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 2 Cl 2 )/methanol
- 55:10:20 or 55:10:30 (v/v/v) isopropanol/NH 4 OH/H 2 O
- Sephadex G‐10, G‐15, or G‐25 resin (Pharmacia; for H 2 O‐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 3 CN
- Mobile phase B: 0.1 M triethylammonium acetate (TEAA) buffer, pH 7 containing 80% CH 3 CN
- 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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