Toxicology Program, Department of Biological Sciences, North Carolina State University, Campus Box 7633, Raleigh, NC 27695, USA.
Int J Mol Sci. 2023 Feb 10;24(4):3604. doi: 10.3390/ijms24043604.
Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein-RNA and protein-DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5'-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization.
研究 RNA 和 DNA 结合蛋白与特定调节序列（如富含 AU 的 RNA 和 DNA 增强子元件）的相互作用对于理解基因调控非常重要。在过去，电泳迁移率变动分析（EMSA）广泛应用于体外结合研究。为了顺应各种生物测定中使用非放射性材料的趋势，末端标记的生物素化 RNA 和 DNA 寡核苷酸可以作为更实用的探针来研究蛋白质-RNA 和蛋白质-DNA 相互作用；因此，结合复合物可以用链霉亲和素偶联树脂拉下，并通过 Western 印迹进行鉴定。然而，在最佳蛋白质结合条件下用生物素化探针设置 RNA 和 DNA 下拉测定仍然具有挑战性。在这里，我们展示了在优化的条件下，用 5' -生物素化茎环 IRE（铁反应元件）RNA、HuR 和 AUF1 与富含 AU 的 RNA 元件以及 Nrf2 与人类铁蛋白 H 基因中的抗氧化反应元件（ARE）增强子结合的 IRP（铁反应元件结合蛋白）的下拉的逐步优化。本研究旨在解决 RNA 和 DNA 下拉测定中的关键技术问题：（1）我们应该使用多少 RNA 和 DNA 探针；（2）可以使用什么结合缓冲液和细胞裂解缓冲液；（3）如何验证特定的相互作用；（4）哪种链霉亲和素树脂（琼脂糖或磁珠）有效；（5）从不同条件到最佳条件，我们可以预期什么样的 Western 印迹结果。我们预计，我们优化的下拉条件可以适用于其他 RNA 和 DNA 结合蛋白以及新兴的非编码小 RNA 结合蛋白，用于它们的体外表征。
