European Biophysics Journal

, Volume 46, Issue 6, pp 561–566 | Cite as

Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy

  • Ying Wang
  • Helene Schellenberg
  • Volker Walhorn
  • Katja Toensing
  • Dario AnselmettiEmail author
Original Article


Fluorescent dyes are broadly used in many biotechnological applications to detect and visualize DNA molecules. However, their binding to DNA alters the structural and nanomechanical properties of DNA and, thus, interferes with associated biological processes. In this work we employed magnetic tweezers and fluorescence spectroscopy to investigate the binding of PicoGreen to DNA at room temperature in a concentration-dependent manner. PicoGreen is an ultrasensitive quinolinium nucleic acid stain exhibiting hardly any background signal from unbound dye molecules. By means of stretching and overwinding single, torsionally constrained, nick-free double-stranded DNA molecules, we acquired force-extension and supercoiling curves which allow quantifying DNA contour length, persistence length and other thermodynamical binding parameters, respectively. The results of our magnetic tweezers single-molecule binding study were well supported through analyzing the fluorescent spectra of stained DNA. On the basis of our work, we could identify a concentration-dependent bimodal binding behavior, where, apparently, PicoGreen associates to DNA as an intercalator and minor-groove binder simultaneously.


PicoGreen DNA Magnetic tweezers Intercalator Minor-groove binder 



We gratefully acknowledge technical support from Christoph Pelargus.


  1. Bouchiat C, Wang MD, Allemand JF, Strick T, Block SM, Croquette V (1999) Estimating the persistence length of a worm-like chain molecule from force-extension measurements. Biophys J 76(1):409–413. doi: 10.1016/S0006-3495(99)77207-3 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Cheng W (2006) Protocol to generate half Lambda DNA for optical/magnetic tweezer. Accessed 5 Sept 2008
  3. Crothers DM (1968) Calculation of binding isotherms for heterogenous polymers. Biopolymers 6(4):575–584. doi: 10.1002/bip.1968.360060411 CrossRefPubMedGoogle Scholar
  4. Dragan AI, Casas-Finet JR, Bishop ES, Strouse RJ, Schenerman MA, Geddes CD (2010) Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding. Biophys J 99(9):3010–3019. doi: 10.1016/j.bpj.2010.09.012 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Glaser T, Fischer von Mollard G, Anselmetti D (2016) Rational design of dinuclear complexes binding at two neighboring phosphate esters of DNA. Inorg Chim Acta 452:62–72. doi: 10.1016/j.ica.2016.02.013 CrossRefGoogle Scholar
  6. Günther K, Mertig M, Seidel R (2010) Mechanical and structural properties of YOYO-1 complexed DNA. Nucleic Acids Res 38(19):6526–6532. doi: 10.1093/nar/gkq434 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Hayashi M, Harada Y (2007) Direct observation of the reversible unwinding of a single DNA molecule caused by the intercalation of ethidium bromide. Nucleic Acids Res 35(19):e125. doi: 10.1093/nar/gkm529 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Jany T, Moreth A, Gruschka C, Sischka A, Spiering A, Dieding M, Wang Y, Haji Samo S, Stammler A, Bögge H, Fischer von Mollard G, Anselmetti D, Glaser T (2015) Rational design of a cytotoxic dinuclear Cu2 complex that binds by molecular recognition at two neighboring phosphates of the DNA backbone. Inorg Chem 54(6):2679–2690. doi: 10.1021/ic5028465 CrossRefPubMedGoogle Scholar
  9. Japaridze A, Benke A, Renevey S, Benadiba C, Dietler G (2015) Influence of DNA binding dyes on bare DNA structure studied with atomic force microscopy. Macromolecules 48(6):1860–1865. doi: 10.1021/ma502537g CrossRefGoogle Scholar
  10. Kleimann C, Sischka A, Spiering A, Tonsing K, Sewald N, Diederichsen U, Anselmetti D (2009) Binding kinetics of bisintercalator Triostin a with optical tweezers force mechanics. Biophys J 97(10):2780–2784. doi: 10.1016/j.bpj.2009.09.001 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Lipfert J, Klijnhout S, Dekker NH (2010) Torsional sensing of small-molecule binding using magnetic tweezers. Nucleic Acids Res 38(20):7122–7132. doi: 10.1093/nar/gkq598 CrossRefPubMedPubMedCentralGoogle Scholar
  12. McGhee JD, von Hippel PH (1974) Theoretical aspects of DNA-protein interactions. Co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. J Mol Biol 86(2):469–489. doi: 10.1016/0022-2836(74)90031-X CrossRefPubMedGoogle Scholar
  13. Singer VL, Jones LJ, Yue ST, Haugland RP (1997) Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal Biochem 249(2):228–238. doi: 10.1006/abio.1997.2177 CrossRefPubMedGoogle Scholar
  14. Smith S, Finzi L, Bustamante C (1992) Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 258(5085):1122–1126. doi: 10.1126/science.1439819 CrossRefPubMedGoogle Scholar
  15. Strick TR, Allemand JF, Bensimon D, Bensimon A, Croquette V (1996) The elasticity of a single supercoiled DNA molecule. Science 271(5257):1835–1837. doi: 10.1126/science.271.5257.1835 CrossRefPubMedGoogle Scholar
  16. Strick TR, Allemand JF, Bensimon D, Croquette V (1998) Behavior of Supercoiled DNA. Biophys J 74(4):2016–2028. doi: 10.1016/S0006-3495(98)77908-1 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Vilfan ID, Lipfert J, Koster DA, Lemay SG, Dekker NH (2009) Magnetic tweezers for single-molecule experiments. In: Hinterdorfer P, van Oijen A (eds) Handbook of single-molecule biophysics, Springer, New YorkGoogle Scholar
  18. Vladescu ID, McCauley MJ, Nunez ME, Rouzina I, Williams MC (2007) Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching. Nat Methods 4(6):517–522. doi: 10.1038/nmeth1044 CrossRefPubMedGoogle Scholar
  19. Wang Y, Sischka A, Walhorn V, Tönsing K, Anselmetti D (2016) Nanomechanics of fluorescent DNA dyes on DNA investigated by magnetic tweezers. Biophys J 111(8):1604–1611. doi: 10.1016/j.bpj.2016.08.042 CrossRefPubMedGoogle Scholar
  20. Williams LD, Egli M, Gao Q, Rich A (1992) Structure & Function: Proceedings of the 7th Conversation in Biomolecular Stereodynamics. In: Sarma RH, Sarma MH (eds) vol 1. Adenine Press, New York, pp 107–125Google Scholar
  21. Zipper H, Brunner H, Bernhagen J, Vitzthum F (2004) Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res 32(12):e103. doi: 10.1093/nar/gnh101 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2017

Authors and Affiliations

  1. 1.Experimental Biophysics and Applied Nanoscience, Physics Department, Physics Faculty, Bielefeld Institute for Nanoscience (BINAS)Bielefeld UniversityBielefeldGermany

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