Synthesis of Oligonucleotide Conjugates via Aqueous Diels‐Alder Cycloaddition

Michael Leuck1, Andreas Wolter1

1 Proligo LLC, Boulder, Colorado
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.18
DOI:  10.1002/0471142700.nc0418s14
Online Posting Date:  November, 2003
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Abstract

The conjugation of maleimide reporter groups to 5′‐diene‐modified oligonucleotides via aqueous Diels‐Alder cycloaddition is described. Detailed basic protocols are provided for the preparation of a diene‐amidite (5′‐diene modifier), for the attachment of a diene group to the 5′‐terminus of oligonucleotides, and for the conjugation of maleimide reporter groups to diene‐modified oligonucleotides.

Keywords: diene‐amidite; 5′‐diene modifier; Diels‐Alder conjugation; 5′‐labeling of oligonucleotides

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of the Diene‐Amidite
  • Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligonucleotides
  • Basic Protocol 3: Labeling of A 5′‐Diene‐Modified Oligonucleotide with Tamra‐5‐Maleimide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of the Diene‐Amidite

  Materials
  • 3‐Cyclohexene‐1‐methanol (S.1; Aldrich)
  • Imidazole
  • Argon source
  • N,N‐Dimethylformamide (DMF)
  • tert‐Butyldimethylsilyl chloride (TBDMS·Cl; Aldrich)
  • Hexanes
  • Ethyl acetate (EtOAc)
  • TLC stain (see recipe)
  • Brine (saturated aqueous NaCl)
  • Magnesium sulfate (MgSO 4)
  • Bromine
  • Dichloromethane (CH 2Cl 2)
  • 10% (w/v) sodium thiosulfate (Na 2S 2O 3)
  • Aliquat 336 (tricaprylylmethylammonium chloride; Aldrich)
  • Tetrahydrofuran (THF)
  • Potassium tert‐butoxide (Aldrich)
  • Saturated ammonium chloride (NH 4Cl)
  • Dowex 50WX4‐50 strongly acidic ion‐exchange resin (Aldrich)
  • Methanol
  • 1,1′‐Carbonyldiimidazole (CDI; Aldrich)
  • 6‐Amino‐1‐hexanol (Aldrich)
  • N,N‐Diisopropylethylamine (DIPEA; Aldrich)
  • 2‐Cyanoethyl diisopropylchlorophosphoramidite (Aldrich)
  • Saturated sodium bicarbonate (NaHCO 3)
  • 100‐ and 250‐mL round‐bottom flasks with outlet stopcock adapters for gas line
  • TLC plates: 7.5 × 5–mm silica gel 60 F 254 precoated aluminum‐backed TLC sheets (EM Science)
  • Heat gun
  • 1‐L and 500‐mL separatory funnels
  • Rotary evaporator equipped with a vacuum pump and vacuum controller
  • Rubber septa
  • 10‐ and 50‐mL vials
  • 3‐ and 5‐mL syringes with 2‐in., 20‐G stainless steel needles
  • Shaker
  • Short‐path distillation apparatus (Aldrich)
  • Biotage Flash 40 chromatography system with Flash 40M silica cartridge
  • Additional reagents and equipment for TLC ( appendix 3D) and flash chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligonucleotides

  Materials
  • Dry acetonitrile (DNA synthesis grade; H 2O content <30 µg/mL)
  • Deblock solution: trichloroacetic acid (TCA) in dichloromethane (Proligo)
  • Cap A solution: acetic anhydride in tetrahydrofuran (Proligo)
  • Cap B solution: 1‐methylimidazole in tetrahydrofuran/pyridine (Proligo)
  • Oxidizer solution: iodine in tetrahydrofuran/water/pyridine (Proligo)
  • Activator: 0.25 M 4,5‐dicyanoimidazole (DCI) in acetonitrile (Proligo)
  • 50 mg/mL standard phosphoramidites [dT, dC(bz), dA(bz), and dG(ib); Proligo] in dry acetonitrile
  • 50 mg/mL diene‐amidite (S.7; see protocol 1) in dry acetonitrile
  • Helium source
  • Concentrated ammonium hydroxide (NH 4OH)
  • 3 M sodium acetate ( appendix 2A)
  • Isopropanol
  • 70% (v/v) ethanol
  • Automated DNA synthesizer (e.g., Expedite 8909, PerSeptive Biosystems)
  • Synthesis column for 1‐µmol scale (Proligo): controlled‐pore glass (CPG; 500 Å for <50‐mers, 1000 Å for ≥50‐mers)
  • Heating block
  • Speedvac evaporator
  • Additional reagents and equipment for quantitating oligonucleotides by OD 260 measurement (unit 10.3)

Basic Protocol 3: Labeling of A 5′‐Diene‐Modified Oligonucleotide with Tamra‐5‐Maleimide

  Materials
  • Diene‐modified oligonucleotide (see protocol 2)
  • 100 mM sodium acetate buffer, pH 4.5 ( appendix 2A)
  • 40 mM TAMRA‐5‐maleimide solution: 5.0 mg TAMRA‐5‐maleimide (Molecular Probes) in 268 µL dimethylformamide (DMF)
  • Heating block
  • NAP‐5 columns (Amersham Pharmacia Biotech)
  • Speedvac evaporator
  • Additional reagents and equipment for quantitating oligonucleotides by OD 260 measurement (unit 10.3)
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Figures

Videos

Literature Cited

Literature Cited
   Bowtell, D. and Sambrook, J. (eds.) 2002. DNA Microarrays: A Molecular Cloning Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Goodchild, J. 1990. Conjugates of oligonucleotides and modified oligonucleotides: A review of their synthesis and properties. Bioconjugate Chem. 1: 165‐187.
   Hill, K.W., Taunton‐Rigby, J., Carter, J.D., Kropp, E., Vagle, K., Pieken, W., McGee, D.P.C., Husar, G., Leuck, M., Anziano, D., and Sebesta, D.P. 2001. Diels‐Alder bioconjugation of diene‐modified oligonucleotides. J. Org. Chem. 66: 5352‐5358.
   Letsinger, R.L., Elghanian, R., Biswanadham, G., and Mirkin, C. 2000. Use of a steroid cyclic disulfide anchor in constructing gold nanoparticle‐oligonucleotide conjugates. Bioconjugate Chem. 11: 289‐291.
   Smith, L.M., Fung, S., Hunkapiller, M.W., Hunkapiller, T.J., and Hoo, L.E. 1985. The synthesis of oligonucleotides containing an aliphatic amino group at the 5′ terminus: Synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucl. Acids Res. 13: 2399‐2412.
   Telser, J., Cruickshank, K.A., Morrison, L.E., and Netzel, T.L. 1989. Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: Comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. Soc. 111: 6966‐6976.
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