Malmqvist Tony, Spickett Carl, Gallo Jean-Marc, Anthony Karen
Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, 125 Coldharbour Lane, London SE5 9NU, UK.
Atlas Antibodies, Voltavägen 13 A, 16869 Bromma, Sweden.
Biol Methods Protoc. 2017 Jun 5;2(1):bpx009. doi: 10.1093/biomethods/bpx009. eCollection 2017 Jan.
Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA-protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [γ-P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioisotopes. Here, we describe a modified UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDye®800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the Odyssey Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the efficiency of transcription. End labelling is also independent of the length of the probe, thus facilitating quantitative comparisons. To validate the method, we have confirmed the binding of HuD to the 3'-UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer's disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant purified glutathione--transferase-HuD fusion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA-protein interactions and their emerging importance in disease.
蛋白质与短RNA寡聚物的光交联是研究RNA-蛋白质相互作用的经典方法，这些相互作用涉及RNA代谢和功能的许多方面。最常见的是，这涉及使用[γ-P]标记的RNA探针。尽管非常灵敏，但这些方法因使用放射性同位素带来的安全问题而变得复杂。在此，我们描述了一种改良的紫外线交联方法，该方法使用末端标记有红外染料IRDye®800的寡核苷酸探针。紫外线交联后，通过SDS-PAGE分离蛋白质，并用奥德赛红外成像系统观察交联产物。这种末端标记方法为随机标记提供了一种简化的替代方法，随机标记会降低转录效率。末端标记也与探针长度无关，因此便于进行定量比较。为了验证该方法，我们证实了HuD与微管相关蛋白tau的mRNA的3'-UTR结合，tau蛋白与阿尔茨海默病的发病机制有关。使用重组纯化的谷胱甘肽转移酶-HuD融合蛋白以及用HuD cDNA转染的CHO细胞裂解物成功地进行了HuD与标记的21聚体探针的紫外线交联。紫外线交联与红外成像相结合，为分析RNA-蛋白质相互作用及其在疾病中的新重要性提供了一种方便且可靠的策略。
