European Biophysics Journal

, 40:1121 | Cite as

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

  • Emma L. Gilroy
  • Søren Vrønning Hoffmann
  • Nykola C. Jones
  • Alison RodgerEmail author
Original Paper


The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)2], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)2] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100 mA beam current of 0.5-μW power varied from 0.6 h−1 for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)2] and high salt poly[(dA-dT)2]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.


YOYO-1 Linear dichroism Synchrotron radiation DNA cleavage Sequence dependence 





Calf thymus DNA




Dimethyl sulfoxide




Linear dichroism


Micrococcus luteus DNA





Support for this work was given by the Engineering and Physical Sciences Research Council, UK (EP/D075750/1), and from the Integrated Infrastructure Initiative (I3), European Light Sources Activities (ELISA), grant agreement no. 226716, under the Research Infrastructure Action of the FP7 EC programme Structuring the European Research Area


  1. Akerman B, Tuite E (1996) Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO. Nucleic Acids Res 24(6):1080–1090PubMedCrossRefGoogle Scholar
  2. Cosa G, Focsaneanu KS et al (2001) Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution. Photochem Photobiol 73(6):585–599PubMedCrossRefGoogle Scholar
  3. Dicko C, Hicks MR et al (2008) Breaking the 200 nm limit for routine flow linear dichroism measurements using UV synchrotron radiation. Biophys J 95(12):5974–5977PubMedCrossRefGoogle Scholar
  4. Eriksson M, Karlsson HJ et al (2003) Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels. Nucleic Acids Res 31(21):6235–6242PubMedCrossRefGoogle Scholar
  5. Flyvbjerg H, Keatch SA et al (2006) Strong physical constraints on sequence-specific target location by proteins on DNA molecules. Nucleic Acids Res 34(9):2550–2557PubMedCrossRefGoogle Scholar
  6. Furstenberg A, Deligeorgiev TG et al (2007) Structure-fluorescence contrast relationship in cyanine DNA intercalators: toward rational dye design. Chemistry 13(30):8600–8609PubMedCrossRefGoogle Scholar
  7. Geron-Landre B, Roulon T et al (2003) Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level. Nucleic Acids Res 31(20):e125PubMedCrossRefGoogle Scholar
  8. Hicks MR, Rodger A et al (2006) Restriction enzyme kinetics monitored by UV linear dichroism. Biochemistry 45(29):8912–8917PubMedCrossRefGoogle Scholar
  9. Hicks MR, Dafforn TR et al (2009) Synchrotron radiation linear dichroism spectroscopy of the antibiotic peptide gramicidin in lipid membranes. Analyst 134(8):1623–1628PubMedCrossRefGoogle Scholar
  10. Keatch SA, Su TJ et al (2004) Alleviation of restriction by DNA condensation and non-specific DNA binding ligands. Nucleic Acids Res 32(19):5841–5850PubMedCrossRefGoogle Scholar
  11. Larsson A, Carlsson C et al (1994) Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarised light spectroscopy. J Am Chem Soc 116:8459–8465CrossRefGoogle Scholar
  12. Li B, Hu J et al (2003) Real time observation of the photocleavage of single DNA molecules. Chin Sci Bull 48:673–675CrossRefGoogle Scholar
  13. Marrington R, Dafforn TR et al (2004) Micro-volume couette flow sample orientation for absorbance and fluorescence linear dichroism. Biophys J 87(3):2002–2012PubMedCrossRefGoogle Scholar
  14. Marrington R, Dafforn TR et al (2005) Validation of new microvolume couette flow linear dichroism cells. Analyst 130(12):1608–1616PubMedCrossRefGoogle Scholar
  15. Marshall KE, Hicks MR et al (2010) Characterizing the assembly of the Sup35 yeast prion fragment, GNNQQNY: structural changes accompany a fiber-to-crystal switch. Biophys J 98(2):330–338PubMedCrossRefGoogle Scholar
  16. Netzel TL, Nafisi K et al (1995) Base-content dependence of emission enhancements, quantum yields, and lifetimes for cyanine dyes bound to double-strand DNA: photophysical properties of monomeric and bichromophoric DNA stains. J Phys Chem 99:12Google Scholar
  17. Nordén B, Rodger A et al (2010) Linear dichroism and circular dichroism: a textbook on polarized spectroscopy. Royal Society of Chemistry, CambridgeGoogle Scholar
  18. Rittman M, Hoffmann SV et al (2010) Probing the structure of long DNA molecules in solution using synchrotron radiation linear dichroism. Awaiting publicationGoogle Scholar
  19. Sischka A, Toensing K et al (2005) Molecular mechanisms and kinetics between DNA and DNA binding ligands. Biophys J 88(1):404–411PubMedCrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2011

Authors and Affiliations

  • Emma L. Gilroy
    • 1
  • Søren Vrønning Hoffmann
    • 2
  • Nykola C. Jones
    • 2
  • Alison Rodger
    • 1
    Email author
  1. 1.Department of Chemistry and Warwick Centre for Analytical ScienceUniversity of WarwickCoventryUK
  2. 2.Institute for Storage Ring Facilities (ISA)Aarhus UniversityAarhus CDenmark

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