Department of Chemistry, The University of Chicago , Chicago, Illinois 60637, United States.
Section of Hematology, Oncology, Stem Cell Transplantation, Department of Pediatrics, The University of Chicago, Comer Children's Hospital , Chicago, Illinois 60637, United States.
J Am Chem Soc. 2017 Aug 30;139(34):11964-11972. doi: 10.1021/jacs.7b06152. Epub 2017 Aug 15.
We report the development of a new technology for monitoring multidimensional protein-protein interactions (PPIs) inside live mammalian cells using split RNA polymerase (RNAP) tags. In this new system, a protein-of-interest is tagged with an N-terminal split RNAP (RNAP), and multiple potential binding partners are each fused to orthogonal C-terminal RNAPs (RNAP). Assembly of RNAP with each RNAP is highly dependent on interactions between the tagged proteins. Each PPI-mediated RNAP-RNAP assembly transcribes from a separate promoter on a supplied DNA substrate, thereby generating a unique RNA output signal for each PPI. We develop and validate this new approach in the context of the Bcl-2 family of proteins. These key regulators of apoptosis are important cancer mediators, but are challenging to therapeutically target due to imperfect selectivity that leads to either off-target toxicity or tumor resistance. We demonstrate binary (1 × 1) and ternary (1 × 2) Bcl-2 PPI analyses by imaging fluorescent protein translation from mRNA outputs. Next, we perform a 1 × 4 PPI network analysis by direct measurement of four unique RNA signals via RT-qPCR. Finally, we use these new tools to monitor pharmacological engagement of Bcl-2 protein inhibitors, and uncover inhibitor-dependent competitive PPIs. The split RNAP tags improve upon other protein fragment complementation (PFC) approaches by offering both multidimensionality and sensitive detection using nucleic acid amplification and analysis techniques. Furthermore, this technology opens new opportunities for synthetic biology applications due to the versatility of RNA outputs for cellular engineering applications.
我们报告了一种使用分裂 RNA 聚合酶 (RNAP) 标签监测活哺乳动物细胞内多维蛋白质-蛋白质相互作用 (PPI) 的新技术的发展。在这个新系统中，感兴趣的蛋白质被标记为 N 端分裂 RNAP (RNAP)，并且多个潜在的结合伙伴各自融合到正交 C 端 RNAP (RNAP)。RNAP 与每个 RNAP 的组装高度依赖于标记蛋白之间的相互作用。每个 PPI 介导的 RNAP-RNAP 组装从供应 DNA 底物上的单独启动子转录，从而为每个 PPI 产生独特的 RNA 输出信号。我们在 Bcl-2 蛋白家族的背景下开发并验证了这种新方法。这些凋亡的关键调节剂是重要的癌症介质，但由于靶向不完全导致非靶毒性或肿瘤耐药性，因此难以进行治疗。我们通过荧光蛋白从 mRNA 输出物翻译的成像来证明二元 (1×1) 和三元 (1×2) Bcl-2 PPI 分析。接下来，我们通过直接测量四个独特的 RNA 信号通过 RT-qPCR 进行 1×4 PPI 网络分析。最后，我们使用这些新工具来监测 Bcl-2 蛋白抑制剂的药理学结合，并发现抑制剂依赖性竞争 PPI。分裂 RNAP 标签通过使用核酸扩增和分析技术提供多维性和灵敏检测，改进了其他蛋白质片段互补 (PFC) 方法。此外，由于 RNA 输出物在细胞工程应用中的多功能性，该技术为合成生物学应用开辟了新的机会。
