![]() ![]() ![]() Analysis of cofactor effects on RNA helicases. ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding. Using fluorophore-labeled oligonucleotides to measure affinities of protein-DNA interactions. Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability. Analysis of duplex unwinding by RNA helicases using stopped-flow fluorescence spectroscopy. Identification and analysis of inhibitors targeting the hepatitis C virus NS3 helicase. Hanson, A.M., Hernandez, J.J., Shadrick, W.R. Molecular beacons: probes that fluoresce upon hybridization. The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities. Monitoring helicase activity with molecular beacons. Spectrophotometric assay for enzyme-mediated unwinding of double-stranded DNA. RecQ helicase-catalyzed DNA unwinding detected by fluorescence resonance energy transfer. Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer. Fluorescence methods to study DNA translocation and unwinding kinetics by nucleic acid motors. Visualizing protein-DNA interactions at the single-molecule level. Insight into helicase mechanism and function revealed through single-molecule approaches. Fluorescence tools to measure helicase activity in real time. ![]() Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B. Ozes, A.R., Feoktistova, K., Avanzino, B.C. Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro. RNA helicases at work: binding and rearranging. Structure and mechanism of helicases and nucleic acid translocases. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h.įairmanWilliams, M.E., Guenther, U.P. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. Complete separation of the C圓-modified reporter strand is thus detected as an increase in total fluorescence. As the helicase unwinds the loading strand, the enzyme displaces the C圓-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. One reporter is modified with cyanine 3 (C圓), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). 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. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. DeltaGraph has advanced as well, and the current version has more types of charts and graphs than any other application, including its direct competitor CA-Cricket Graph III 1.5 (Computer Associates).ĭeltaGraph Pro 3 has moved beyond charting and graphing, and it now competes with presentation programs such as PowerPoint 3.0 (Microsoft), Aldus Persuasion 2.1, More 3.1 (Symmantec), and Clarislmpact (Claris).Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Current versions of Excel and other Macintosh spreadsheet and statistics programs offer charting features that rival those in the original version of DeltaGraph. This endeared DeltaGraph to users who needed Microsoft Excel's data analysis and manipulation features but were dissatisfied with its limited charting capabilities. The first version of DeltaGraph filled an important niche by providing better and more numerous charting options compared with existing spreadsheet and statistics applications. DeltaGraph Pro 3 is the most recent version of the premier Apple Macintosh charting application. ![]()
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