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The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
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Protein-responsive ribozyme switches in eukaryotic cells.

Andrew B Kennedy1, James V Vowles2, Leo d'Espaux2

  • 1Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA.

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Scientists created new ribozyme-based genetic devices that detect protein levels inside cells. These synthetic biology tools work in yeast and mammalian cells, enabling gene control for biomarker detection.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Biochemistry

Background:

  • Genetic devices are crucial for synthetic biology, enabling targeted cellular responses.
  • Ribozyme-based devices offer a platform for sensing and regulating gene expression based on intracellular conditions.

Purpose of the Study:

  • To develop novel ribozyme-based genetic devices for detecting intracellular protein concentrations.
  • To enable both gene activation (ON) and repression (OFF) in response to protein ligands.
  • To validate device performance in eukaryotic hosts like yeast and mammalian cells.

Main Methods:

  • Design and construction of ribozyme switches responsive to protein ligands.
  • In vitro characterization pipeline for prescreening device designs using magnesium concentration gradients.
  • In vivo testing of gene-regulatory activities in yeast and mammalian cells.
  • Ligand localization studies to determine cellular compartment activity (nucleus/cytoplasm).

Main Results:

  • Developed functional ribozyme devices for gene regulation in yeast and mammalian cells.
  • Demonstrated both gene-ON and gene-OFF responses to protein ligands.
  • Correlated in vitro cleavage activity with in vivo gene-regulatory function across different magnesium concentrations.
  • Confirmed ribozyme switch activity in both nuclear and cytoplasmic cellular compartments.

Conclusions:

  • Ribozyme-based devices are effective tools for sensing protein concentrations and regulating gene expression in eukaryotic cells.
  • The developed in vitro characterization pipeline aids in efficient screening of synthetic gene-regulatory devices.
  • These findings advance the application of ribozyme switches for detecting protein biomarkers and engineering cellular functions.