Direct Attachment of Conjugate Groups to the 5′ Terminus of Oligodeoxyribonucleotides

Ulysse Asseline1, Nguyen T. Thuong1

1 Centre de Biophysique Moléculaire, CNRS, Orléans, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.3
DOI:  10.1002/0471142700.nc0403s00
Online Posting Date:  May, 2001
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Abstract

This unit gives protocols for the attachment of intercalating and photoreactive conjugate groups to oligodeoxyribonucleotides. Protocols are given for acridine‐ and psoralen‐conjugated oligonucleotides, and include attachment of the linker, preparation of the phosphoramidite, coupling to the oligonucleotide, deprotection, purification, and characterization.

     
 
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Table of Contents

  • Basic Protocol 1: Direct Addition of Acridine Derivatives to the 5′ End of Oligodeoxyribonucleotides
  • Support Protocol 1: Purification and Characterization of Oligonucleotide‐Acridine Conjugates
  • Basic Protocol 2: Direct Addition of Psoralen Derivatives to the 5′ End of Oligodeoxyribonucleotides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Direct Addition of Acridine Derivatives to the 5′ End of Oligodeoxyribonucleotides

  Materials
  • 6‐Amino‐1‐hexanol
  • 6,9‐Dichloro‐2‐methoxyacridine
  • Phenol
  • Dichloromethane (CH 2Cl 2), distilled from P 2O 5 and passed over activated, basic aluminum oxide
  • Distilled methanol (MeOH)
  • 2 M sodium hydroxide (NaOH)
  • Acetonitrile (CH 3CN), HPLC grade
  • 2,6‐Dibromo‐4‐benzoquinone‐N‐chloroimine (DBPNC; Prolabo)
  • Ethanol, distilled
  • P 2O 5
  • Acetonitrile (CH 3CN), DNA synthesis grade, anhydrous and stored over 3‐Å molecular sieves
  • N,N‐Diisopropylethylamine (DIEA), distilled from KOH
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphite
  • Ethyl acetate, distilled
  • Hexane, distilled
  • Triethylamine (Et 3N), distilled from KOH
  • 10% (w/v) Na 2CO 3
  • Saturated NaCl (brine)
  • MgSO 4
  • Oligodeoxyribonucleotide bound to solid supports, such as controlled‐pore glass (CPG; unit 3.1), synthesized by classic phosphoramidite chemistry (1‐µmol scale; unit 3.3 and appendix 3C)
  • DNA synthesis reagents recommended by the synthesizer's manufacturer
  • 0.4 M sodium hydroxide (NaOH) in 50:50 (v/v) MeOH/H 2O
  • Dowex 50 resin, pyridinium form (Aldrich)
  • Round‐bottomed flasks (various sizes) with rubber septa and glass stoppers
  • Drying tube containing CaCl 2
  • Reflux condenser
  • Analytical thin‐layer chromatography (TLC) setup and silica‐gel plates: e.g., Merck 5554 Kieselgel 60F plates, including UV lamp for detection
  • Desiccator
  • Beakers, various sizes
  • Silica gel for column chromatography: Merck 9387 Kieselgel 60 or Merck 7734 Kieselgel 60
  • Chromatographic column (3 cm × 45 cm)
  • Argon
  • Nitrogen gas
  • Spectrophotometer
  • Rotary evaporator with water bath
  • Chemically inert syringes with replaceable needles
  • Funnel
  • Separatory funnels (various sizes)
  • Filter (porosity 4)
  • Vials and Teflon‐faced septa
  • Disposable filters for syringes and for filtration of HPLC buffers
  • Liquid chromatography apparatus equipped with multiwavelength detector
  • Melting‐point apparatus

Support Protocol 1: Purification and Characterization of Oligonucleotide‐Acridine Conjugates

  Materials
  • 0.01 bis‐Tris, pH 6 or 0.01 M NaH 2PO 4, pH 6.8 ( appendix 2A), each containing 10% or 10% HPLC‐grade methanol (MeOH)
  • Sodium chloride (NaCl)
  • 0.025 M Tris⋅Cl, pH 7 ( appendix 2A)
  • Acetonitrile (CH 3CN)
  • 1 M triethylammonium acetate (TEAA) buffer, pH 7 (stock solution; see recipe)
  • 0.01 M Tris⋅Cl, pH 8 ( appendix 2A)
  • 1 U snake venom phosphodiesterase (3′ exonuclease)
  • 10 µg alkaline phosphatase
  • Ion‐exchange columns: Mono Q HR 5/5 or HR 10/10 (Amersham Pharmacia Biotech) or DEAE (Waters)
  • Reversed‐phase columns: e.g., Lichrospher 100 RP 18 (5 µm, 125 × 4 mm, or 10 µm, 250 ×10 mm; Merck) or CC Nucleosil 100‐5 C18 (125/4) column (Macherey‐Nagel)
  • Desalting columns: HR 10/10 columns (Amersham Pharmacia Biotech) packed with Lichroprep RP 18 (Merck) or Sephadex G‐10 or G‐25 resin

Basic Protocol 2: Direct Addition of Psoralen Derivatives to the 5′ End of Oligodeoxyribonucleotides

  Materials
  • 5‐Methoxypsoralen (S.2a)
  • Pyridine hydrochloride
  • CaCl 2
  • Dichloromethane (CH 2Cl 2), distilled from P 2O 5 and passed over activated, basic aluminum oxide
  • Methanol (MeOH), distilled
  • P 2O 5
  • N,N‐Dimethylformamide, redistilled under vacuum over ninhydrin and stored over 4‐Å molecular sieves
  • 6‐Bromo‐1‐hexanol
  • Potassium carbonate, anhydrous
  • Ethyl acetate
  • Pyridine, anhydrous
  • N,N‐Diisopropylethylamine (DIEA), distilled from KOH
  • Sodium sulfate, anhydrous
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphite
  • Triethylamine (Et 3N)
  • 10% (w/v) aqueous sodium carbonate (NaHCO 3)
  • Cold saturated aqueous NaCl
  • Acetonitrile (CH 3CN), DNA synthesis grade, anhydrous and stored over 3 Å‐molecular sieves
  • Oligodeoxyribonucleotides bound to solid supports, such as controlled‐pore glass (CPG; unit 3.1), synthesized by classic phosphoramidite chemistry (1‐µmol scale)
  • DNA synthesis reagents recommended by synthesizer manufacturer
  • Concentrated (25%) aqueous ammonia
  • 25‐mL round‐bottomed flask
  • Reflux condenser
  • Drying tube
  • Nitrogen gas
  • Oven
  • Analytical thin‐layer chromatography (TLC) setup and silica‐gel plates: e.g., Merck 5554 Kieselgel 60F plates, including UV lamp for detection
  • Filter funnel, porosity 4
  • Desiccator
  • Argon
  • Silica gel for column chromatography: Merck 9387 Kieselgel 60 or Merck 7734 Kieselgel 60
  • Chromatographic columns: (3 cm × 50 cm) and (1.5 cm × 40 cm)
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Figures

Videos

Literature Cited

Literature Cited
   Asseline, U., Thuong, N.T., and Hélène, C. 1986. Oligothymidylates substitutés en position 3′ par un dérivè de l' acridine. Nucleosides Nucleotides 5:45‐63.
  The authors would like to express their appreciation to their collaborators M. Chassignol, V. Roig, and Y. Aubert for their contribution to the development of varied oligonucleotide sequences linked to acridine and psoralen derivatives. This work was supported by Rhône‐Poulenc, the Agence Nationale de Recherches sur le SIDA, and bio‐Mérieux.
   Asseline, U. and Thuong, N.T. 1988. Oligothymidylates substitués par un dérive de l' acridine en position 5′, à la fois en position 5′ et 3′ ou sur un phosphate internucleotidique. Nucleosides Nucleotides 7:431‐455.
   Asseline, U., Toulmé, F., Thuong, N. T., Delarue, M., Montenay‐Garestier, T., and Hélène, C. 1984. Oligodeoxynucleotides covalently linked to intercalating dyes as base sequence–specific ligands. Influence of dye attachment site. EMBO J. 3:795‐800.
   Asseline, U., Bonfils, E., Dupret, D., and Thuong, N.T. 1996. Synthesis and binding properties of oligonucleotides covalently linked to an acridine derivative: A new study of the influence of the dye attachment site. Bioconjugate Chemistry 7:369‐379.
   Costes, B., Girodon, E., Ghanem, N., Chassignol, M., Thuong, N.T., Dupret, D., and Goossens, M. 1993. Psoralen‐modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC‐clamping. Hum. Mol. Genet. 2:393‐397.
   Dupret, D., Gossens, M., Chassignol, M., and Thuong, N.T. 1994. European Patent No. 0596028 A1, 1994,05,11.
   Giovannangeli, C., Thuong, N.T., and Hélène, C. 1992. Oligodeoxynucleotide‐directed photo‐induced cross‐linking of HIV proviral DNA via triple‐helix formation. Nucleic Acids Res. 20:4275‐4281.
   Giovannangeli, C., Perrouault, L., Escudé, C., Thuong, N.T., and Hélène, C. 1996. Specific inhibition of in vitro transcription elongation by triplex‐forming oligonucleotide‐intercalator conjugates targeted to HIV proviral DNA. Biochemistry 35:10539‐10548.
   Grigoriev, M., Praseuth, D., Guieysse, A.L., Robin, P., Thuong, N.T., Hélène, C., and Harel‐Bellan, A. 1993. Inhibition of gene expression by triple helix–directed DNA cross‐linking at specific sites. Proc. Natl. Acad. Sci. USA. 90:3501‐3505.
   Kurfürst, R., Roig, V., Chassignol, M., Asseline, U., and Thuong, N.T. 1993. Oligo‐α‐Deoxyribonucleotides with a modified nucleic base and covalently linked to reactive agents. Tetrahedron 49:6975‐6990.
   Raynaud, F., Asseline, U., Roig, V., and Thuong, N.T. 1996. Synthesis and characterization of O6‐modified deoxyguanosine‐containing oligodeoxyribonucleotides for triplex helix formation. Tetrahedron 52:2047‐2064.
   Sun, J.S., François, J.C., Montenay‐Garestier, T., Saison‐Behmoaras, T., Roig, V., Thuong, N.T., and Hélène, C. 1989. Sequence‐specific intercalating agents. Intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide‐intercalator conjugates. Proc. Natl. Acad. Sci. USA. 86:9198‐9202.
   Takasugi, M., Guendouz, A., Chassignol, M., Decout, J.L., Lhomme, J., Thuong, N.T., and Hélène, C. 1991. Sequence‐specific photo‐induced cross‐linking of the two strands of double‐helical DNA by a psoralen covalently linked to a triple helix forming oligonucleotide. Proc. Natl. Acad. Sci. USA. 88:5602‐5606.
   Thuong, N.T. and Chassignol, M. 1988. Solid phase synthesis of oligo‐α‐ and oligo‐β‐deoxynucleotides covalently linked to an acridine. Tetrahedron Lett. 29:5905‐5908.
   Thuong, N.T., Hélène, C., and Asseline, U. 1984. European Patent No. 84‐400143‐8.
   1989. U.S. Patent No. 4‐835‐263.
   Thuong, N.T., Asseline, U., Roig, V., Takasugi, M., and Hélène, C. 1987. Oligo(α‐deoxynucleotide)s covalently linked to intercalating agents: Differential binding to ribo‐ and deoxyribopolynucleotides and stability towards nuclease digestion. Proc. Natl. Acad. Sci. USA. 84:5129‐5133.
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