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BioNanoScience

, 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
Article
  • 180 Downloads

Abstract

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.

Keywords

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

Notes

Acknowledgments

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.

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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

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