3′‐Modified Oligonucleotides and their Conjugates

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Solid?phase synthesis of 3??aminoalkyl, 3??thioalkyl, and 3??polyethyleneglycol are described. The first two are important as specific points of attachment for a large variety of reporter groups; the latter is important because of its increased cell membrane permeability, which aids in the development of antisense drugs. These protocols cover details of the synthetic organic chemistry needed to make each solid support, as well as DNA synthesis, workup, characterization, and conjugation.

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Basic Protocol 1: Preparation of 3′‐Aminoalkyl‐Functionalized DNA Oligonucleotides
Alternate Protocol 1: Preparation of 3′‐Thioalkyl‐Functionalized DNA Oligonucleotides
Alternate Protocol 2: Preparation of 3′‐Polyethylene‐Glycol‐Functionalized DNA Oligonucleotides
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Preparation of 3′‐Aminoalkyl‐Functionalized DNA Oligonucleotides

Materials

1000 Å aminopropyl‐conjugated controlled‐pore glass (aminopropyl‐CPG; Biosearch Technologies)
Pyridine
Methylene chloride
Trimellitic anhydride chloride (Aldrich)
100% and 20% (v/v) acetonitrile
6‐Amino‐1‐hexanol
N ,N ‐Dimethylformamide (DMF)
4,4′‐Dimethoxytrityl chloride (DMTr chloride; Aldrich)
3% (v/v) dichloroacetic acid in methylene chloride
28% (v/v) aqueous ammonia
5‐ and 6‐Carboxy fluorescein succinimide ester (Molecular Probes)
Dimethyl sulfoxide (DMSO)
1 M sodium carbonate/1 M sodium bicarbonate solution
0.05 M aqueous ammonium acetate
Sephadex G‐25 resin (Amersham Pharmacia Biotech)
1 M triethylammonium acetate (TEAA)
14% (v/v) aqueous ammonia (1 part concentrated ammonium hydroxide, 1 part water)
2.8% (v/v) aqueous ammonia (1 part concentrated ammonium hydroxide, 9 parts water)
Buffer A: 0.025 M Tris⋅Cl and 0.01 M Tris base
Buffer B: 0.025 M Tris⋅Cl, 0.01 M Tris base, and 1 M NaCl
125‐mL Erlenmeyer flasks with stoppers
150‐mL sintered glass funnels, coarse frit
1‐liter side‐arm filter flask with rubber gasket
Water aspirator
Heavy‐walled vacuum tubing
3‐way valve
Speedvac (Savant)
Spectrophotometer and 1‐cm path length glass cuvettes (e.g., Beckman)
Automated DNA synthesizer and reagents (e.g., PE Biosystems)
1.5‐mL plastic screw‐cap tubes
55°C water bath
Reversed‐phase DNA purification cartridges and reagents (e.g., Biosearch Technologies)
1 × 30–cm glass tube with a frit and valve on bottom
10‐mL syringes
High‐performance liquid chromatograph (HPLC) with anion‐exchange column
Additional reagents and equipment for DNA purification with a reversed‐phase cartridge (unit 10.7 )

Alternate Protocol 1: Preparation of 3′‐Thioalkyl‐Functionalized DNA Oligonucleotides

6‐Mercapto‐1‐O ‐DMTr hexanol (Biosearch Technologies)
Dithiothreitol (DTT)
Triethylamine
Fluorescein‐5‐maleimide (Molecular Probes)
Sodium phosphate/sodium chloride buffer: 50 mM sodium phosphate and 150 mM sodium chloride, adjust to pH 7.2 (if necessary)

Alternate Protocol 2: Preparation of 3′‐Polyethylene‐Glycol‐Functionalized DNA Oligonucleotides

Triethylene glycol (Aldrich)
N‐ Methylimidazole (Aldrich)

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Figures

Figure 4.6.1 An anhydride‐functionalized solid support for the synthesis of 3′‐modified DNA.

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Videos

Literature Cited

Literature Cited
	Gupta, K.C., Sharma, P., Kumar, P., and Sathyanarayana, S. 1991. A general method for the synthesis of 3′‐sulfhydryl and phosphate group containing oligonucleotides. Nucl. Acids Res. 19:3019‐3025.
	Lyttle, M.H., Adams, H., Hudson, D., and Cook, R.M. 1997. Versatile linker chemistry for synthesis of 3′‐modified DNA. Bioconjug. Chem. 8:193‐198.
	Stewart, J. and Young, J. 1984. Laboratory techniques in solid phase peptide synthesis. In Solid Phase Peptide Synthesis, pp. 105‐107. Pierce Chemical Company, Rockford, Ill.

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3′‐Modified Oligonucleotides and their Conjugates

Abstract

Table of Contents

Materials

Basic Protocol 1: Preparation of 3′‐Aminoalkyl‐Functionalized DNA Oligonucleotides

Alternate Protocol 1: Preparation of 3′‐Thioalkyl‐Functionalized DNA Oligonucleotides

Alternate Protocol 2: Preparation of 3′‐Polyethylene‐Glycol‐Functionalized DNA Oligonucleotides

Figures

Videos

Literature Cited