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Temperature-dependent FRET spectroscopy for the high-throughput analysis of self-assembled DNA nanostructures in real time

Nature Protocols volume 4, pages 271–285 (2009)Cite this article

Abstract

We describe a method for the real-time and high-throughput monitoring of the self-assembly and disassembly of complex DNA superstructures, using temperature-dependent Förster resonance energy transfer (FRET) spectroscopy. Compared with other spectroscopic approaches, such as UV-visible and circular dichroism, the method described has advantages in terms of sensitivity, feasibility for high-throughput analysis and applicability to virtually any kind of supramolecular structure. To this end, two oligonucleotides out of the entire set building up the superstructure are labeled with a fluorescein and a tetramethylrhodamine, as FRET donor and acceptor, respectively. Correct assembly of the superstructure induces maximum FRET efficiency, whereas complete dissociation leads to minimal FRET. Monitoring of temperature-dependent FRET efficiency yields a thermal profile that is used for thermodynamic analysis. In the case of reversible and cooperative assembly/disassembly of the DNA superstructure, application of the van't Hoff law allows for the determination of the thermodynamic parameters of the process. Owing to slow temperature ramping, the entire assay requires about 17 h. The protocol allows to simultaneously analyze up to 384 samples with only 30 μl sample volume each.

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Figure 1
Figure 2: Characterization of two different 4 × 4 DNA tiles by gel electrophoresis and FRET spectroscopy.
Figure 3: Labeling strategies used for the FRET thermal analysis of the self-assembly of DNA superstructures.
Figure 4: Schematic representation of the one-tile labeling strategy used to monitor the self-assembly of a DNA nanoarray.
Figure 5: Characterization of the A2B3 nanoarray by AFM, using tapping mode in TAEMg 1 ×.
Figure 6: Raw and elaborated FRET-thermal curves obtained from the self-assembly of DNA-superstructures.

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Acknowledgements

Our work has been financially supported by the Zentrum für Angewandte Chemische Genomik, a joint research initiative founded by the European Union and the Ministry of Innovation and Research of the state Northrhine Westfalia. We thank Dr Udo Feldkamp for the design of the DNA structures used in this work and Dr Hendrick Schoeder for his competent support in the setup of the thermocycler. C.M.N. thanks Max-Planck Society for financial support of a Max-Planck Fellow research group at the Max Planck Institute of Molecular Physiology, Dortmund.

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Authors and Affiliations

  1. Technische Universität Dortmund, Fakultät Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, Dortmund, D-44227, Germany

    Barbara Saccà, Rebecca Meyer & Christof M Niemeyer

Authors
  1. Barbara Saccà
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  2. Rebecca Meyer
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  3. Christof M Niemeyer
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Contributions

B.S. designed experiments, analyzed the data, prepared figures and wrote the manuscript; R.M. carried out experiments, analyzed the data and wrote the manuscript; C.M.N. designed and coordinated the research, analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Barbara Saccà or Christof M Niemeyer.

Supplementary information

Supplementary Data 1: Organization of the raw data. (PDF 60 kb)

Supplementary Data 2: Calculation of the FRET efficiency and related parameters for Van´t Hoff analysis. (PDF 73 kb)

41596_2009_BFnprot2008220_MOESM393_ESM.pdf

Supplementary Data 3: Arrhenius plot and determination of the thermodynamic parameters of the thermal process. (PDF 87 kb)

Supplementary Data 4: Temperature scan program of the real-time thermocycler. (PDF 64 kb)

41596_2009_BFnprot2008220_MOESM395_ESM.pdf

Supplementary Table 1: Raw data (fluorescence emission intensity of fluorescein vs. temperature) obtained from a FRET-thermal experiment performed on a 4x4 DNA-tile. (PDF 102 kb)

41596_2009_BFnprot2008220_MOESM396_ESM.pdf

Supplementary Table 2: Temperature dependence of the FRET efficiency E, assembled fraction θ and equilibrium constant Kass for the self-assembly of the single-tile. (PDF 149 kb)

41596_2009_BFnprot2008220_MOESM397_ESM.pdf

Supplementary Table 3: Values of the slope a and intercept b of the Arrhenius plot and thermodynamic parameters obtained from one single experiment of FRET-thermal self-assembly of the DNA-nanostructure. (PDF 70 kb)

Supplementary Fig. 1: Temperature program used for the assembly/disassembly of the DNA-superstructures. (PDF 51 kb)

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Saccà, B., Meyer, R. & Niemeyer, C. Temperature-dependent FRET spectroscopy for the high-throughput analysis of self-assembled DNA nanostructures in real time. Nat Protoc 4, 271–285 (2009). https://doi.org/10.1038/nprot.2008.220

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