides to PNAs

  • Biotin (Sigma)
  • 42°C water bath
  • 1‐mL syringe

Support Protocol 2: Addition of Biotin

  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 protocol 5)
  • BOC‐PNA monomers (Fig. ; Applied Biosystems):
  • 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 protocol 6)
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.

Basic Protocol 2: Manual Synthesis of Peptide Nucleic Acids

  • 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

Support Protocol 3: Preparation of Carrier Resin for Manual PNA Synthesis

  Materials
  • PNA sample solution (see protocol 1Basic Protocol 1 and protocol 42)
  • 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)
  • α‐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
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Figures

Videos

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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