Cléry Antoine, Sohier Thibault J M, Welte Thomas, Langer Andreas, Allain Frédéric H T
Institute of Molecular Biology and Biophysics, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland.
Institute of Molecular Biology and Biophysics, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland.
Methods. 2017 Apr 15;118-119:137-145. doi: 10.1016/j.ymeth.2017.03.004. Epub 2017 Mar 9.
Characterization of RNA-binding protein interactions with RNA became inevitable to properly understand the cellular mechanisms involved in gene expression regulation. Structural investigations bring information at the atomic level on these interactions and complementary methods such as Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) are commonly used to quantify the affinity of these RNA-protein complexes and evaluate the effect of mutations affecting these interactions. The switchSENSE technology has recently been developed and already successfully used to investigate protein interactions with different types of binding partners (DNA, protein/peptide or even small molecules). In this study, we show that this method is also well suited to study RNA binding proteins (RBPs). We could successfully investigate the binding to RNA of three different RBPs (Fox-1, SRSF1 and Tra2-β1) and obtained K values very close to the ones determined previously by SPR or ITC for these complexes. These results show that the switchSENSE technology can be used as an alternative method to study protein-RNA interactions with K values in the low micromolar (10) to nanomolar (10-10) and probably picomolar (10-10) range. The absence of labelling requirement for the analyte molecules and the use of very low amounts of protein and RNA molecules make the switchSENSE approach very attractive compared to other methods. Finally, we discuss about the potential of this approach in obtaining more sophisticated information such as structural conformational changes upon RBP binding to RNA.
为了正确理解基因表达调控所涉及的细胞机制，对RNA结合蛋白与RNA的相互作用进行表征变得势在必行。结构研究能在原子水平上提供有关这些相互作用的信息，诸如等温滴定量热法（ITC）和表面等离子体共振（SPR）等互补方法通常用于量化这些RNA-蛋白质复合物的亲和力，并评估影响这些相互作用的突变效应。开关传感技术最近已被开发出来，并已成功用于研究蛋白质与不同类型结合伴侣（DNA、蛋白质/肽甚至小分子）的相互作用。在本研究中，我们表明该方法也非常适合研究RNA结合蛋白（RBP）。我们能够成功研究三种不同RBP（Fox-1、SRSF1和Tra2-β1）与RNA的结合，并获得了与先前通过SPR或ITC测定的这些复合物的K值非常接近的K值。这些结果表明，开关传感技术可以用作研究蛋白质-RNA相互作用的替代方法，其K值在低微摩尔（10⁻⁶）到纳摩尔（10⁻⁹）甚至可能皮摩尔（10⁻¹²）范围内。与其他方法相比，分析物分子无需标记以及使用极少量的蛋白质和RNA分子使得开关传感方法极具吸引力。最后，我们讨论了这种方法在获取更复杂信息（如RBP与RNA结合时的结构构象变化）方面的潜力。
