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Building an RNA-Based Toggle Switch Using Inhibitory RNA Aptamers.

Alicia Climent-Catala1,2, Thomas E Ouldridge1,3, Guy-Bart V Stan1,3

  • 1Imperial College Centre for Synthetic Biology, London, SW7 2AZ, U.K.

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This study presents an in vitro RNA toggle switch using inhibitory RNA aptamers. This synthetic biology tool offers programmable control for nucleic acid networks, demonstrating robust function even under degrading conditions.

Keywords:
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Area of Science:

  • Synthetic biology
  • Molecular systems biology
  • Biochemistry

Background:

  • Protein-based biological networks face limitations in response time and specificity.
  • Synthetic RNA systems offer advantages like faster response, increased specificity, and programmability.
  • RNA aptamers provide a versatile platform for molecular recognition and control.

Purpose of the Study:

  • To demonstrate an in vitro RNA-based toggle switch.
  • To utilize RNA aptamers for mutual inhibition of RNA polymerases.
  • To explore the programmability and robustness of RNA systems in synthetic biology.

Main Methods:

  • Constructed a toggle switch using T7 and SP6 RNA polymerases and their specific inhibitory aptamers.
  • Employed Broccoli and malachite green light-up aptamer systems for simultaneous polymerase activity monitoring.
  • Utilized DNA sequences to sequester RNA aptamers for toggling switch states.
  • Assessed toggle switch performance under controlled RNA degradation using RNases.

Main Results:

  • Successfully demonstrated a functional RNA-based toggle switch with two distinct states.
  • Showcased the ability to switch between states by adding DNA sequestering agents.
  • Confirmed the RNA toggle switch's operational capacity in the presence of RNase-induced degradation.
  • Validated the use of light-up aptamer systems for real-time monitoring of polymerase activity.

Conclusions:

  • Developed a novel RNA-based toggle switch for synthetic biology applications.
  • Highlighted the potential of RNA aptamers as programmable control elements in nucleic acid networks.
  • Demonstrated the robustness of the RNA toggle switch in challenging, degrading environments.