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Transcriptional Interference in Toehold Switch-Based RNA Circuits.

Elisabeth Falgenhauer1, Andrea Mückl1, Matthaeus Schwarz-Schilling1

  • 1Physics Department - E14 and ZNN/WSI, TU Munich, Am Coulombwall 4a, 85748 Garching, Germany.

ACS Synthetic Biology
|April 12, 2022
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Summary

This study explores antisense transcription for compact synthetic biology circuits using toehold switches. Antisense RNA enables precise gene expression control, leading to a two-input, two-output logic gate.

Keywords:
antisense RNAgene regulationtranscriptional interference

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Area of Science:

  • Synthetic biology
  • Gene regulation
  • RNA-based circuits

Background:

  • Regulatory RNA is crucial for cellular mechanisms and synthetic biology.
  • Toehold switches are post-transcriptional riboregulators controlling protein production.
  • Antisense RNA can inhibit gene expression by targeting trigger RNAs.

Purpose of the Study:

  • To investigate the utility of antisense transcription for compact gene regulation circuits.
  • To design and analyze synthetic gene circuits using toehold switches, trigger RNAs, and antisense RNAs.
  • To explore the impact of promoter arrangement and transcriptional interference on circuit dynamics.

Main Methods:

  • Constructed synthetic gene circuits with inducible promoters for trigger and antisense RNA transcription.
  • Utilized constitutive promoters for toehold switch transcription regulating reporter protein expression.
  • Varied promoter arrangements to study transcriptional interference (TI) effects.

Main Results:

  • Promoter proximity and strength influence transcriptional activity due to RNA polymerase concentration.
  • Transcriptional interference effects were observed and characterized based on promoter distance.
  • Successfully designed a two-input, two-output logic gate using two toehold switches, triggers, and antitriggers.

Conclusions:

  • Antisense transcription offers a compact strategy for designing regulatory RNA circuits.
  • Transcriptional interference is a significant factor influencing the dynamic behavior of these circuits.
  • The developed logic gate demonstrates the potential of these systems for complex synthetic biology applications.