, Volume 7, Issue 1, pp 132–147 | Cite as

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

  • Tatiana A. Nevzorova
  • Qingze Zhao
  • Yakov A. Lomakin
  • Anastasia A. Ponomareva
  • Alexander R. Mukhitov
  • Prashant K. Purohit
  • John W. Weisel
  • Rustem I. LitvinovEmail author


Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibody-coated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface. After careful discrimination of non-specific interactions, we showed that the DNA-antibody rupture force spectra had two regimes, reflecting formation of weaker (20–40 pN) and stronger (>40 pN) immune complexes that implies the existence of at least two bound states with different mechanical stability. The two-dimensional force-free off-rate for the DNA-antibody complexes was ∼2.2 × 10−3 s−1, the transition state distance was ∼0.94 nm, the apparent on-rate was ∼5.26 s−1, and the stiffness of the DNA-antibody complex was characterized by a spring constant of 0.0021 pN/nm, suggesting that the DNA-antibody complex is a relatively stable, but soft and deformable macromolecular structure. The stretching elasticity of the DNA molecules was characteristic of single-stranded DNA, suggesting preferential binding of the MRL4 antibody to one strand of DNA. Collectively, the results provide fundamental characteristics of formation and forced dissociation of DNA-antibody complexes that help to understand principles of DNA-protein interactions and shed light on the molecular basis of autoimmune diseases accompanied by formation of anti-DNA antibodies.


DNA Anti-DNA antibody Single-molecule force spectroscopy Optical trap Two-dimensional kinetics Nanomechanics 



This work was supported by NIH grants U01-HL116330, R56 HL090774 and PO1-HL110860, NSF grant DMR 1505662, and the Program for Competitive Growth at Kazan Federal University.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Pisetsky, D. S. (1998). Antibody responses to DNA in normal immunity and aberrant immunity. Clinical and Diagnostic Laboratory Immunology, 5, 1–6.Google Scholar
  2. 2.
    Williams, W. M., & Isenberg, D. A. (1996). A cross-sectional study of anti-DNA antibodies in the serum and IgG and IgM fraction of healthy individuals, patients with systemic lupus erythematosus and their relatives. Lupus, 5, 576–586.CrossRefGoogle Scholar
  3. 3.
    Uchida, K. (2014). Natural antibodies as a sensor of electronegative damage-associated molecular patterns (DAMPs). Free Radical Biology and Medicine, 72, 156–161.CrossRefGoogle Scholar
  4. 4.
    Lora, V., Bonaguri, C., Gisondi, P., Sandei, F., Battistelli, L., Russo, A., et al. (2013). Autoantibody induction and adipokine levels in patients with psoriasis treated with infliximab. Immunologic Research, 56, 382–389.CrossRefGoogle Scholar
  5. 5.
    Quan, C. P., Watanabe, S., Pamonsinlapatham, P., Bouvet, J.-P. (2001). Different dysregulations of the natural antibody repertoire in treated and untreated HIV-1 patients. Journal of Autoimmunity, 17, 81–87.CrossRefGoogle Scholar
  6. 6.
    Williamson, R. A., Burgoon, M. P., Owens, G. P., Ghausi, O., Leclerc, E., Firme, L., et al. (2001). Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis. Proceedings of the National Academy of Sciences of the United States of America, 98, 1793–1798.CrossRefGoogle Scholar
  7. 7.
    Compagno, M., Rekvig, O. P., Bengtsson, A. A., Sturfelt, G., Heegaard, N. H., Jönsen, A., et al. (2014). Clinical phenotype associations with various types of anti-dsDNA antibodies in patients with recent onset of rheumatic symptoms. Results from a multicentre observational study. Lupus Science and Medicine, 1, e000007.CrossRefGoogle Scholar
  8. 8.
    Gorny, M. K., Lawniczak, M., Jenek, R., Słowik-Gabryelska, A., Kaczmarek, E., Zeromski, J. (1988). Alloantibodies, autoantibodies, and immune complexes in patients with lung cancer. Lung, 166, 97–105.CrossRefGoogle Scholar
  9. 9.
    Barbas, S. M., Ditzel, H. J., Saloneh, E. M., Yang, W.-P., Silverman, G. J., Burton, D. R. (1995). Human autoantibody recognition of DNA. Proceedings of the National Academy of Sciences of the United States of America, 92, 2529–2533.CrossRefGoogle Scholar
  10. 10.
    Suenaga, R., & Abdou, N. I. (1993). Cationic and high affinity serum IgG anti-dsDNA antibodies in active lupus nephritis. Clinical and Experimental Immunology, 94, 418–422.CrossRefGoogle Scholar
  11. 11.
    Deocharan, B., Qing, X., Beger, E., Putterman, C. (2002). Antigenic triggers and molecular targets for anti-double-stranded DNA antibodies. Lupus, 11, 865–871.CrossRefGoogle Scholar
  12. 12.
    Madaio, M. P., & Yanase, K. (1998). Cellular penetration and nuclear localization of anti-DNA antibodies: mechanisms, consequences, implications and applications. Journal of Autoimmunity, 11, 535–538.CrossRefGoogle Scholar
  13. 13.
    Putterman, C. (2004). New approaches to the renal pathogenicity of anti-DNA antibodies in systemic lupus erythematosus. Autoimmunity Reviews, 3, 7–11.CrossRefGoogle Scholar
  14. 14.
    Ullala, A. J., Marion, T. N., Pisetsky, D. S. (2014). The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells. Clinical Immunology, 154, 178–187.CrossRefGoogle Scholar
  15. 15.
    Foster, M. H., Kieber-Emmons, T., Ohliger, M., Madaio, M. P. (1994). Molecular and structural analysis of nuclear localizing anti-DNA lupus antibodies. Immunologic Research, 13, 186–206.CrossRefGoogle Scholar
  16. 16.
    Aranow C, Zhou D, Diamond B (2011) Anti-DNA antibodies: structure, regulation and pathogenicity. In: Lahita RG, Tsokos G, Buyon JP, Koike T (eds.) Systemic lupus erythematosus, Fifth Ed. Academic Press, pp 235–258.Google Scholar
  17. 17.
    Shlomchik, M., Mascelli, M., Shan, H., Radic, M. Z., Pisetsky, D., Marshak-Rothstein, A., et al. (1990). Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation. Journal of Experimental Medicine, 171, 265–292.CrossRefGoogle Scholar
  18. 18.
    Uccellini, M. B., Busto, P., Debatis, M., Marshak-Rothstein, A., Viglianti, G. A. (2012). Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence. Immunology Letters, 143, 85–91.CrossRefGoogle Scholar
  19. 19.
    Jin, H., Sepulveda, J., Burrone, O. R. (2004). Specific recognition of a dsDNA sequence motif by an immunoglobulin VH homodimer. Protein Science, 13, 3222–3229.CrossRefGoogle Scholar
  20. 20.
    Eivazova, E. R., McDonnell, J. M., Sutton, B. J., Staines, N. A. (2000). Specificity and binding kinetics of murine lupus anti-DNA monoclonal antibodies implicate different stimuli for their production. Immunology, 101, 371–377.CrossRefGoogle Scholar
  21. 21.
    Chaurasiya, K. R., Paramanathan, T., McCauley, M. J., Williams, M. C. (2010). Biophysical characterization of DNA binding from single molecule force measurements. Physics of Life Reviews, 7, 299–341.CrossRefGoogle Scholar
  22. 22.
    Noy, A. (2011). Force spectroscopy 101: how to design, perform, and analyze an AFM-based single molecule force spectroscopy experiment. Current Opinion in Chemical Biology, 15, 710–718.CrossRefGoogle Scholar
  23. 23.
    Rocha, M. S. (2015). Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments. Integrative Biology (Camb), 7, 967–986.CrossRefGoogle Scholar
  24. 24.
    Chung, J. W., Shin, D., Kwak, J. M., Seog, J. (2013). Direct force measurement of single DNA-peptide interactions using atomic force microscopy. Journal of Molecular Recognition, 26, 268–275.CrossRefGoogle Scholar
  25. 25.
    Bartels, F., McIntosh, M., Fuhrmann, A., Metzendorf, C., Plattner, P., Sewald, N., et al. (2007). Effector-stimulated single molecule protein–DNA interactions of a quorum-sensing system in Sinorhizobium meliloti. Biophysical Journal, 92, 4391–4400.CrossRefGoogle Scholar
  26. 26.
    Wollschlager, K., Gaus, K., Kornig, A., Eckel, R., Wilking, S., McIntosh, M., et al. (2009). Single-molecule experiments to elucidate the minimal requirement for DNA recognition by transcription factor epitopes. Small, 5, 484–495.CrossRefGoogle Scholar
  27. 27.
    Koch, S., & Wang, M. (2003). Dynamic force spectroscopy of protein-DNA interactions by unzipping DNA. Physical Review Letters, 91, 028103.CrossRefGoogle Scholar
  28. 28.
    Krasnoslobodtsev, A. V., Shlyakhtenko, L. S., Lyubchenko, Y. L. (2007). Probing interactions within the synaptic DNA–SfiI complex by AFM force spectroscopy. Journal of Molecular Biology, 365, 1407–1416.CrossRefGoogle Scholar
  29. 29.
    Sanchez, H., Suzuki, Y., Yokokawa, M., Takeyasu, K., Wyman, C. (2011). Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54. Integrative Biology, 3, 1127–1134.CrossRefGoogle Scholar
  30. 30.
    Lionnet, T., Allemand, J. F., Revyakin, A., Strick, T. R., Saleh, O. A., Bensimon, D., et al. (2012). Single-molecule studies using magnetic traps. Cold Spring Harbor Protocols, 2012, 34–49.Google Scholar
  31. 31.
    Limmer, K., Pippig, D. A., Aschenbrenner, D., Gaub, H. E. (2014). A force-based, parallel assay for the quantification of protein-DNA interactions. PloS One, 9, e89626.CrossRefGoogle Scholar
  32. 32.
    Kofler, R., Strohal, R., Balderas, R. S., Johnson, M. E., Noonan, D. J., Duchosal, M. A., et al. (1988). Immunoglobulin kappa light chain variable region gene complex organization and immunoglobulin genes encoding anti-DNA autoantibodies in lupus mice. Journal of Clinical Investigation, 82, 852–860.CrossRefGoogle Scholar
  33. 33.
    Litvinov, R. I., Gorkun, O. V., Owen, S. F., Shuman, H., Weisel, J. W. (2005). Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the single-molecule level. Blood, 106, 2944–2951.CrossRefGoogle Scholar
  34. 34.
    Litvinov, R. I., Yarovoi, S. V., Rauova, L., Barsegov, V., Sachais, B. S., Rux, A. H., et al. (2013). Distinct specificity and single-molecule kinetics characterize the interaction of pathogenic and non-pathogenic antibodies against platelet factor 4-heparin complexes with platelet factor 4. Journal of Biological Chemistry, 288, 33060–33070.CrossRefGoogle Scholar
  35. 35.
    Litvinov, R. I., Shuman, H., Bennett, J. S., Weisel, J. W. (2002). Binding strength and activation state of single fibrinogen-integrin pairs on living cells. Proceedings of the National Academy of Sciences of the United States of America, 99, 7426–7431.CrossRefGoogle Scholar
  36. 36.
    Litvinov, R. I., Mekler, A., Shuman, H., Bennett, J. S., Barsegov, V., Weisel, J. W. (2012). Resolving two-dimensional kinetics of the integrin αIIbβ3-fibrinogen interactions using binding-unbinding correlation spectroscopy. Journal of Biological Chemistry, 287, 35275–35285.CrossRefGoogle Scholar
  37. 37.
    Evans, E., & Ritchie, K. (1997). Dynamic strength of molecular adhesion bonds. Biophysical Journal, 72, 1541–1555.CrossRefGoogle Scholar
  38. 38.
    Tees, D. F. J., Woodward, J. T., IV, Hammer, D. A. (2001). Reliability theory for receptor-ligand bond dissociation. Journal of Chemical Physics, 114, 7483–7496.CrossRefGoogle Scholar
  39. 39.
    Bell, G. I. (1978). Models for the specific adhesion of cells to cells. Science, 200, 618–627.CrossRefGoogle Scholar
  40. 40.
    Bura, E., Zhmurov, A., Barsegov, V. (2009). Nonparametric density estimation and optimal bandwidth selection for protein unfolding and unbinding data. Journal of Chemical Physics, 130, 015102.CrossRefGoogle Scholar
  41. 41.
    Nelson P (2004) Biological physics. In: Freeman WH and company, New York.Google Scholar
  42. 42.
    Zhang, Y., Zhou, H., Ou-Yang, Z. C. (2001). Stretching single-stranded DNA: interplay of electrostatic, base-pairing, and base-pair stacking interactions. Biophysical Journal, 81, 1133–1143.CrossRefGoogle Scholar
  43. 43.
    Jorgensen, M. H., Rekvig, O. P., Jacobsen, R. S., Jacobsen, S., Fenton, K. A. (2011). Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus. Immunology Letters, 138, 179–186.CrossRefGoogle Scholar
  44. 44.
    Spatz, L., Iliev, A., Saenko, V., Jones, L., Irigoyen, M., Manheimer-Lory, A., et al. (1997). Studies on the structure, regulation, and pathogenic potential of anti-dsDNA antibodies. Methods, 11, 70–78.CrossRefGoogle Scholar
  45. 45.
    Kozyr, A. V., Kolesnikov, A. V., Khlyntseva, A. E., Bogun, A. G., Savchenko, G. A., Shemyakin, I. G., et al. (2012). Role of structure-based changes due to somatic mutation in highly homologous DNA-binding and DNA-hydrolyzing autoantibodies exemplified by A23P substitution in the VH domain. Autoimmune Diseases, 2012, 683829.CrossRefGoogle Scholar
  46. 46.
    Lomakin, Y. A., Zakharova, M. Y., Stepanov, A. V., Dronina, M. A., Smirnov, I. V., Bobik, T. V., et al. (2014). Heavy-light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation. Molecular Immunology, 62, 305–314.CrossRefGoogle Scholar
  47. 47.
    Pavlovic, M., Kats, A., Cavallo, M., Chen, R., Hartmann, J. X., Shoenfeld, Y. (2010). Pathogenic and epiphenomenal anti-DNA antibodies in SLE. Autoimmune Diseases, 2010, 462841.CrossRefGoogle Scholar
  48. 48.
    Liu, Y. Y., Guthold, M., Snyder, M. J., Lu, H. F. (2015). AFM of self-assembled lambda DNA-histone networks. Colloids and Surfaces, B: Biointerfaces, 134, 17–25.CrossRefGoogle Scholar
  49. 49.
    Nawas, S., Sanches, P., Bodensiek, K., Li, S., Simons, M., Schaap, I. A. (2012). Cell visco-elasticity measured with AFM amp optical trapping at sub-micrometer deformations. PloS One, 7, e45297.CrossRefGoogle Scholar
  50. 50.
    Weisel, J. W., Shuman, H., Litvinov, R. I. (2003). Protein-protein unbinding induced by force: single-molecule studies. Current Opinion in Structural Biology, 13, 227–235.CrossRefGoogle Scholar
  51. 51.
    Goldsmith, H. L., McIntosh, F. A., Shahin, J., Frojmovic, M. M. (2000). Time and force dependence of the rupture of glycoprotein IIb-IIIa-fibrinogen bonds between latex spheres. Biophysical Journal, 78, 1195–1206.CrossRefGoogle Scholar
  52. 52.
    Lee, I., & Marchant, R. E. (2001). Force measurements on the molecular interactions between ligand (RGD) and human platelet αIIbβ3 receptor system. Surface Science, 491, 433–443.CrossRefGoogle Scholar
  53. 53.
    Zhu, C., Long, M., Chesla, S. E., Bongrand, P. (2002). Measuring receptor/ligand interaction at the single-bond level: experimental and interpretative issues. Annals of Biomedical Engineering, 30, 305–314.CrossRefGoogle Scholar
  54. 54.
    Litvinov, R. I., Bennett, J. S., Weisel, J. W., Shuman, H. (2005). Multi-step fibrinogen binding to the integrin αIIbβ3 detected using force spectroscopy. Biophysical Journal, 89, 2824–2834.CrossRefGoogle Scholar
  55. 55.
    Litvinov, R. I., Gorkun, O. V., Galanakis, D. K., Yakovlev, S., Medved, L., Shuman, H., et al. (2007). Polymerization of fibrin: direct observation and quantification of individual B:b knob-hole interactions. Blood, 109, 130–138.CrossRefGoogle Scholar
  56. 56.
    Chilkoti, A., & Stayton, P. S. (1995). Molecular origins of the slow streptavidin-biotin dissociation kinetics. Journal of the American Chemical Society, 117, 10622–10628.CrossRefGoogle Scholar
  57. 57.
    Poongavanam, M.-V., Kisley, L., Kourentzi, K., Landes, C. F., Willson, R. C. (2016). Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer-IgE interactions. Biochimica et Biophysica Acta, Proteins Proteomics, 1864, 154–164.CrossRefGoogle Scholar
  58. 58.
    Schwesinger, F., Ros, R., Strunz, T., Anselmetti, D., Güntherodt, H. J., Honegger, A., et al. (2000). Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates. Proceedings of the National Academy of Sciences of the United States of America, 97, 9972–9977.CrossRefGoogle Scholar
  59. 59.
    Giuntoli, R. D., Linzer, N. B., Banigan, E. J., Sing, C. E., de la Cruz, M. O., Graham, J. S., et al. (2015). DNA-segment-facilitated dissociation of Fis and NHP6A from DNA detected via single-molecule mechanical response. Journal of Molecular Biology, 427, 3123–3136.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Tatiana A. Nevzorova
    • 1
    • 2
  • Qingze Zhao
    • 3
  • Yakov A. Lomakin
    • 4
  • Anastasia A. Ponomareva
    • 2
    • 5
  • Alexander R. Mukhitov
    • 1
  • Prashant K. Purohit
    • 3
  • John W. Weisel
    • 1
  • Rustem I. Litvinov
    • 1
    • 2
    Email author
  1. 1.Department of Cell and Developmental BiologyUniversity of Pennsylvania School of MedicinePhiladelphiaUSA
  2. 2.Institute of Fundamental Medicine and BiologyKazan Federal UniversityKazanRussian Federation
  3. 3.Department of Mechanical Engineering and Applied MechanicsUniversity of Pennsylvania School of Engineering and Applied SciencePhiladelphiaUSA
  4. 4.Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussian Federation
  5. 5.Kazan Institute of Biochemistry and BiophysicsRussian Academy of SciencesKazanRussian Federation

Personalised recommendations