Purification of Synthetic Oligonucleotides via Catching by Polymerization

Shiyue Fang1, Suntara Fueangfung1, Yinan Yuan2

1 Department of Chemistry, Michigan Technological University, Houghton, Michigan, 2 School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.14
DOI:  10.1002/0471142700.nc1014s49
Online Posting Date:  June, 2012
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Abstract

This unit describes the purification of synthetic oligodeoxyribonucleotides (ODN) using a catching‐by‐polymerization approach. In a crude ODN, the major impurity is the failure sequences generated in the coupling step of each synthetic cycle. They are difficult to remove due to the similarity of their physical properties to the full‐length sequences. Two non‐chromatographic methods are described in the unit to solve the problem. In the first one, during automated synthesis, the failure sequences are tagged with a methacrylamide group, which is polymerizable and can participate in acrylamide radical polymerization reactions; the full‐length sequences are not tagged. After synthesis, the crude mixture is subjected to polymerization. The failure sequences are incorporated into an insoluble polymer; the full‐length sequences are extracted with water. In the second method, the full‐length sequences are tagged with a methacrylamide group via a cleavable linker; the failure sequences are not tagged. After synthesis, the full‐length sequences are incorporated into a polymer; the failure sequences are washed away with water. Pure full‐length sequences are cleaved from the polymer. The two methods are complementary. Curr. Protoc. Nucleic Acid Chem. 49:10.14.1‐10.14.21. © 2012 by John Wiley & Sons, Inc.

Keywords: oligonucleotide; purification; catching by polymerization; scalable purification; acrylamide radical polymerization; full‐length sequence; failure sequence

     
 
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Table of Contents

  • Basic Protocol 1: Purification of ODN via Catching Failure Sequences by Polymerization
  • Basic Protocol 2: Purification of ODN via Catching Full‐Length Sequences by Polymerization
  • Support Protocol 1: Synthesis of ODN with Failure Sequences Being Tagged with a Polymerizable Group
  • Support Protocol 2: Synthesis of ODN with Full‐Length Sequences Being Tagged with a Polymerizable Group
  • Support Protocol 3: Synthesis of Polymerizable Phosphoramidite for Tagging Failure Sequences
  • Support Protocol 4: Synthesis of Polymerizable Phosphoramidite for Tagging Full‐Length Sequences
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Purification of ODN via Catching Failure Sequences by Polymerization

  Materials
  • Nitrogen gas (tank with regulator)
  • Sample ODN ( S.1) with failure sequences tagged with a methacrylamide polymerizable group (see protocol 3 for automated synthesis)
  • Polymerization solution [N,N‐dimethylacrylamide/N,N′‐methylenebis(acrylamide); see recipe]
  • 10% ammonium persulfate [(NH 4) 2S 2O 8] solution (see recipe)
  • N,N,N′,N′‐tetramethylethylenediamine (TMEDA, ≥99.5%, Aldrich)
  • Concentrated ammonium hydroxide (NH 4OH) solution (∼28%, ACS reagent grade, Aldrich)
  • n‐Butanol (n‐BuOH; HPLC grade, Aldrich)
  • T‐shaped glass tube with oil bubbler (or Schlenk inert gas/vacuum line)
  • 25‐mL 2‐necked round‐bottom flask
  • Magnetic stirring bar and stirring plate
  • Adaptor and septa (to fit the neck of the 25‐mL 2‐necked round‐bottom flask)
  • Spatula
  • SpeedVac vacuum concentrator with high‐vacuum oil pump
  • Heating block, sand bath, or oil bath
  • Shaker (optional)
  • Additional reagents and equipment for analytical reversed‐phase HPLC (unit 10.5, optional)

Basic Protocol 2: Purification of ODN via Catching Full‐Length Sequences by Polymerization

  Materials
  • Nitrogen gas (tank with regulator)
  • Sample ODN ( S.4) with full‐length sequences tagged with a methacrylamide polymerizable group via a cleavable silylacetal linker (see protocol 4 for automated synthesis)
  • Polymerization solution [N,N‐dimethylacrylamide/N,N′‐methylenebis(acrylamide); see recipe]
  • 10% ammonium persulfate [(NH 4) 2S 2O 8] solution (see recipe)
  • N,N,N′,N′‐tetramethylethylenediamine (TMEDA, ≥99.5%, Aldrich)
  • Dimethylformamide (DMF; anhydrous, 99.8%, Aldrich)
  • Hydrogen fluoride–pyridine (HF‐pyridine; HF ∼70%, pyridine ∼30%; Aldrich)
  • Methoxytrimethylsilane (Me 3SiOMe, 99%, Aldrich)
  • T‐shaped glass tube with oil bubbler or a Schlenk inert gas/vacuum line
  • 25‐mL 2‐necked round‐bottom flask
  • Magnetic stirring bar and stirring plate
  • Adaptor and septa (to fit to the neck of the 25‐mL 2‐necked round‐bottom flask)
  • Spatula
  • SpeedVac vacuum concentrator and high‐vacuum oil pump
  • Shaker (optional)
  • Additional reagents and equipment for analytical reversed‐phase HPLC (unit 10.5, optional)

Support Protocol 1: Synthesis of ODN with Failure Sequences Being Tagged with a Polymerizable Group

  Materials
  • 0.2 M polymerizable phosphoramidite S.8 capping solution (from protocol 5)
  • UltraMild phosphoramidites:
    • Pac‐dA‐CE phosphoramidite (Glen Research, Inc.)
    • Ac‐dC‐CE phosphoramidite (Glen Research, Inc.)
    • iPr‐Pac‐dG‐CE phosphoramidite (Glen Research, Inc.)
  • Concentrated ammonium hydroxide (NH 4OH) solution (∼28%, ACS reagent grade, Aldrich)
  • ABI 394 DNA synthesizer (or other synthesizer; see appendix 3C)
  • SpeedVac vacuum concentrator and high‐vacuum oil pump
  • Additional reagents for automated solid‐phase DNA synthesis ( appendix 3C)

Support Protocol 2: Synthesis of ODN with Full‐Length Sequences Being Tagged with a Polymerizable Group

  Materials
  • 0.1 M polymerizable phosphoramidite S.9 tagging solution (from protocol 6)
  • UltraMild phosphoramidites:
    • Pac‐dA‐CE phosphoramidite (Glen Research Inc.)
    • Ac‐dC‐CE phosphoramidite (Glen Research Inc.)
    • iPr‐Pac‐dG‐CE phosphoramidite (Glen Research Inc.)
  • UltraMild capping reagents:
    • Cap mix A, THF/pyridine/Pac 2O (Glen Research Inc.)
    • Cap mix B, 16% Melm in THF (Glen Research Inc.)
  • Concentrated ammonium hydroxide (NH 4OH) solution (∼28%, ACS reagent grade, Aldrich)
  • ABI 394 DNA synthesizer (or other synthesizer; see appendix 3C)
  • SpeedVac vacuum concentrator and high‐vacuum oil pump
  • Additional reagents for automated solid‐phase DNA synthesis ( appendix 3C)

Support Protocol 3: Synthesis of Polymerizable Phosphoramidite for Tagging Failure Sequences

  Materials
  • 6‐amino‐1‐hexanol ( S.10, 97%, Aldrich)
  • Saturated sodium carbonate (Na 2CO 3) solution
  • Methylene chloride (CH 2Cl 2)
  • Nitrogen gas (tank with regulator)
  • Methacryloyl chloride ( S.11, 97%, contains 200 ppm monomethyl ether hydroquinone as inhibitor, Aldrich)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Hexanes (mixture of isomers is OK)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphoramidite (97%, Aldrich)
  • 1H‐tetrazole acetonitrile solution (0.45 M, Aldrich)
  • Ethyl acetate (EtOAc)
  • Triethylamine (Et 3N, 99.5%, Aldrich)
  • Dry acetonitrile (freshly distilled over calcium hydride)
  • Drierite
  • 1‐ And 2‐necked round‐bottom flasks
  • Magnetic stirring bars and stirring plates
  • 100‐mL pressure‐equalizing addition funnel
  • Nitrogen gas line with oil bubbler (or a standard Schlenk inert gas/vacuum line)
  • Syringes
  • 100‐mL graduated cylinder
  • 500‐mL separatory funnel
  • Erlenmeyer flasks
  • Filter paper and funnel
  • High‐vacuum oil pump and vacuum desiccator
  • Septa
  • Rotary evaporator equipped with a water aspirator
  • 5 × 15–cm silica gel flash chromatography column ( appendix 3E)
  • Used phosphoramidite bottle that can fit to the phosphoramidite position on a DNA synthesizer
  • Additional regents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 4: Synthesis of Polymerizable Phosphoramidite for Tagging Full‐Length Sequences

  Materials
  • Nitrogen gas (tank with regulator)
  • Succinic anhydride (97%, Aldrich)
  • THF (freshly distilled from sodium benzophenone ketyl)
  • Ethyl magnesium bromide solution in diethyl ether (3.0 M, Aldrich)
  • Acetic acid (glacial, 99.8%, Aldrich)
  • Methylene chloride (CH 2Cl 2)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • 1,12‐diaminododecane (98%, Aldrich)
  • Diethyl ether (Et 2O, Aldrich)
  • Methanol (CH 3OH; anhydrous, 99.8%, Aldrich)
  • Acetonitrile (CH 3CN; freshly distilled from calcium hydride)
  • Triethylamine (Et 3N, 99.5%, Aldrich)
  • Diisopropylethylamine (99%, Aldrich)
  • Methacryloyl chloride ( S.11, 97%, contains 200 ppm monomethyl ether hydroquinone as inhibitor, Aldrich)
  • N,N‐Dimethylformamide (DMF, anhydrous, 99.8%, Aldrich)
  • Diisopropylsilyl(trifluoromethanesulfonate; Gelest, http://www.gelest.com/)
  • Thymidine (≥99%, Aldrich)
  • Ethyl acetate (EtOAc)
  • Sodium bicarbonate solution (NaHCO 3, 5%)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphoramidite (97%, Aldrich)
  • 1H‐tetrazole in acetonitrile (0.45 M, Aldrich)
  • Triethyl amine (Et 3N, 99.5%, Aldrich)
  • Hexanes (mixture of isomers is OK)
  • Dry acetonitrile (freshly distilled over calcium hydride)
  • Drierite
  • Schlenk inert gas/vacuum line
  • 1‐ And 2‐necked round‐bottom flasks
  • Magnetic stirring bars and plates
  • Condenser
  • Septa
  • Syringes
  • Rotary evaporator equipped with a water aspirator
  • High‐vacuum oil pump
  • Oil heating bath
  • Separatory funnels
  • 5 × 20–cm and 2.5 × 12–cm silica‐gel‐packed flash chromatography columns ( appendix 3E)
  • Shaker
  • Filtration funnel and paper
  • Erlenmeyer flasks
  • Cannula (optional)
  • Used phosphoramidite bottle that can fit to the phosphoramidite position on a DNA synthesizer
  • Vacuum desiccator
  • Additional regents and equipment for TLC ( appendix 3D), column chromatography ( appendix 3E), and DNA synthesis ( appendix 3C)
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Figures

Videos

Literature Cited

Literature Cited
   Beller, C. and Bannwarth, W. 2005. Noncovalent attachment of nucleotides by fluorous fluorous interactions: Application to a simple purification principle for synthetic DNA fragments. Helv. Chim. Acta 88:171‐179.
   Fang, S. and Bergstrom, D.E. 2003a. Fluoride‐cleavable biotinylation phosphoramidite for 5′‐end‐labeling and affinity purification of synthetic oligonucleotides. Nucleic Acids Res. 31:708‐715.
   Fang, S. and Bergstrom, D.E. 2003b. Reversible biotinylation phosphoramidite for 5′‐end‐labeling, phosphorylation, and affinity purification of synthetic oligonucleotides. Bioconjugate Chem. 14:80‐85.
   Fang, S. and Fueangfung, S. 2010. Scalable synthetic oligodeoxynucleotide purification with use of a catching by polymerization, washing, and releasing approach. Org. Lett. 12:3720‐3723.
   Fang, S., Fueangfung, S., Lin, X., Zhang, X., Mai, W., Bi, L., and Green, S.A. 2011. Synthetic oligodeoxynucleotide purification by polymerization of failure sequences. Chem. Commun. 47:1345‐1347.
   Gupta, A.P. and Will, S.G. 2008. Compounds and methods for the synthesis and purification of oligodeoxyribonucleotides. PCT Int. Appl. WO 2008077600, A1.
   Kitano, H., Nakada, H., and Mizukami, K. 2008. Interaction of wheat germ agglutinin with an n‐acetylglucosamine‐carrying telomer brush accumulated on a colloidal gold monolayer. Colloids Surf. B Biointerfaces 61:17‐24.
   Natt, F. and Haner, R. 1997. Lipocap: A lipophilic phosphoramidite‐based capping reagent. Tetrahedron 53:9629‐9636.
   Pearson, W.H., Berry, D.A., Stoy, P., Jung, K.Y., and Sercel, A.D. 2005. Fluorous affinity purification of oligonucleotides. J. Org. Chem. 70:7114‐7122.
   Sawadogo, M. and Vandyke, M.W. 1991. A rapid method for the purification of deprotected oligodeoxynucleotides. Nucleic Acids Res. 19:674.
   Sproat, B.S., Rupp, T., Menhardt, N., Keane, D., and Beijer, B. 1999. Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag. Nucleic Acids Res. 27:1950‐1955.
   Zhu, Z., Wu, C.C., Liu, H.P., Zou, Y., Zhang, X.L., Kang, H.Z., Yang, C.J., and Tan, W.H. 2010. An aptamer cross‐linked hydrogel as a colorimetric platform for visual detection. Angew. Chem. Int. Ed. 49:1052‐1056.
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