Solid‐Phase Oligonucleotide Labeling with DOTA

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes a method to construct oligonucleotide conjugates labeled with 1,4,7,10?tetraazacyclododecane?1,4,7,10?tetraacetic acid (DOTA) on a solid support. A nucleosidic phosphoramidite that contains a protected DOTA ligand compatible with normal chain assembly is prepared first. As the chain assembly is completed, the oligonucleotide is deprotected and converted to the corresponding gadolinium(III) chelate, resulting in an oligonucleotide conjugate containing the chelate at the 5??terminus.

Keywords: solid support; oligonucleotides; DOTA; MRI; molecular imaging; phosphoramidites

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Basic Protocol 1: Preparation of the Nucleosidic Phosphoramidite Tethered to DOTA
Basic Protocol 2: Synthesis of Oligonucleotide‐DOTA Conjugates
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Preparation of the Nucleosidic Phosphoramidite Tethered to DOTA

Materials

1,4,7,10‐Tetraazacyclododecane‐1‐carboxymethylbenzyl ester (S.1 ; Heppeler et al., )
Dry acetonitrile, ≥99.5% pure, ≤0.005% H₂ O (Merck)
Anhydrous potassium carbonate, p.a. (Merck)
Methyl bromoacetate, 99% pure (Acros Organics)
Silica gel 60, 0.063 to 0.200 nm (Merck)
Methanol (MeOH), ≥99.9% pure (Merck)
Dichloromethane (CH₂ Cl₂ ), 99% pure (Lab‐Scan)
10% palladium on activated carbon (Pd/C; Aldrich)
Hydrogen gas
Celite 521 (Aldrich)
5′‐O ‐(4,4′‐Dimethoxytrityl)‐2′‐deoxyuridine (S.4 ; Sigma)
N ‐Trifluoroacetyl‐6‐aminohexan‐1‐ol (Sinha and Striepeke, )
Triphenylphosphine, 99% pure (Aldrich)
Dry tetrahydrofuran (THF), ≥99.5% pure, ≤0.0075% H₂ O (Merck)
Diisopropyl azodicarboxylate (DIAD), 95% pure (Aldrich)
Diethyl ether, ≥99.7% pure (Merck)
Aqueous ammonia, p.a. 28% to 30% (Merck)
Sodium sulfate (Na₂ SO₄ ), anhydrous, ≥99.0% pure (Merck)
N ,N ‐Diisopropylethylamine (DIPEA), ≥99.5% pure (Aldrich)
N,N ‐Dimethylformamide (DMF), dry, 99.8% pure, ≤0.01% H₂ O (Lab‐Scan)
O ‐(7‐Azabenzotriazol‐1‐yl)‐N,N,N′,N′ ‐tetramethyluronium hexafluorophosphate (HATU; Applied Biosystems)
Sodium hydrogen carbonate (NaHCO₃ ) solutions, saturated and 5% (w/v)
2‐Cyanoethyl‐N,N,N′,N′ ‐tetraisopropylphosphordiamidite, 97% pure (Aldrich)
0.45 M 1H ‐tetrazole in acetonitrile (Applied Biosystems)
Triethylamine (TEA), ≥99% pure (Merck)

Glass filters (3‐µm pore size)
Rotary evaporator equipped with an oil pump
Chromatography columns: 18 × 4 cm, 5 × 30 cm, 4 × 15 cm, and 4 × 2.5 cm
TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F₂₅₄ )
Hydrogenation apparatus (Parr Instruments Company)
Ultrasonic bath
254‐nm UV lamp
Reflux condenser

Additional reagents and equipment for TLC ( appendix 3D ) and column chromatography ( appendix 3E )

Basic Protocol 2: Synthesis of Oligonucleotide‐DOTA Conjugates

Materials

DOTA‐labeled phosphoramidite (S.8 ; see protocol 1 )
Dry acetonitrile, ≥99.5%, ≤0.005% H₂ O (Merck)
Deoxyribonucleoside phosphoramidites (e.g., Proligo)
0.1 M NaOH
1 M NH₄ Cl
Aqueous ammonia, p.a. 28% to 30% (Merck)
Gadolinium(III) citrate: 0.1 M Gd(III)Cl₃ /0.2 M citric acid
20% polyacrylamide gel containing 7 M urea ( appendix 3B )
1 M Na₂ CO₃ , pH 9.8
1‐Butanol, ≥99.5% pure (Merck)
Sterile, nuclease‐free, deionized water

Automated DNA/RNA synthesizer (e.g., Applied Biosystems)
0.2‐µmol DNA synthesis column (1000 Å CPG; Applied Biosystems)
1‐mL syringes
2‐mL vials
Speedvac evaporator (e.g., SPD121P, Savant)
NAP5 gel filtration columns (GE Healthcare)
Centrifuge (e.g., BR4i, JOUAN)

Additional reagents and equipment for automated DNA synthesis ( appendix 3C ) and urea‐PAGE ( appendix 3B )

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Figures

Figure 4.31.1 Synthesis of the DOTA‐labeled nucleosidic phosphoramidite (see ). Bn, benzyl; DIAD, diisopropyl azodicarboxylate; DIPEA, N,N ‐diisopropylethylamine; DMF, N,N ‐dimethylformamide; DMTr, 4,4′‐dimethoxytrityl; HATU, O ‐(7‐azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate. Modified from Jaakkola et al. () with permission from the American Chemical Society.

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Figure 4.31.2 Synthesis of the oligonucleotide conjugate tethered to DOTA (see ). CPG, controlled‐pore glass; DMTr, 4,4′‐dimethoxytrityl. Modified from Jaakkola et al. () with permission from the American Chemical Society.

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Literature Cited

Literature Cited
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	Jaakkola, L., Ylikoski, A., and Hovinen, J. 2006. Simple synthesis of a building block for solid phase labeling of oligonucleotides with 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA). Bioconjug. Chem. 17:1105‐1107.
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