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SYBR Green I: Fluorescence Properties and Interaction with DNA

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Abstract

In this study, we have investigated the fluorescence properties of SYBR Green I (SG) dye and its interaction with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techniques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence enhancement in complex with DNA can be explained by a dampening of its intra-molecular motions. Analysis of the obtained SG/DNA binding isotherms in solutions of different ionic strength and of SG/DNA association in the presence of a DNA minor groove binder, Hoechst 33258, revealed multiple modes of interaction of SG inner groups with DNA. In addition to interaction within the DNA minor groove, both intercalation between base pairs and stabilization of the electrostatic SG/DNA complex contributed to increased SG affinity to double-stranded DNA. We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100 % glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe. A proposed structural model of the SG/DNA complex is compared and discussed with results recently reported for the closely related PicoGreen chromophore.

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Abbreviations

SG:

SYBR Green I

PG:

PicoGreen

MEF:

Metal-Enhanced Fluorescence

FCS:

Fluorescence Correlation Spectroscopy

FRET:

Fluorescence Resonance Energy Transfer

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Acknowledgments

The authors would like to thank MedImmune for financial support. Support from the Institute of Fluorescence, Department of Chemistry and Biochemistry and UMBC is also gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Fluorescence, University of Maryland Baltimore County, 701 East Pratt Street, Baltimore, MD, 21202, USA

    A. I. Dragan & R. Pavlovic

  2. MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD, 20878, USA

    J. B. McGivney, J. R. Casas-Finet, R. J. Strouse & M. A. Schenerman

  3. Cygnus Technologies, 4705 Southport Supply Road, Southport, NC, 28461, USA

    E. S. Bishop

  4. Institute of Fluorescence and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 701 East Pratt Street, Baltimore, MD, 21202, USA

    C. D. Geddes

Authors
  1. A. I. Dragan
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  2. R. Pavlovic
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  3. J. B. McGivney
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  4. J. R. Casas-Finet
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  5. E. S. Bishop
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  6. R. J. Strouse
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  7. M. A. Schenerman
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  8. C. D. Geddes
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Correspondence to C. D. Geddes.

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Dragan, A.I., Pavlovic, R., McGivney, J.B. et al. SYBR Green I: Fluorescence Properties and Interaction with DNA. J Fluoresc 22, 1189–1199 (2012). https://doi.org/10.1007/s10895-012-1059-8

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  • DOI: https://doi.org/10.1007/s10895-012-1059-8

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