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Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases

Abstract

Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes 4 h.

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Figure 1: Representation of the two fluorescence-based approaches to probing RNA unwinding reactions described in the INTRODUCTION.
Figure 2: Monitoring reporter strand annealing to the loading strand.
Figure 3: Evaluating duplex melting by eIF4A and its accessory proteins with the double-reporter fluorescence unwinding assay.
Figure 4: Determining the rate of phosphate release during duplex unwinding.

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Acknowledgements

We thank B. Rad and S. Kowalczykowski and members of the Kowalczykowski laboratory for expert help and advice with developing this protocol. We also thank J. Hershey for his many insightful comments throughout the protocol development. We gratefully acknowledge M. Webb for his generous gift of PBP-MDCC and A. Do and E. Tuvshintogs for expert technical assistance. This work was supported by the US National Institutes of Health (NIH) through an NIHR01 grant (R01GM092927; to C.S.F.), an NIH training grant (T32 GM007377; to K.F.) and an American Heart Association and Myocarditis Foundation predoctoral fellowship (B.C.A.).

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A.R.O., K.F., B.C.A., E.P.B. and C.S.F. designed the study. A.R.O., K.F. and B.C.A. performed the experiments. A.R.O., K.F., B.C.A., E.P.B. and C.S.F. wrote the manuscript.

Corresponding author

Correspondence to Christopher S Fraser.

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Özeş, A., Feoktistova, K., Avanzino, B. et al. Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases. Nat Protoc 9, 1645–1661 (2014). https://doi.org/10.1038/nprot.2014.112

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