Conjugation of 5′‐Functionalized Oligodeoxyribonucleotides with Properly Functionalized Ligands

Ulysse Asseline1, Nguyen T. Thuong1

1 Centre de Biophysique Moleculaire, CNRS Orléans, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.10
DOI:  10.1002/0471142700.nc0410s07
Online Posting Date:  February, 2002
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Abstract

This unit reports the synthesis of oligodeoxyribonucleotides covalently linked via their 5′ termini to various ligands such as intercalating agents, reactive groups, or labels. Methods for incorporation of halogenoalkyl, isothiocyanate, and 2‐pyridyldisulfide linkers onto ligands, and addition of amino, carboxyl, thiophosphate, phosphate, and masked thiol groups at the 5 terminus of an oligodeoxyribonucleotide are described elsewhere in the series. This unit reports procedures for coupling the ligands and oligonucleotides, as well as details for purification and characterization of the final products.

     
 
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Table of Contents

  • Basic Protocol 1: Conjugation of 5′‐Carboxylated Oligodeoxyribonucleotides to Aminoalkylated Ligands
  • Basic Protocol 2: Conjugation of 5′‐Aminoalkylated Oligodeoxyribonucleotides to Ligands Functionalized with an Isothiocyanate or N‐Hydroxysuccinimidyl Group
  • Basic Protocol 3: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphorothioates to Ligands Functionalized with Halogenoalkyl, 2‐Pyridyldithio, or Iodoacetamidyl Groups
  • Basic Protocol 4: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphates to Ligands Functionalized with Amino Groups
  • Basic Protocol 5: Conjugation of 5′‐Mercaptoalkylated Oligodeoxyribonucleotides to Ligands Functionalized with Halogenoalkyl, Iodoacetamidyl, or 2‐Pyridyldithio Groups
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Conjugation of 5′‐Carboxylated Oligodeoxyribonucleotides to Aminoalkylated Ligands

  Materials
  • Purified 5′‐carboxylated oligodeoxyribonucleotide (unit 4.9)
  • 0.5 M pyridine buffer, pH 5.5 to 6 (use pyridine redistilled from p‐toluenesulfonyl chloride and stored over 3A molecular sieves; adjust pH with HCl)
  • 10 to 20 mg/mL aminoalkylated ligand (unit 4.8; e.g., Lucifer Yellow hydrazine derivative) dissolved in water, dimethylsulfoxide (DMSO), or N,N‐dimethylformamide (DMF; redistilled in vacuo over ninhydrin)
  • 1‐[3‐(Dimethylamino)propyl]‐3‐ethylcarbodiimide hydrochloride (EDC)
  • 80:20 or 50:50 (v/v) dichloromethane (CH 2Cl 2)/methanol
  • 55:10:20 or 55:10:30 (v/v/v) isopropanol/NH 4OH/H 2O
  • Sephadex G‐10, G‐15, or G‐25 resin (Pharmacia; for H 2O‐soluble ligands)
  • 0.5 M triethylammonium acetate (TEAA), pH 7
  • Dichloromethane or ethyl acetate (for very lipophilic ligands)
  • 4% (w/v) LiClO 4 in distilled acetone, or n‐butanol (for ligands of medium lipophilicity)
  • Mobile phase A: 5% (v/v) acetonitrile in 0.1 M aqueous TEAA, pH 7, all HPLC grade
  • Mobile phase B: 80% (v/v) acetonitrile in 0.1 M aqueous TEAA, pH 7, all HPLC grade
  • 2‐mL vial equipped with Teflon‐faced septum
  • Analytical silica gel TLC plates (e.g., 5554 Kieselgel 60F plates; Merck)
  • 5‐mL column
  • UV lamp and viewing box
  • Rotary evaporator with water bath and vacuum pump
  • High‐performance liquid chromatography (HPLC) system, including:
  •  Chemically inert syringes with replaceable needles
  •  Reversed‐phase column: 125‐mm × 4‐mm, 5‐µm Lichrospher 100 RP18 (Merck), CC Nucleosil 100‐5 C18 (125/4; Macherey‐Nagel), or polystyrene  reversed‐phase (PRP‐1; Hamilton)
  •  Multiwavelength detector capable of measuring UV‐Vis absorption between 230 and 600 nm
  • Spectrophotometer
  • Lyophilizer
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and purification and characterization of oligonucleotide‐acridine conjugates (unit 4.3)
NOTE: All buffered solutions used for HPLC purifications should be filtered through a 0.22‐ or 0.45‐µm disposable filter.

Basic Protocol 2: Conjugation of 5′‐Aminoalkylated Oligodeoxyribonucleotides to Ligands Functionalized with an Isothiocyanate or N‐Hydroxysuccinimidyl Group

  Materials
  • Purified 5′‐aminoalkylated oligodeoxyribonucleotide (unit 4.9)
  • 0.5 M sodium bicarbonate (NaHCO 3) buffer, pH 9.5
  • 5 to 10 mg/mL functionalized ligand (select one) in N,N‐dimethylformamide (DMF):
  •  Orthophenanthroline (OP) isothiocyanate (unit 4.8, structure 3d)
  • N‐Succinimidyl ester of 1‐pyrene butanoic acid (Molecular Probes)
  •  Oregon Green (OR) 5‐ and 6‐isothiocyanate (mixed isomers; Molecular Probes)
  • Ninhydrin
  • 25 mM Tris⋅Cl, pH 7 ( appendix 2A), containing 10% (v/v) methanol:
  •  without NaCl and
  •  with 1 M NaCl (for ion‐exchange with a Mono Q column) or
  •  with 1.5 M NaCl (for ion‐exchange with a DEAE column)
  • 2‐mL vial equipped with Teflon‐faced septum
  • Mono Q HR 5/5 or HR 10/10 (Pharmacia; for OP isothiocyanate ion‐exchange HPLC)
  • 100‐mm × 10‐mm, 8‐µm DEAE (Waters; for OP isothiocyante ion‐exchange HPLC)
  • HR 10/10 column packed with Lichroprep PR 18 (Art 13900, Merck) or Sephadex G‐10 or G‐25 (for desalting)
  • Additional reagents and equipment for TLC (see protocol 1 and appendix 3D) and for isolating, purifying, and characterizing the final conjugate (see protocol 1)

Basic Protocol 3: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphorothioates to Ligands Functionalized with Halogenoalkyl, 2‐Pyridyldithio, or Iodoacetamidyl Groups

  Materials
  • Purified lyophilized oligodeoxyribonucleotide 5′‐phosphorothioates (Na+ or K+ salt; unit 4.9)
  • 12.5 mg/mL 15‐crown‐5 or 18‐crown‐6 (Aldrich) in methanol
  • Functionalized ligand (select one):
  •  Halogenoalkylated acridine, psoralen, orthophenanthroline, or thiazole orange (unit 4.8, structures 1e, 2f, 3c, and 4e, respectively)
  •  2‐Pyridyldisulfide psoralen (unit 4.8, structure 2h)
  •  Iodoacetamidylated fluorescein (Aldrich)
  • Methanol (for halogenoalkylated ligands and iodoacetamidylated fluorescein)
  • Dichloromethane
  • 0.5 M sodium or potassium phosphate buffer, pH 7 ( appendix 2A)
  • Sephadex G‐10 or G‐15 column
  • 0.1 M triethylammonium acetate (TEAA) buffer, pH 7
  • 2‐mL vial equipped with a Teflon‐faced septum
  • 30° to 35°C water bath (optional; for halogenoalkylated ligands)
  • UV‐Vis spectrophotometer
  • Additional reagents and equipment for TLC (see protocol 1 and appendix 3D) and for isolating, purifying, and characterizing the final conjugate (see protocol 1)
NOTE: In order to prevent chelation of phosphorothioates and phenanthrolines attached to oligodeoxyribonucleotides, all solvents and buffer solutions used for their purification must be passed through a column of Chelex 100 resin to remove divalent cations.

Basic Protocol 4: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphates to Ligands Functionalized with Amino Groups

  Materials
  • Purified oligodeoxyribonucleotide 5′‐phosphate (unit 4.9)
  • 0.5 M aqueous 1,5‐diaminopentane, pH 4.5
  • 1‐[3‐(Dimethylamino)propyl]‐3‐ethylcarbodiimide hydrochloride (EDC)
  • 4% (w/v) LiClO 4 in acetone
  • Mobile phase A: 0.1 M triethylammonium acetate buffer pH 7, containing 5% CH 3CN
  • Mobile phase B: 0.1 M triethylammonium acetate (TEAA) buffer, pH 7 containing 80% CH 3CN
  • 2‐mL vial equipped with a Teflon‐faced septum
  • HPLC system (also see protocol 1)
  •  Column: 150‐mm × 3‐mm, 5‐µm RP8 glass cartridge system (Merck)
  • Additional reagents and equipment for TLC (see protocol 1 and appendix 3D) and for isolating, purifying, and characterizing the final conjugate (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   Asseline, U., Bonfils, E., Dupret, D., and Thuong, N.T. 1996. Synthesis and binding properties of oligonucleotides covalently linked to an acridine derivative: New study of the influence of the dye attachment site. Bioconjugate Chem. 7:369‐379.
  The authors would like to express their appreciation to past and present collaborators for their contribution to the development of varied families of oligonucleotide conjugates over the past years. This work was supported by Rhône‐Poulenc, Agence Nationale de Recherche contre le SIDA, and bioMérieux.
   Aubert, Y., Bourgerie, S., Meunier, L., Mayer, R., Roche, A.‐C., Monsigny, M., Thuong, N.T., and Asseline, U. 2000. Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5′‐protected thiol function and a 3′‐amino group. Nucl. Acids Res. 28:818‐825.
   Aubert, Y., Perrouault, L., Hélene, C., Giovannangeli, C., and Asseline, U. 2001. Synthesis and properties of triple helix‐forming oligodeoxyribonucleotides containing 7‐chloro‐7‐deaza‐2′‐deoxyguanosine. Bioorg. Med. Chem. 9:1617‐1624.
   Ivanovskaya, M.G., Gottihk, M.B., and Shabarova, Z.A. 1987. Modification of Oligo(poly)nucleotide Phosphomonoester groups in aqueous solutions. Nucleosides‐Nucleotides 6:913‐934.
   Raymond, F., Asseline, U., Roig, V., and Thuong, N.T. 1996. Synthesis and characterization of O6 modified deoxyguanosine‐containing oligodeoxyribonucleotides for triple helix formation. Tetrahedon 52:2047‐2064.
   Smith, L.M., Fung, S., Hunkapiller, M.W., Hunkapiller, T.J., and Hood, 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 analysis. Nucl. Acids. Res. 13:2399‐2412.
   Thuong, N.T. and Asseline, U. 1991. Oligodeoxyribonucleotides attached to intercalators, photoreactive and cleavage agents. In Oligodeoxyribonucleotides and Analogues: A Practical Approach (F. Eckstein, ed.) pp.283‐308. IRL Press, Oxford.
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