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Split T7 RNA polymerase biosensors to study multiprotein interaction dynamics.

Jeffrey A Dewey1, Bryan C Dickinson1

  • 1Department of Chemistry, The University of Chicago, Chicago, IL, United States.

Methods in Enzymology
|July 28, 2020
PubMed
Summary
This summary is machine-generated.

This study introduces split T7 RNA polymerase (RNAP) biosensors to measure multiple protein-protein interactions (PPIs) in live cells. This method allows for the analysis of complex PPI networks and their modulation by small molecules.

Keywords:
Directed evolutionMultiplexedPhage assisted continuous evolutionProtein-protein interactionsSplit biosensorSynthetic biologyT7 RNA polymerase

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Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biophysics

Background:

  • Protein-protein interactions (PPIs) are fundamental to cellular processes and signaling pathways.
  • Understanding PPI networks is crucial for basic research and therapeutic development.
  • Existing methods for studying PPIs have limitations in capturing complex network dynamics.

Purpose of the Study:

  • To present proximity-dependent split T7 RNA polymerase (RNAP) biosensors as a method for measuring multidimensional PPIs in live cells.
  • To enable the study of multiple, competitive PPIs within complex cellular environments.
  • To provide protocols for researchers to detect and analyze PPIs in mammalian cells.

Main Methods:

  • Utilized evolved, proximity-dependent split T7 RNAP biosensors.
  • Employed orthogonal split RNAP tags to generate unique RNA signals for each PPI.
  • Quantified RNA signals using established RNA analysis methods.
  • Applied the method to detect multiple PPIs in mammalian cells, including their dynamic interplay with small molecule inhibitors.

Main Results:

  • Demonstrated the ability of split RNAP biosensors to measure multidimensional PPIs in live cells.
  • Enabled the simultaneous study of multiple competitive PPIs through unique RNA signals.
  • Facilitated the analysis of PPI dynamics in response to small molecule inhibitors.

Conclusions:

  • Split RNAP biosensors offer a powerful tool for dissecting complex PPI networks in real-time.
  • This method advances the study of cellular regulation and the development of targeted therapeutics.
  • The provided protocols will aid researchers in applying this technique to mammalian cell systems.