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

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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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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
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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)

^a Sequences 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., H^P ‐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

Figure 12.7.1 Schematic representation of the modular preparation of functional conjugates employed in the DDI‐method. (A ) Self‐assembly of DNA‐STV conjugate S.1 with biotinylated antibody S.2 to give the DNA‐antibody conjugate S.3. (B ) Site‐specific immobilization of the conjugates S.3A‐D on their complementary oligonucleotides on a solid support.

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Figure 12.7.2 Covalent crosslinking of STV and 5′‐thiolated oligonucleotides.

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Figure 12.7.3 Chromatograph of conjugate purification by anion‐exchange chromatography.

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Figure 12.7.4 Gel pictures of DNA‐STV conjugate. M: 123‐bp DNA ladder; lane 1: 1:1 DNA:STV; lane 2: 2:1 DNA:STV (also containing 3:1); lane 3: 3:1 and 4:1 DNA:STV.

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Figure 12.7.5 Schematic drawing of the DDI method.

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Figure 12.7.6 Fluorescent images of the microarrays obtained from DDI.

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Figure 12.7.7 Molecular structure of STV (Weber et al., ) and D ‐biotin.

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Videos

Literature Cited

Literature Cited
	Angenendt, P., Glokler, J., Murphy, D., Lehrach, H., and Cahill, D.J. 2002. Toward optimized antibody microarrays: A comparison of current microarray support materials. Anal. Biochem. 309:253‐260.
	Angenendt, P., Glokler, J., Sobek, J., Lehrach, H., and Cahill, D.J. 2003. Next generation of protein microarray support materials: Evaluation for protein and antibody microarray applications. J. Chromatogr. 1009:97‐104.
	de Wildt, R.M., Mundy, C.R., Gorick, B.D., and Tomlinson, I.M. 2000. Antibody arrays for high‐throughput screening of antibody‐antigen interactions. Nat. Biotechnol. 18:989‐994.
	Feldkamp, U., Wacker, R., Banzhaf, W., and Niemeyer, C.M. 2004. Microarray‐based in vitro evaluation of DNA oligomer libraries designed in silico. Chem. Phys. Chem. 5:367‐372.
	Kusnezow, W. and Hoheisel, J.D. 2002. Antibody microarrays: Promises and problems. BioTechniques Suppl:14‐23.
	Lueking, A., Horn, M., Eickhoff, H., Bussow, K., Lehrach, H., and Walter, G. 1999. Protein microarrays for gene expression and antibody screening. Anal. Biochem. 270:103‐111.
	MacBeath, G. and Schreiber, S.L. 2000. Printing proteins as microarrays for high‐throughput function determination. Science 289:1760‐1763.
	Niemeyer, C.M. 2002. The developments of semisynthetic DNA‐protein conjugates. Trends Biotechnol. 20:395‐401.
	Niemeyer, C.M., Sano, T., Smith, C.L., and Cantor, C.R. 1994. Oligonucleotide‐directed self‐assembly of proteins: Semisynthetic DNA–streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. Nucl. Acids Res. 22:5530‐5539.
	Niemeyer, C.M., Bürger, W., and Hoedemakers, R.M. 1998. Hybridization characteristics of biomolecular adaptors, covalent DNA–streptavidin conjugates. Bioconjugate Chem. 9:168‐175.
	Niemeyer, C.M., Boldt, L., Ceyhan, B., and Blohm, D. 1999. DNA‐directed immobilization: Efficient, reversible, and site‐selective surface binding of proteins by means of covalent DNA‐streptavidin conjugates. Anal. Biochem. 268:54‐63.
	Parker, J. 2003. Computing with DNA. EMBO Rep. 4:7‐10.
	Pirrung, M.C. 2002. How to make a DNA chip. Angew. Chem. Int. Ed. Engl. 41:1276‐1289; Angew. Chem. 114: 326‐1341.
	Schweitzer, B. and Kingsmore, S.F. 2002. Measuring proteins on microarrays. Curr. Opin. Biotechnol. 13:14‐19.
	Seeman, N.C. 2003. DNA in a material world. Nature 421:427‐431.
	Templin, M.F., Stoll, D., Schrenk, M., Traub, P.C., Vohringer, C.F., and Joos, T.O. 2002. Protein microarray technology. Trends Biotechnol. 20:160‐166.
	Wacker, R. and Niemeyer, C.M. 2004. DDI‐µFIA—A readily configurable microarray‐fluorescence immunoassay based on DNA‐directed immobilization of proteins. Chem. Bio. Chem. 5:453‐459.
	Wacker, R., Schroeder, H., and Niemeyer, C.M. 2004. Performance of antibody‐microarrays fabricated by either DNA‐directed immobilization, direct spotting or streptavidin‐biotin attachment: A comparative study. Anal. Biochem. 330:281‐287.
	Weber, P.C., Ohlendorf, D.H., Wendoloski, J.J., and Salemme, F.R. 1989. Structural origins of high‐affinity biotin binding to streptavidin. Science 243:85‐88.
	Winssinger, N., Harris, J.L., Backes, B.J., and Schultz, P.G. 2001. From split‐pool libraries to spatially addressable microarrays and its application to functional proteomic profiling. Angew. Chem. Int. Ed. Engl. 40:3152‐3155.

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited