School of Pharmacy, University of Nottingham, Nottingham, UK.
School of Science and Technology, Nottingham Trent University, Nottingham, UK.
Methods Mol Biol. 2024;2723:55-68. doi: 10.1007/978-1-0716-3481-3_4.
Deadenylase enzymes play a key role in mRNA degradation and RNA processing. In this chapter, we describe two activity assays for the quantitative biochemical analysis of deadenylase enzymes, which can easily be adapted for other nuclease enzymes. The assays use distinct principles of detection, which are based on differential annealing of a probe complementary to the substrate RNA or detection of adenosine monophosphate (AMP). The assays are sensitive, flexible, and can be used in low-throughput tube-based formats and 96-well or 384-well plate formats. The assays rely on plate reader detection and can be carried out using manual pipetting or robotic liquid handling equipment. In addition to two activity assays, we describe differential scanning fluorimetry (thermal shift assay) as a complementary assay that allows the direct characterization of ligand binding to deadenylase enzymes. The assays can be useful for the characterization of deadenylase variants and are particularly suitable for the discovery and development of small-molecule inhibitors of deadenylase enzymes.
脱腺苷酶在 mRNA 降解和 RNA 加工中发挥着关键作用。在本章中，我们将描述两种用于定量生化分析脱腺苷酶的活性测定法，这些方法可以轻松适应其他核酸酶。这些测定法使用不同的检测原理，基于与底物 RNA 互补的探针的差异退火或腺苷一磷酸 (AMP) 的检测。这些测定法灵敏、灵活，可用于低通量管格式以及 96 孔或 384 孔板格式。这些测定法依赖于平板读数器检测，可使用手动移液或机器人液体处理设备进行。除了两种活性测定法外，我们还描述了差示扫描荧光法（热位移测定法）作为一种补充测定法，可直接表征配体与脱腺苷酶的结合。这些测定法可用于脱腺苷酶变体的表征，特别适合于脱腺苷酶的小分子抑制剂的发现和开发。
