ArticleNature protocols2014
Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases.
Article in Nature protocols, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed, 53 citations in OpenAlex.
- Nonequilibrium phases of a biomolecular condensate facilitated by enzyme activity.Soft matter · 2026Article
- Assembly of a homohexameric minichromosome maintenance complex is dependent on ATP and DNA.The Journal of biological chemistry · 2026Article
- HELZ2 RegulatesCirculation · 2026Article
- Structure, Function and Inhibition of Helicases Involved in Virus Infection.Biomolecules · 2026Review
- Chemical Probe Discovery for DEAD-Box RNA-Binding Protein DDX21 Using Small-Molecule Microarrays.ACS chemical biology · 2025Article
- Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria.Nucleic acids research · 2024Article
- A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes.Biochemistry and biophysics reports · 2023Article
- Coronaviruses SARS-CoV, MERS-CoV, and SARS-CoV-2 helicase inhibitors: a systematic review ofJournal of virus eradication · 2023Review
- Extended DNA threading through a dual-engine motor module of the activating signal co-integrator 1 complex.Nature communications · 2023Article
- Measuring the impact of cofactors on RNA helicase activities.Methods (San Diego, Calif.) · 2022Article
- Insight into the biochemical mechanism of DNA helicases provided by bulk-phase and single-molecule assays.Methods (San Diego, Calif.) · 2022Review
- Structural basis for RNA surveillance by the human nuclear exosome targeting (NEXT) complex.Cell · 2022Article
- The stalk domain of SARS-CoV-2 NSP13 is essential for its helicase activity.Biochemical and biophysical research communications · 2022Article
- Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins.Methods in enzymology · 2022Article
- Methods to assess helicase and translocation activities of human nuclear RNA exosome and RNA adaptor complexes.Methods in enzymology · 2022Article
- Monitoring helicase-catalyzed unwinding of multiple duplexes simultaneously.Methods in enzymology · 2022Article
- Quantitative methods to study helicase, DNA polymerase, and exonuclease coupling during DNA replication.Methods in enzymology · 2022Article
- Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Targeting eIF4A-Dependent Translation of KRAS Signaling Molecules.Cancer research · 2021Article
- A skipping rope translocation mechanism in a widespread family of DNA repair helicases.Nucleic acids research · 2021Article
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
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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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.