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 language | English (US) |
|---|---|
| Pages (from-to) | 1645-1661 |
| Number of pages | 17 |
| Journal | Nature Protocols |
| Volume | 9 |
| Issue number | 7 |
| DOIs | |
| State | Published - 2014 |
Fingerprint
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Medicine(all)
Cite this
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 journal › Article
}
TY - JOUR
T1 - Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases
AU - Özeş, Ali R.
AU - Feoktistova, Kateryna
AU - Avanzino, Brian C.
AU - Baldwin, Enoch P.
AU - Fraser, Christopher S.
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84908211387&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84908211387&partnerID=8YFLogxK
U2 - 10.1038/nprot.2014.112
DO - 10.1038/nprot.2014.112
M3 - Article
C2 - 24945382
AN - SCOPUS:84908211387
VL - 9
SP - 1645
EP - 1661
JO - Nature Protocols
JF - Nature Protocols
SN - 1754-2189
IS - 7
ER -