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Fluorescence Resonance Energy Transfer Studies of Structure and Dynamics in Nucleic Acids

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Book cover Biophysics and Structure to Counter Threats and Challenges

Abstract

Fluorescence spectroscopy is highly sensitive, and can be performed on single molecules. Using fluorescence resonance energy transfer (FRET) distances can be estimated in biological macromolecules. This has provided significant structural and dynamic information of DNA and RNA molecules. It has even been able to allow us to observe the catalytic function of a ribozyme in real time.

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Acknowledgements

I thank my co-workers in Dundee over a number of years, especially Tim Wilson, Jo Ouellet, Alastair Murchie and Asif Iqbal, and our collaborators in the University of Illinois Taekjip Ha, Sean McKinney, Sungchul Hohng and Michelle Nahas. We thank Cancer Research UK for financial support.

Note Added in Proof

In the 2 years since this article was written there has been significant progress in the analysis of orientation effects using the cyanine fluorophores attached to double-stranded nucleic acids. It was found that the orientation effects were not lost when Cy3 and Cy5 were attached by long, 13-atom tethers, suggesting that these fluorophores possess an intrinsic tendency to undergo terminal stacking (A). However, repeating the experiment with a series of DNA duplexes of varying length led to a phase shift (e.g. compared to the data shown in Fig. 5.3) that was interpreted in terms of a reorientation of each fluorophore by 30∘, so that the transition moment lay parallel to the long axis of the terminal basepair. This was subsequently confirmed in NMR studies (B), providing proof of principle that such FRET data can provide reliable orientational information in nucleic acids.

(A)Ouellet J, Schorr S, Iqbal A, Wilson TJ, Lilley DMJ (2011) Orientation of cyanine fluorophoresterminally attached to DNA via long, flexible tethers. Biophys J 101:1148–1154

(B) Urnavicius L, McPhee SA, Lilley DMJ, Norman DG (2012) The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether. Biophys J 102:561–569

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Authors and Affiliations

  1. Cancer Research UK Nucleic Acid Structure Research Group, The University of Dundee, MSI/WTB Complex, Dundee, DD1 5EH, UK

    David M. J. Lilley

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  1. David M. J. Lilley
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Correspondence to David M. J. Lilley .

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Editors and Affiliations

  1. , Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, D105a Fairchild Sciences Building, Stanford, 94305-5126, USA

    Joseph D. Puglisi

  2. , Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Fairchild Science Building D100E, Stanford, 94305-5126, USA

    Manolia V. Margaris

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Lilley, D.M.J. (2012). Fluorescence Resonance Energy Transfer Studies of Structure and Dynamics in Nucleic Acids. In: Puglisi, J., Margaris, M. (eds) Biophysics and Structure to Counter Threats and Challenges. NATO Science for Peace and Security Series B: Physics and Biophysics. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4923-8_5

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  • DOI: https://doi.org/10.1007/978-94-007-4923-8_5

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