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Protein-responsive ribozyme switches in eukaryotic cells.

Kennedy AB, Vowles JV, d'Espaux L, Smolke CD - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA.

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In vivo activity of protein-responsive ribozyme switches in HEK293 cells. (A) Schematic of the protein-responsive ribozyme switch characterization system for human cells. The first construct encodes the expression of the ribozyme switch in the 3′ UTR of a fluorescent reporter (BFP) from a constitutive promoter. The second construct encodes the expression of the protein ligand from a tetracycline-responsive CMV-TetO2 promoter. Both expression constructs are placed on a single plasmid, which is transfected into a Flp-In T-REx HEK293 cell line that stably expresses TetR. TetR repression is relieved by the addition of doxycycline. (B) Gene-regulatory activity of MS2-responsive ribozyme switches in human cells. Relative BFP levels are reported for transiently transfected cells harboring the indicated ribozyme switch constructs or controls in the absence and presence of MS2 (1 mg/l doxycycline). (C) Gene-regulatory activity of MS2-responsive ribozyme switches to optimized ligand in human cells. Relative BFP levels are reported for transiently transfected constructs encoding ribozyme switch sequences cotransfected with a transfection control plasmid. Reported values are the geometric mean ± SD from biological duplicates and normalized to the non-cleaving control (sTRSVctrl).
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Figure 5: In vivo activity of protein-responsive ribozyme switches in HEK293 cells. (A) Schematic of the protein-responsive ribozyme switch characterization system for human cells. The first construct encodes the expression of the ribozyme switch in the 3′ UTR of a fluorescent reporter (BFP) from a constitutive promoter. The second construct encodes the expression of the protein ligand from a tetracycline-responsive CMV-TetO2 promoter. Both expression constructs are placed on a single plasmid, which is transfected into a Flp-In T-REx HEK293 cell line that stably expresses TetR. TetR repression is relieved by the addition of doxycycline. (B) Gene-regulatory activity of MS2-responsive ribozyme switches in human cells. Relative BFP levels are reported for transiently transfected cells harboring the indicated ribozyme switch constructs or controls in the absence and presence of MS2 (1 mg/l doxycycline). (C) Gene-regulatory activity of MS2-responsive ribozyme switches to optimized ligand in human cells. Relative BFP levels are reported for transiently transfected constructs encoding ribozyme switch sequences cotransfected with a transfection control plasmid. Reported values are the geometric mean ± SD from biological duplicates and normalized to the non-cleaving control (sTRSVctrl).

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.


Protein-responsive ribozyme switches in eukaryotic cells.

Kennedy AB, Vowles JV, d'Espaux L, Smolke CD - Nucleic Acids Res. (2014)

In vivo activity of protein-responsive ribozyme switches in HEK293 cells. (A) Schematic of the protein-responsive ribozyme switch characterization system for human cells. The first construct encodes the expression of the ribozyme switch in the 3′ UTR of a fluorescent reporter (BFP) from a constitutive promoter. The second construct encodes the expression of the protein ligand from a tetracycline-responsive CMV-TetO2 promoter. Both expression constructs are placed on a single plasmid, which is transfected into a Flp-In T-REx HEK293 cell line that stably expresses TetR. TetR repression is relieved by the addition of doxycycline. (B) Gene-regulatory activity of MS2-responsive ribozyme switches in human cells. Relative BFP levels are reported for transiently transfected cells harboring the indicated ribozyme switch constructs or controls in the absence and presence of MS2 (1 mg/l doxycycline). (C) Gene-regulatory activity of MS2-responsive ribozyme switches to optimized ligand in human cells. Relative BFP levels are reported for transiently transfected constructs encoding ribozyme switch sequences cotransfected with a transfection control plasmid. Reported values are the geometric mean ± SD from biological duplicates and normalized to the non-cleaving control (sTRSVctrl).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231745&req=5

Figure 5: In vivo activity of protein-responsive ribozyme switches in HEK293 cells. (A) Schematic of the protein-responsive ribozyme switch characterization system for human cells. The first construct encodes the expression of the ribozyme switch in the 3′ UTR of a fluorescent reporter (BFP) from a constitutive promoter. The second construct encodes the expression of the protein ligand from a tetracycline-responsive CMV-TetO2 promoter. Both expression constructs are placed on a single plasmid, which is transfected into a Flp-In T-REx HEK293 cell line that stably expresses TetR. TetR repression is relieved by the addition of doxycycline. (B) Gene-regulatory activity of MS2-responsive ribozyme switches in human cells. Relative BFP levels are reported for transiently transfected cells harboring the indicated ribozyme switch constructs or controls in the absence and presence of MS2 (1 mg/l doxycycline). (C) Gene-regulatory activity of MS2-responsive ribozyme switches to optimized ligand in human cells. Relative BFP levels are reported for transiently transfected constructs encoding ribozyme switch sequences cotransfected with a transfection control plasmid. Reported values are the geometric mean ± SD from biological duplicates and normalized to the non-cleaving control (sTRSVctrl).
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA.

Show MeSH
Related in: MedlinePlus