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

Ali R. Özeş, Kateryna Feoktistova, Brian C. Avanzino, Enoch P. Baldwin, Christopher S. Fraser

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1645-1661
Number of pages17
JournalNature Protocols
Volume9
Issue number7
DOIs
StatePublished - 2014

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Adenosine Triphosphatases
Assays
Fluorescence
Adenosine Triphosphate
RNA
Biosensing Techniques
Oligonucleotides
Nucleic Acids
Hydrolysis
Biosensors
DNA
Enzymes
Kinetics
Proteins

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Özeş, A. R., Feoktistova, K., Avanzino, B. C., Baldwin, E. P., & Fraser, C. S. (2014). Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases. Nature Protocols, 9(7), 1645-1661. https://doi.org/10.1038/nprot.2014.112

Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases. / Özeş, Ali R.; Feoktistova, Kateryna; Avanzino, Brian C.; Baldwin, Enoch P.; Fraser, Christopher S.

In: Nature Protocols, Vol. 9, No. 7, 2014, p. 1645-1661.

Research output: Contribution to journalArticle

Özeş, AR, Feoktistova, K, Avanzino, BC, Baldwin, EP & Fraser, CS 2014, 'Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases', Nature Protocols, vol. 9, no. 7, pp. 1645-1661. https://doi.org/10.1038/nprot.2014.112
Özeş, Ali R. ; Feoktistova, Kateryna ; Avanzino, Brian C. ; Baldwin, Enoch P. ; Fraser, Christopher S. / Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases. In: Nature Protocols. 2014 ; Vol. 9, No. 7. pp. 1645-1661.
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