5 years ago

RNA Polymerase Tags To Monitor Multidimensional Protein–Protein Interactions Reveal Pharmacological Engagement of Bcl-2 Proteins

RNA Polymerase Tags To Monitor Multidimensional Protein–Protein Interactions Reveal Pharmacological Engagement of Bcl-2 Proteins
Jinyue Pu, Bryan C. Dickinson, Jeffrey A. Dewey, Abbas Hadji, James L. LaBelle
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 (RNAPN), and multiple potential binding partners are each fused to orthogonal C-terminal RNAPs (RNAPC). Assembly of RNAPN with each RNAPC is highly dependent on interactions between the tagged proteins. Each PPI-mediated RNAPN-RNAPC 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.

Publisher URL: http://dx.doi.org/10.1021/jacs.7b06152

DOI: 10.1021/jacs.7b06152

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.