A UV cross-linking method combined with infrared imaging to analyse RNA–protein interactions

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 [c-32 P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioiso-topes. Here, we describe a modified UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDyeV R 800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the Odyssey V R Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the efficiency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facili-tating quantitative comparisons. To validate the method, we have confirmed the binding of HuD to the 3 0 -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-S-transferase–HuD fu-sion 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