3′‐Modified Oligonucleotides and their Conjugates

Matthew H. Lyttle1

1 Biosearch Technologies, Inc., Novato, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.6
DOI:  10.1002/0471142700.nc0406s03
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • 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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library