Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Quantifying DNA–protein interactions by double-stranded DNA arrays

Nature Biotechnology volume 17, pages 573–577 (1999)Cite this article

Abstract

We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA–protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2´-deoxyribonucleoside 5´-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Generalized array strand (not to scale).
Figure 2: Alternative methods for second-strand labeling.
Figure 3: (A) Schematic of RsaI digestion of an array of dsDNA oligonucleotides labeled with fluorescein-12-dATP.
Figure 4: RsaI and DpnI digestions of labeled dsDNA arrays.
Figure 5: z scores (normalized signal intensity differences) at 5´-GTAC-3´ sites before and after RsaI digestion of a dsDNA array designed to vary the density and accessibility of the DNA strands.

Similar content being viewed by others

References

  1. Pabo, C.O. & Sauer, R.T. Transcription factors: structural families and principles of DNA recognition. Annu. Rev. Biochem. 61, 1053–1095 ( 1992).

    Article  CAS  Google Scholar 

  2. Craig, N.L. The mechanism of conservative site-specific recombination. Annu. Rev. Genet. 22, 77–105 ( 1988).

    Article  CAS  Google Scholar 

  3. Pingoud, A. & Jeltsch, A. Recognition and cleavage of DNA by type-II restriction endonucleases. Eur. J. Biochem. 246, 1–22 (1997).

    Article  CAS  Google Scholar 

  4. Margulies, C. & Kaguni, J.M. Ordered and sequential binding of DNA protein to oriC, the chromosomal origin of Escherichia coli. J. Biol. Chem. 271, 17035–17040 (1996).

    Article  CAS  Google Scholar 

  5. Woodbury, C.P. & Hippel, P.H.V. On the determination of deoxyribonucleic acid–protein interaction parameters using the nitrocellulose filter-binding assay. Biochemistry 22, 4730– 4737 (1983).

    Article  CAS  Google Scholar 

  6. Jansen, C., Gronenborn, A.M. & Clore, G.M. The binding of the cyclic AMP receptor protein to synthetic DNA sites containing permutations in the consensus sequence TGTGA. Biochem. J. 246, 227–232 (1987).

    Article  CAS  Google Scholar 

  7. Bowen, B., Steinberg, J., Laemmli, U.K. & Weintraub, H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8, 1–20 ( 1980).

    Article  CAS  Google Scholar 

  8. Miskimins, W.K., Roberts, M.P., McClelland, A. & Ruddle, F.H. Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. Proc. Natl. Acad. Sci. USA 82, 6741– 6744 (1985).

    Article  CAS  Google Scholar 

  9. Hanes, S.D. & Brent, R. A genetic model for interaction of the homeodomain recognition helix with DNA. Science 251, 426–430 (1991).

    Article  CAS  Google Scholar 

  10. Lockhart, D.J. et al. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 14, 1675– 1680 (1996).

    Article  CAS  Google Scholar 

  11. Roth, F.P., Hughes, J.D., Estep, P.W. & Church, G.M. Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation. Nat. Biotechnol. 16, 939–945 (1998).

    Article  CAS  Google Scholar 

  12. Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 ( 1995).

    Article  CAS  Google Scholar 

  13. Chee, M. et al. Accessing genetic information with high-density DNA arrays. Science 274, 610–614 ( 1996).

    Article  CAS  Google Scholar 

  14. Gunderson, K.L. et al. Mutation detection by ligation to complete N-mer DNA arrays. Genome Res. 8, 1142–1153 (1998).

    Article  CAS  Google Scholar 

  15. Hacia, J.G., Brody, L.C., Chee, M.S., Fodor, S.P.A. & Collins, F.S. Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis. Nat.Genet. 14, 441–447 (1996).

    Article  CAS  Google Scholar 

  16. Wang, D.G. et al. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280, 1077– 1082 (1998).

    Article  CAS  Google Scholar 

  17. Shoemaker, D.D., Lashkari, D.A., Morris, D., Mittmann, M. & Davis, R.W. Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy. Nat. Genet. 14, 450–456 (1996).

    Article  CAS  Google Scholar 

  18. Cho, R.J. et al. Parallel analysis of genetic selections using whole genome oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95, 3752 –3757 (1998).

    Article  CAS  Google Scholar 

  19. Lockhart, D.J., Vetter, D. & Diggelmann, M. Surface-bound, unimolecular, double-stranded DNA. (Affymetrix, Inc., USA). US patent # 5556752, issue date 9/17/96.

  20. Pease, A.C. et al. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. USA 91, 5022– 5026 (1994).

    Article  CAS  Google Scholar 

  21. McGall, G.H. et al. The efficiency of light-directed synthesis of DNA arrays on glass substrates. J. Amer. Chem. Soc. 119, 5081 –5090 (1997).

    Article  CAS  Google Scholar 

  22. Polisky, B. et al. Specificity of substrate recognition by the EcoRI restriction endonuclease. Proc. Natl. Acad. Sci. USA 72, 3310–3314 (1975).

    Article  CAS  Google Scholar 

  23. Thielking, V., Alves, J., Fliess, A., Maass, G. & Pingoud, A. Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry 29, 4682–4691 (1990).

    Article  CAS  Google Scholar 

  24. Engler, L.E., Welch, K.K. & Jen-Jacobson, L. Specific binding by EcoRV endonuclease to its DNA recognition site GATATC. J. Mol. Biol. 269, 82–101 (1997).

    Article  CAS  Google Scholar 

  25. Lesser, D.R., Kurpiewski, M.R. & Jen-Jacobson, L. The energetic basis of specificity in the EcoRI endonuclease–DNA interaction. Science 250, 776–786 (1990).

    Article  CAS  Google Scholar 

  26. McClarin, J.A. et al. Structure of the DNA–EcoRI endonuclease recognition complex at 3 A resolution. Science 234, 1526–1541 (1986).

    Article  CAS  Google Scholar 

  27. Wah, D.A., Hirsch, J.A., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of the multimodular endonuclease FokI bound to DNA. Nature 388, 97–100 (1997).

    Article  CAS  Google Scholar 

  28. Winkler, F.K. et al. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 12, 1781–1795 (1993).

    Article  CAS  Google Scholar 

  29. Newman, M., Strzelecka, T., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of BamHI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 269, 656–663 ( 1995).

    Article  CAS  Google Scholar 

  30. Cheng, X., Balendiran, K., Schildkraut, I. & Anderson, J.E. Structure of PvuII endonuclease with cognate DNA. EMBO J. 13, 3927–3935 ( 1994).

    Article  CAS  Google Scholar 

  31. Newman, M. et al. Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence. EMBO J. 17,5466–5476 (1998).

    Article  CAS  Google Scholar 

  32. Terry, B.J., Jack, W.E., Rubin, R.A. & Modrich, P. Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences. J. Biol. Chem. 258, 9820–9825 (1983).

    CAS  PubMed  Google Scholar 

  33. Kuz'min, N.P., Loseva, S.P., Beliaeva, R.K., Kravets, A.N. & Solonin, A.S. EcoRV restrictase: physical and catalytic properties of homogenous enzyme. Mol. Biol. (Mosk) 18, 197–204 (1984).

    CAS  Google Scholar 

  34. George, J., Blakesley, R.W. & Chirikjian, J.G. Sequence-specific endonuclease BamHI. Effect of hydrophobic reagents on sequence recognition and catalysis. J. Biol. Chem. 255, 6521–6524 (1980).

    CAS  PubMed  Google Scholar 

  35. Nasri, M. & Thomas, D. Alteration of the specificity of PvuII restriction endonuclease. Nucleic Acids Res. 15, 7677–7687 (1987).

    Article  CAS  Google Scholar 

  36. Escherichia coli and salmonella. Cellular and molecular biology Vol. 1. (ed. Neidhardt, F.C.) (ASM, Washington, DC; 1996).

  37. Swartz, D.R. Covalent labeling of proteins with fluorescent compounds for imaging applications. Scanning Microsc. Suppl. 10, 273– 284 (1996).

    CAS  PubMed  Google Scholar 

  38. Kim, Y.-G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).

    Article  CAS  Google Scholar 

  39. Panayotatos, N. & Backman, S. A site-directed recombinant nuclease probe of DNA structure. J. Biol. Chem. 264, 15070–15073 (1989).

    CAS  PubMed  Google Scholar 

  40. Tavazoie, S. & Church, G.M. Quantitative whole-genome analysis of DNA–protein interactions by in vivo methylase protection in E. coli. Nat. Biotechnol. 16, 566– 571 (1998).

    Article  CAS  Google Scholar 

  41. Robison, K., McGuire, A.M. & Church, G.M. A comprehensive library of DNA-binding site matrices for 55 proteins applied to the complete Escherichia coli K-12 genome. J. Mol. Biol. 284, 241– 254 (1998).

    Article  CAS  Google Scholar 

  42. Desjarlais, J.R. & Berg, J.M. Length-encoded multiplex binding site determination: application to zinc finger proteins. Proc. Natl. Acad. Sci. USA 91, 11099– 11103 (1994).

    Article  CAS  Google Scholar 

  43. Harrison, S.C. & Aggarwal, A.K. DNA recognition by proteins with the helix-turn-helix motif. Annu. Rev. Biochem. 59, 933–969 ( 1990).

    Article  CAS  Google Scholar 

  44. Suzuki, M., Yagi, N. & Gerstein, M. DNA recognition and superstructure formation by helix-turn-helix proteins. Protein Eng. 8, 329– 338 (1995).

    Article  CAS  Google Scholar 

  45. Harrison, S.C. A structural taxonomy of DNA-binding domains. Nature 353, 715–719 (1991).

    Article  CAS  Google Scholar 

  46. Liu-Johnson, H.-N., Garterberg, M.R. & Crothers, D.M. The DNA binding domain and bending angle of E. coli CAP protein. Cell 47, 995– 1005 (1986).

    Article  CAS  Google Scholar 

  47. Wodicka, L., Dong, H., Mittmann, M., Ho, M.-H. & Lockhart, D.J. Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat. Biotechnol. 15, 1359 –1366 (1997).

    Article  CAS  Google Scholar 

  48. Molecular probes: handbook of fluorescent probes and research chemicals. (ed. Haugland, R.P.) (Molecular Probes, Inc., Eugene, Oregon, 1996).

Download references

Acknowledgements

We thank John Aach and Keith Robison for help with Perl. We also thank Mark Chee, Rich Baldarelli, as well as members of the Church lab for helpful discussions and critical reading of the manuscript. This work was supported by the US Department of Energy (grant no. DE-FG02-87-ER60565). M.B. was supported by an NSF Graduate Fellowship. G.M.C. was partially supported by the Howard Hughes Medical Institute. This article is dedicated to my father, Roman P. Bulyk, who passed away while this work was being completed.

Author information

Authors and Affiliations

  1. Harvard University Graduate Biophysics Program and Harvard Medical School Department of Genetics, Boston, 02115, MA

    Martha L. Bulyk & George M. Church

  2. Affymetrix, Santa Clara, 95051, CA

    Erik Gentalen & David J. Lockhart

Authors
  1. Martha L. Bulyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erik Gentalen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David J. Lockhart
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. George M. Church
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to George M. Church.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bulyk, M., Gentalen, E., Lockhart, D. et al. Quantifying DNA–protein interactions by double-stranded DNA arrays . Nat Biotechnol 17, 573–577 (1999). https://doi.org/10.1038/9878

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/9878

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing