Özeş Ali R, Feoktistova Kateryna, Avanzino Brian C, Baldwin Enoch P, Fraser Christopher S
1] Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California, USA. [2].
Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California, USA.
Nat Protoc. 2014 Jul;9(7):1645-61. doi: 10.1038/nprot.2014.112. Epub 2014 Jun 19.
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.
解旋酶的许多生理功能取决于它们以ATP依赖方式解开核酸双链体的能力。确定这些过程的动力学框架对于理解这些蛋白质的功能至关重要。我们最近开发了一种荧光测定法，用于实时监测DEAD盒解旋酶解开RNA双链体的过程。在该测定法中，将两个荧光修饰的短报告寡核苷酸与任意长度的未修饰RNA加载链退火，使得两个报告分子的荧光部分彼此靠近，荧光被淬灭。一个报告分子用花青素3（Cy3）修饰，而另一个用光谱配对的黑洞猝灭剂（BHQ）修饰。随着解旋酶解开加载链，该酶取代Cy3修饰的报告分子，该报告分子将与捕获或竞争DNA链结合，使其与BHQ修饰的报告分子永久分离。因此，Cy3修饰的报告链的完全分离被检测为总荧光的增加。该测定法与无试剂生物传感器兼容，以监测ATP酶活性，从而可以确定ATP水解与双链体解旋之间的偶联。按照所述方案，获得解旋和ATP酶测定的数据并分析结果大约需要4小时。
