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.

Protein detection using proximity-dependent DNA ligation assays

Abstract

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe . The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 × 10−21 mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Schematic view of the homodimeric PDGF-BB (ref. 23) bound by two aptamer-based proximity probes, A1 and A2, for detection by proximity ligation.
Figure 2: Optimization of reaction conditions for the proximity ligation assay.
Figure 3: Detection of a dilution series of PDGF-BB by proximity ligation (solid line) and by sandwich ELISA (dashed line).
Figure 4: Homogeneous detection of PDGF-BB in the presence of complex biological fluids and cell culture media: FCS, EMEM, and human CSF.
Figure 5: Detection of human α-thrombin by homogeneous proximity ligation.
Figure 6: Dilution series of PDGF-BB analyzed in a solid-phase assay, either by proximity ligation (circles) or by aptamer-based immuno-PCR (squares).

References

  1. 1

    Saiki, R.K. et al. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354 (1985).

    CAS  Article  Google Scholar 

  2. 2

    Hartwell, L.H., Hopfield, J.J., Leibler S. & Murray A.W. From molecular to modular cell biology. Nature 402, c47–52 (1999).

    CAS  Article  Google Scholar 

  3. 3

    Heldin, C.H. & Westermark, B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol. Rev. 79, 1283–1316 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    CAS  Article  Google Scholar 

  5. 5

    Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    CAS  Article  Google Scholar 

  6. 6

    Jayasena, S.D. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin. Chem. 45, 1628–1650 (1999).

    CAS  PubMed  Google Scholar 

  7. 7

    Ellington, A.D. & Robertsson, M.P. In vitro selection of an allosteric ribozyme that transduces analytes to amplicons. Nat. Biotechnol. 17, 62–66 (1999).

    Article  Google Scholar 

  8. 8

    Green, L.S. et al. Inhibitory DNA ligands to platelet-derived growth factor B-chain. Biochemistry 45, 14413–14424 (1996).

    Article  Google Scholar 

  9. 9

    Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. Real time quantitative PCR. Genome Res. 6, 986–994 (1996).

    CAS  Article  Google Scholar 

  10. 10

    Li, X. et al. PDGF-C is a new protease-activated ligand for the PDGF α-receptor. Nat. Cell Biol. 2, 302–309 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Bergsten, E. et al. PDGF-D is a specific, protease-activated ligand for the PDGF β-receptor. Nat. Cell Biol. 5, 512–516 (2001).

    Article  Google Scholar 

  12. 12

    LaRochelle, W.J. et al. PDGF-D, a new protease-activated growth factor. Nat. Cell Biol. 5, 517–521 (2001).

    Article  Google Scholar 

  13. 13

    Leppänen, O. et al. Predimerization of recombinant platelet-derived growth factor receptor extracellular domains increases antagonistic potency. Biochemistry 39, 2370–2375 (2000).

    Article  Google Scholar 

  14. 14

    Dahlman, T. et al. Fibrosis in undifferentiated (anaplastic) thyroid carcinomas: evidence for a dual action of tumor cells in collagen type I synthesis. J. Pathol. 191, 376–386 (2000).

    CAS  Article  Google Scholar 

  15. 15

    Macaya, R.F. et al. Structural and functional characterization of potent antithrombotic oligonucleotides possessing both quadruplex and duplex motifs. Biochemistry 34, 4478–4492 (1995).

    CAS  Article  Google Scholar 

  16. 16

    Tasset, D.M., Kubik, M.F. & Steiner, W. Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J. Mol. Biol. 272, 688–698 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Sano, T., Smith, C.L. & Cantor, C.R. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258, 120–122 (1992).

    CAS  Article  Google Scholar 

  18. 18

    Drolet, D.W., Jenison, R.D., Smith, D.E., Pratt, D. & Hicke, B.J. A high throughput platform for systematic evolution of ligands by exponential enrichment (SELEX). Comb. Chem. High Throughput Screen. 2, 271–278 (1999).

    CAS  PubMed  Google Scholar 

  19. 19

    Brody, E.N. et al. The use of aptamers in large arrays for molecular diagnostics. Mol. Diagn. 4, 381–388 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Li, M. Applications of display technology in protein analysis. Nat. Biotechnol. 18, 1251–1256 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Bonner, J.C. & Osornio-Vargas, A.R. Differential binding and regulation of platelet-derived growth factor A and B chain isoforms by α2-macroglobulin. J. Biol. Chem. 27, 16236–16242 (1995).

    Article  Google Scholar 

  22. 22

    Thyberg, J., Ostman, A., Backstrom, G., Westermark, B. & Heldin, C.H. Localization of platelet-derived growth factor (PDGF) in CHO cells transfected with PDGF A- or B-chain cDNA: retention of PDGF-BB in the endoplasmic reticulum and Golgi complex. J. Cell Sci. 97, 219–229 (1990).

    CAS  PubMed  Google Scholar 

  23. 23

    Oefner, C., D'Arcy, A., Winkler, F.K., Eggimann, B. & Hosang, M. Crystal structure of human platelet-derived growth factor BB. EMBO J. 11, 3921–3926 (1992).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Mats Nilsson and Carl-Henrik Heldin offered valuable comments on the manuscript. Olli Leppänen and Nils-Erik Heldin kindly supplied the PDGF β-receptor fragment and the SW-1736 cell line, respectively. Frida Berg contributed to the connector oligonucleotide studies. S.M.G. was funded by a Norfa stipend. The work was supported by the Beijer and Wallenberg Foundations, the Technical and Medical Research Councils of Sweden, the Swedish Cancer Fund, and by Polysaccharide Research AB (Uppsala).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Ulf Landegren.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fredriksson, S., Gullberg, M., Jarvius, J. et al. Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20, 473–477 (2002). https://doi.org/10.1038/nbt0502-473

Download citation

Further reading

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