Protein detection using proximity-dependent DNA ligation assays

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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.

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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).


  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).

  2. 2

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

  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).

  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).

  5. 5

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

  6. 6

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

  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).

  8. 8

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

  9. 9

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

  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).

  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).

  12. 12

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

  13. 13

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

  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).

  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).

  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).

  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).

  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).

  19. 19

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

  20. 20

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

  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).

  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).

  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).

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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).

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Correspondence to Ulf Landegren.

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