Protein-responsive ribozyme switches in eukaryotic cells.
Bottom Line: The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations.Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action.By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers.
Affiliation: Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA.Show MeSH
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Mentions: We also tested the in vitro identified MS2-responsive ribozyme switch designs for gene-regulatory and ligand-responsive activities in a human cell line. A ribozyme switch characterization construct was designed in which the ribozyme switches and non-switch controls were placed in the 3′ UTR of a reporter gene encoding BFP (Figure 5A). A doxycycline-inducible expression cassette for MS2 was located on the same plasmid, in which MS2 expression was under the control of a CMV promoter with two downstream tetracycline operator (TetO) sites (CMV-TetO2). All constructs were characterized in a Flp-In T-REx HEK293 cell line, which stably expresses the tetracycline repressor (TetR). Thus, transcription of the protein ligand is inhibited by TetR and the addition of doxycycline to the cell culture media activates transcription of the protein ligand.
Affiliation: Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA.