Synthesis of Covalent Oligonucleotide‐Streptavidin Conjugates and Their Application in DNA‐Directed Immobilization (DDI) of Proteins

Ron Wacker1, Christof M. Niemeyer2

1 Chimera Biotec GmbH, Dortmund, 2 Universität Dortmund, Fachbereich Chemie, Dortmund
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 12.7
DOI:  10.1002/0471142700.nc1207s22
Online Posting Date:  October, 2005
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Abstract

Semisynthetic DNA‐streptavidin conjugates are synthesized by covalent coupling of thiol‐modified DNA oligonucleotides and streptavidin (STV). The resulting conjugates have binding capacities for four equivalents of biotin and a complementary nucleic acid sequence. The conjugates are purified to homogeneity by ultrafiltration and chromatography, and are characterized by spectrophotometry and gel electrophoresis. Subsequently, the conjugates are applied as molecular connectors in the DNA‐directed immobilization (DDI) of biotinylated antibodies using DNA microarrays as immobilization matrices. The results are protein microarrays that can be used for the multiplexed detection of various antigens.

Keywords: biotin‐streptavidin interaction; DNA conjugation; bioconjugate purification; electrophoretic characterization of DNA‐protein conjugates; protein biotinylation; DNA‐directed immobilization; DNA and protein microarray technology

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of Semisynthetic DNA‐Streptavidin Conjugates by Covalent Coupling of Thiol‐Modified Oligonucleotides
  • Basic Protocol 2: Semisynthetic DNA‐STV Conjugates as Molecular Linkers in DNA‐Directed Immobilization of Biotinylated Antibodies on DNA Microarrays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Semisynthetic DNA‐Streptavidin Conjugates by Covalent Coupling of Thiol‐Modified Oligonucleotides

  Materials
  • 1 M dithiothreitol (DTT)
  • Thiolated oligonucleotides (Table 12.7.1, e.g., Thermo Electron)
  • TE buffer, pH 7.5 ( appendix 2A)
  • PBSE buffer (see recipe)
  • Sulfosuccinimidyl‐4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate (sSMCC; Pierce)
  • Dimethylformamide (DMF)
  • 100 µM streptavidin, recombinant (STV; Roche) in PBS1 (see recipe)
  • 1 M 2‐mercaptoethanol
  • Tris⋅Cl ( appendix 2A): 20 mM at pH 6.3, 1.5 M at pH 8.8, and 1 M at pH 6.8
  • TBSE (see recipe)
  • 37.5%:1% (w/v) acrylamide/bisacrylamide solution
  • 10% (w/v) ammonium persulfate
  • Tetramethylethylenediamine (TEMED)
  • 1‐Butanol (pure)
  • Running buffer (see recipe)
  • Loading buffer (see recipe)
  • 123‐bp DNA ladder (Invitrogen)
  • SybrGold (Molecular Probes)
  • 37°C heating block or water bath
  • Fast protein liquid chromatography (FPLC) system (Amersham Biosciences) with Superdex peptide column, fraction collector, and detector
  • Gel filtration columns (e.g., NAP5 and NAP10, Amersham Biosciences)
  • Molecular cut‐off ultrafiltration unit (e.g., Centricon 30, Millipore)
  • Anion‐exchange column (e.g., MonoQ HR5/5, Amersham Biosciences)
  • Photometer (e.g., BioPhotometer, Eppendorf)
  • Electrophoresis device (e.g., Bio‐Rad)
  • Container for gel staining
  • Transilluminator
  • SybrGold camera filter (Biozym)
  • AlphaImager 2200 gel documentation device (Biozym)
    Table 2.7.1   Materials   Sequences of Thiolated Oligonucleotide Library for the Generation of DNA‐Protein Conjugates and Complementary Amino‐Modified Capture Oligomers a   Sequences of Thiolated Oligonucleotide Library for the Generation of DNA‐Protein Conjugates and Complementary Amino‐Modified Capture Oligomers

    Name Sequence Modification
    Thiolated oligonucleotide library
    tA TCC TGT GTG AAA TTG TTA TCC GCT 5′ Thiolink (C6)
    tB ACC TCA AGT GAT CTA CCT ACC TCA G 5′ Thiolink (C6)
    tC CTC ACA TCC AAC AAT ACA GGT CAC AT 5′ Thiolink (C6)
    tD TGA GCG TTC GTG GGA TAG T 5′ Thiolink (C6)
    Complementary amino‐modified capture oligomers
    cA AGC GGA TAA CAA TTT CAC ACA GGA 5′ Aminolink (C6)
    cB CTG AGG TAG GTA GAT CAC TTG AGG T 5′ Aminolink (C6)
    cC ATG TGA CCT GTA TTG TTG GAT GTG AG 5′ Aminolink (C6)
    cD ACT ATC CCA CGA ACG CTC A 5′ Aminolink (C6)

     aSequences are shown in 5′‐ 3′ direction.

Basic Protocol 2: Semisynthetic DNA‐STV Conjugates as Molecular Linkers in DNA‐Directed Immobilization of Biotinylated Antibodies on DNA Microarrays

  Materials
  • PBS2 (see recipe)
  • Antibodies (Table 12.7.4, Sigma‐Aldrich)
  • Sulfo‐(N‐hydroxysuccinimid)biotin (sulfoNHS‐biotin; Pierce)
  • Dimethylformamide (DMF)
  • Blocking solution (Chimera Biotec)
  • DNA‐STV conjugates S.1 (Table 12.7.4; see protocol 1 or Chimera Biotec)
  • TE buffer, pH 7.5 ( appendix 2A)
  • Conjugate dilution buffer (Chimera Biotec)
  • TETBS (see recipe)
  • Rabbit anti–goat IgG conjugated with Cy5 (e.g., Chimera Biotec)
  • Gel filtration columns (NAP10, Amersham Biosciences)
  • Molecular cut‐off ultrafiltration unit (Centricon 30, Millipore)
  • Photometer (e.g., BioPhotometer, Eppendorf)
  • Microarray slides (e.g., HP‐slides; Chimera Biotec) functionalized with covalently coupled oligonucleotides complementary to DNA‐STV conjugates (see bottom of Table 12.7.1)
  • Slide boxes (e.g., Chimera Biotec)
  • Microplate centrifuge (e.g., 5804R with rotor A‐2‐DWP, Eppendorf)
  • Adhesive hybridization chambers, 25 µL (e.g., Chimera Biotec)
  • Microscope slide holder (for centrifuge; e.g., Chimera Biotec)
  • Microarray laser scanning system (e.g., Axon 4000B, Axon)
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Figures

Videos

Literature Cited

Literature Cited
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