Reversible Biotinylation of the 5′‐Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification

Shiyue Fang1, Donald E. Bergstrom1

1 Walther Cancer Institute, Indianapolis, Indiana
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.20
DOI:  10.1002/0471142700.nc0420s14
Online Posting Date:  November, 2003
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Abstract

The preparation of two reversible biotinylation phosphoramidites and their application in labeling and affinity purification of synthetic oligodeoxyribonucleotides will be described. In both cases, the biotin is linked to the 5′‐terminus of DNA through a diisopropyl silyl acetal functionality. This linkage is completely stable under certain postsynthetic cleavage/deprotection conditions, but can be readily broken by fluoride ions, releasing unmodified 5′‐OH and 5′‐phosphate DNA, respectively. To demonstrate the use of these reversible biotinylation methods, crude DNA was incubated with NeutrAvidin‐coated microspheres, full‐length biotinylated DNA was efficiently attached to the solid phase, and nonbiotinylated failure sequences and other impurities were readily removed by washing with buffer. Cleavage of the silyl acetal linkage afforded high‐quality, full‐length, unmodified 5′‐OH and 5′‐phosphate DNA, respectively, depending on which of the two phosphoramidites was used. It is anticipated that this method will find applications in areas that require efficient isolation of DNA from a complex mixture.

Keywords: DNA; Phosphoramidite; Biotinylation; Fluoride Cleavage; Silyl Acetal; Avidin; Affinity Purification

     
 
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Table of Contents

  • Basic Protocol 1: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐OH DNA
  • Basic Protocol 2: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐Phosphate DNA
  • Support Protocol 1: Application of the Reversible Biotinylation Method in Affinity Purification Using Avidin‐Coated Microspheres
  • Support Protocol 2: Synthesis of Biotinyl Alcohol
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐OH DNA

  Materials
  • Biotinyl alcohol S.1 (see protocol 4 for preparation)
  • Imidazole (99%)
  • Argon and nitrogen gas
  • N,N‐Dimethylformamide (DMF, anhydrous)
  • Diisopropylethylamine (DIEA)
  • Diisopropyldichlorosilane (Fluka)
  • Thymidine (99%+)
  • Ethyl acetate
  • 5% (w/v) sodium bicarbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • Chloroform (CHCl 3)
  • Methanol
  • 40‐µm silica gel (Baker)
  • TLC plates: silica gel on aluminum (60F‐254, 200‐µm thickness)
  • Methylene chloride (CH 2Cl 2, anhydrous)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphoramidite (2‐cyanoethyl‐ tetraisopropylphosphorodiamidite, 97%)
  • 1H‐Tetrazole (99%+, sublimed)
  • Tetrahydrofuran (THF, 99%+)
  • 5′‐DMTr, 2‐cyanoethyl phosphoramidite monomers:
    • 5′‐O‐dimethoxytrityl‐N6‐benzoyl‐2′‐deoxyadenosine‐3′‐(2‐cyanoethyl‐N,N‐di‐isopropyl)phosphoramidite (dABz)
    • 5′‐O‐dimethoxytrityl‐N2‐isobutyryl‐2′‐deoxyguanosine‐3′‐(2‐cyanoethyl‐N,N‐di‐isopropyl)phosphoramidite (dGi‐Bu)
    • 5′‐O‐dimethoxytrityl‐N4‐acetyl‐2′‐deoxycytidine‐3′‐(2‐cyanoethyl‐N,N‐diisopro‐pyl)phosphoramidite (dCAc)
    • 5′‐O‐dimethoxytrityl‐2′‐deoxythymidine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl)phos‐phoramidite (T)
  • Acetonitrile (CH 3CN, anhydrous)
  • ∼29% (v/v) ammonium hydroxide (NH 4OH)
  • ∼40% (v/v) methylamine (CH 3NH 2)
  • 50‐mL one‐neck round‐bottomed flasks (oven dried)
  • Vacuum pump
  • 10‐, 5‐, 1‐, 0.25‐mL graduated glass syringes
  • 20‐G, 5‐cm stainless steel needles
  • Septa (14/20, 24/40)
  • 250‐mL separatory funnel
  • Filter funnel and Whatman no. 1 filter paper
  • Rotary evaporator equipped with a water aspirator
  • Flash chromatography columns (4.5 × 12–cm, 3.0 × 12–cm)
  • UV lamp
  • 5‐mL screw‐cap vials
  • Speedvac evaporator
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D), column chromatography ( appendix 3E), solid‐phase automatic DNA synthesis ( appendix 3C) using classical phosphoramidite method (unit 3.3), and purification of oligonucleotides by SDS‐PAGE (unit 10.4) and reversed‐phase HPLC (unit 10.5)

Basic Protocol 2: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐Phosphate DNA

  Materials
  • Biotinyl alcohol S.1 (see protocol 4 for preparation)
  • Imidazole (99%)
  • Argon and nitrogen gas
  • N,N‐Dimethylformamide (DMF, anhydrous)
  • Diisopropylethylamine (DIEA)
  • Diisopropyldichlorosilane (Fluka)
  • Diethyl bis(hydroxymethyl)malonate (97%)
  • Methylene chloride (CH 2Cl 2)
  • 5% (v/v) citric acid
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • Chloroform (CHCl 3)
  • Methanol
  • 40‐µm silica gel (Baker)
  • TLC plates: silica gel on aluminum (60F‐254, 200‐µm thickness)
  • Acetonitrile (distilled over CaH 2)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphoramidite (2‐cyanoethyl‐ tetraisopropylphosphorodiamidite, 97%)
  • 0.45 M 1H‐tetrazole (99%+, sublimed) in acetonitrile
  • 5% (w/v) sodium bicarbonate (NaHCO 3)
  • Tetrahydrofuran (THF, 99%+)
  • Triethylamine (TEA)
  • 50‐mL one‐neck, round‐bottomed flasks (oven dried)
  • Vacuum pump
  • 20‐G, 5‐cm stainless steel needles
  • 5‐, 1‐, and 0.5‐mL graduated glass syringes
  • Septa (14/20, 24/40)
  • 250‐mL separatory funnels
  • Filter funnel and Whatman no. 1 filter paper
  • Rotary evaporator equipped with a water aspirator
  • Flash chromatography columns (3.0 × 12–cm, 3.0 × 10–cm)
  • UV lamp
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D), column chromatography ( appendix 3E), and incorporation of biotinylated phosphoramidite into oligonucleotide (see protocol 1)

Support Protocol 1: Application of the Reversible Biotinylation Method in Affinity Purification Using Avidin‐Coated Microspheres

  Materials
  • UltraLink Immobilized NeutrAvidin (1000‐Å pore size; 50‐ to 80‐µm particle size; 0.08 µmol biotin/ml gel binding capacity; Pierce), prepared as 50% (v/v) slurry containing 0.02% (w/v) sodium azide
  • PBS ( appendix 2A)
  • Biotinylated DNA (see protocol 1 for S.4 or protocol 2 for S.8)
  • Acetone (dried over anhydrous sodium sulfate)
  • Tetrahydrofuran (THF, distilled over sodium/benzophenone ketyl)
  • 70:30 (v/v) hydrogen fluoride in pyridine (HF/pyridine)
  • Methoxytrimethylsilane
  • ∼40% (v/v) methylamine (CH 3NH 2; for 5′‐phosphate DNA S.8 only)
  • 10‐ and 1.5‐mL centrifuge tubes
  • Lyophilizer or Speedvac evaporator

Support Protocol 2: Synthesis of Biotinyl Alcohol

  • Levulinic acid (98%)
  • Dry ice/acetone
  • 3.0 M methyl magnesium bromide in ether
  • Acetic acid (99.7%+)
  • Magnesium sulfate (MgSO 4, anhydrous)
  • 2,2′‐(Ethylenedioxy)bis(ethylamine) (98%)
  • Ether
  • Triethylamine
  • Ninhydrin
  • Ethanol (anhydrous)
  • Biotin (AnaSpec)
  • 4‐(Dimethylamino)pyridine (DMAP)
  • Pyridine (distilled from CaH 2)
  • t‐Butylchlorodiphenylsilane (98%)
  • 4‐t‐Butylbenzoylchloride (98%)
  • Potassium carbonate (K 2CO 3)
  • 5% citric acid
  • Isobutylchloroformate (98%)
  • 100‐, 20‐, 10‐, 5‐, and 1‐mL graduated glass syringes
  • 1000‐mL two‐neck round‐bottomed flask, oven dried
  • Condenser
  • 500‐ and 250‐mL separatory funnels
  • Aldrich short‐path distillation apparatus
  • 50‐, 100‐ and 250‐mL one‐neck round‐bottomed flasks, oven dried
  • Flash chromatography columns (4.5 × 30–cm and 4.5 × 10–cm)
  • Heat gun
  • Rubber septum
  • 2‐ and 6‐cm stainless steel needles
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Figures

Videos

Literature Cited

Literature Cited
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   Cadet, J. and Vigny, P. 1990. The photochemistry of nucleic acids. In Bioorganic Photochemistry (H. Morrison, ed.) Vol. 1, pp. 170‐184. John Wiley & Sons, New York.
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   De Vos, M.J., Van Elsen, A., and Bollen, A. 1994. New non‐nucleosidic phosphoramidites for the solid phase multi‐labeling of oligonucleotides: Comb‐ and multifork‐like structure. Nucleosides Nucleotides 13:2245‐2265.
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   Fang, S. and Bergstrom, D.E. 2003b. Fluoride‐cleavable biotinylation phosphoramidite for 5′‐end‐labeling, affinity purification of synthetic oligonucleotides. Nucl. Acids Res. 31:708‐715.
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