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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 vitro gel-based cleavage activity characterizations of protein-responsive RNA devices. (A) Cleavage rate constants for MS2-responsive RNA devices at 500 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 500 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are determined by one phase exponential fit (R2 > 0.99) of the cleavage kinetics of each assay (Supplementary Figure S6). Reported kobs are the mean ± SD of at least three independent cleavage assays performed for each device at the indicated ligand concentrations. (B) Correlation analysis of RNA device in vivo gene-regulatory activities in yeast (Figure 4; ‘No MS2’ and ‘+MS2’ conditions) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.89, slope = 0.006 ± 0.0007. (C and D) Concentration dependence of cleavage rate constants for (C) MS2-A1 and (D) MS2-A2 RNA devices. Cleavage rate constants (kobs) were determined at MS2 protein concentrations spanning five orders of magnitude and fit to a three-parameter logistic equation (R2 > 0.96). Dotted lines indicate IC50, the MS2 protein concentration at which the RNA device cleavage rate constant is halfway between maximum and minimum values. Cleavage rate constants reported at each MS2 concentration are the mean ± SD of at least three independent assays. Representative cleavage kinetics are presented in Supplementary Figure S8 and Supplementary Figure S9. (E) Cleavage rate constants for MS2-responsive RNA devices at 200 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 200 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are reported as the mean ± SD from fit of cleavage kinetics (Supplementary Figure S10) to one phase exponential (R2 > 0.99) of at least three independent assays for each device. (F) Correlation analysis of RNA device in vivo basal level gene-regulatory activities in mammalian cells (Figure 5; ‘No MS2’ condition) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.97, slope = 0.008 ± 0.001.
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Figure 6: In vitro gel-based cleavage activity characterizations of protein-responsive RNA devices. (A) Cleavage rate constants for MS2-responsive RNA devices at 500 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 500 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are determined by one phase exponential fit (R2 > 0.99) of the cleavage kinetics of each assay (Supplementary Figure S6). Reported kobs are the mean ± SD of at least three independent cleavage assays performed for each device at the indicated ligand concentrations. (B) Correlation analysis of RNA device in vivo gene-regulatory activities in yeast (Figure 4; ‘No MS2’ and ‘+MS2’ conditions) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.89, slope = 0.006 ± 0.0007. (C and D) Concentration dependence of cleavage rate constants for (C) MS2-A1 and (D) MS2-A2 RNA devices. Cleavage rate constants (kobs) were determined at MS2 protein concentrations spanning five orders of magnitude and fit to a three-parameter logistic equation (R2 > 0.96). Dotted lines indicate IC50, the MS2 protein concentration at which the RNA device cleavage rate constant is halfway between maximum and minimum values. Cleavage rate constants reported at each MS2 concentration are the mean ± SD of at least three independent assays. Representative cleavage kinetics are presented in Supplementary Figure S8 and Supplementary Figure S9. (E) Cleavage rate constants for MS2-responsive RNA devices at 200 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 200 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are reported as the mean ± SD from fit of cleavage kinetics (Supplementary Figure S10) to one phase exponential (R2 > 0.99) of at least three independent assays for each device. (F) Correlation analysis of RNA device in vivo basal level gene-regulatory activities in mammalian cells (Figure 5; ‘No MS2’ condition) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.97, slope = 0.008 ± 0.001.

Mentions: The gel-based cleavage assays were performed on radiolabeled transcripts at physiologically relevant reaction conditions at 37°C in the presence and absence of 2 μM MS2 protein (Figure 6A, Supplementary Figure S6). The cleavage rate constants for the theophylline-responsive control device (L2b8) were comparable in the absence and presence of MS2 protein, indicating that MS2 protein has no non-specific effect on the cleavage activity. In contrast, for almost all the MS2-responsive ON switches, kobs values measured in the presence of MS2 protein were lower than those measured in the absence of MS2 protein, indicating that MS2 protein binding to the aptamer shifts the distribution between the two functional conformations to the ribozyme-inactive state, resulting in slower cleavage activity as expected. For the OFF switch (MS2-B1), kobs in the presence of MS2 protein is higher, indicating that MS2 protein binding shifts the conformational distribution toward the ribozyme-active state, resulting in faster cleavage activity as expected. The MS2-A8 device cleavage activity (kobs) slightly increases, from 0.08 to 0.10 min−1 in the presence of MS2 protein, supporting the observed OFF-switch behavior in yeast (Figure 4B). We compared the corresponding cleavage time constants (kobs−1) to the yeast gene-regulatory activities (Figure 4B; ‘No MS2’ and ‘+MS2’ condition). The cleavage time constants and in vivo yeast gene-regulatory activities exhibit a strong linear correlation (Figure 6B; Pearson product-moment correlation coefficient (Pearson r): 0.89). The in vivo mammalian gene-regulatory activities (Figure 5C) also correlated to the cleavage time constants, but this correlation was not significant at a P-value of 0.01 (Supplementary Figure S7; Pearson r: 0.58). These results suggest that the in vitro gel-based cleavage assay conditions are more reflective of in vivo RNA device cleavage in yeast cells than mammalian cells.


Protein-responsive ribozyme switches in eukaryotic cells.

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

In vitro gel-based cleavage activity characterizations of protein-responsive RNA devices. (A) Cleavage rate constants for MS2-responsive RNA devices at 500 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 500 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are determined by one phase exponential fit (R2 > 0.99) of the cleavage kinetics of each assay (Supplementary Figure S6). Reported kobs are the mean ± SD of at least three independent cleavage assays performed for each device at the indicated ligand concentrations. (B) Correlation analysis of RNA device in vivo gene-regulatory activities in yeast (Figure 4; ‘No MS2’ and ‘+MS2’ conditions) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.89, slope = 0.006 ± 0.0007. (C and D) Concentration dependence of cleavage rate constants for (C) MS2-A1 and (D) MS2-A2 RNA devices. Cleavage rate constants (kobs) were determined at MS2 protein concentrations spanning five orders of magnitude and fit to a three-parameter logistic equation (R2 > 0.96). Dotted lines indicate IC50, the MS2 protein concentration at which the RNA device cleavage rate constant is halfway between maximum and minimum values. Cleavage rate constants reported at each MS2 concentration are the mean ± SD of at least three independent assays. Representative cleavage kinetics are presented in Supplementary Figure S8 and Supplementary Figure S9. (E) Cleavage rate constants for MS2-responsive RNA devices at 200 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 200 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are reported as the mean ± SD from fit of cleavage kinetics (Supplementary Figure S10) to one phase exponential (R2 > 0.99) of at least three independent assays for each device. (F) Correlation analysis of RNA device in vivo basal level gene-regulatory activities in mammalian cells (Figure 5; ‘No MS2’ condition) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.97, slope = 0.008 ± 0.001.
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Figure 6: In vitro gel-based cleavage activity characterizations of protein-responsive RNA devices. (A) Cleavage rate constants for MS2-responsive RNA devices at 500 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 500 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are determined by one phase exponential fit (R2 > 0.99) of the cleavage kinetics of each assay (Supplementary Figure S6). Reported kobs are the mean ± SD of at least three independent cleavage assays performed for each device at the indicated ligand concentrations. (B) Correlation analysis of RNA device in vivo gene-regulatory activities in yeast (Figure 4; ‘No MS2’ and ‘+MS2’ conditions) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.89, slope = 0.006 ± 0.0007. (C and D) Concentration dependence of cleavage rate constants for (C) MS2-A1 and (D) MS2-A2 RNA devices. Cleavage rate constants (kobs) were determined at MS2 protein concentrations spanning five orders of magnitude and fit to a three-parameter logistic equation (R2 > 0.96). Dotted lines indicate IC50, the MS2 protein concentration at which the RNA device cleavage rate constant is halfway between maximum and minimum values. Cleavage rate constants reported at each MS2 concentration are the mean ± SD of at least three independent assays. Representative cleavage kinetics are presented in Supplementary Figure S8 and Supplementary Figure S9. (E) Cleavage rate constants for MS2-responsive RNA devices at 200 μM MgCl2. Gel-based cleavage assays were performed at 37°C in 200 μM MgCl2, 150 mM NaCl, 1 mM DTT and 10 mM HEPES (pH 7.4). Cleavage rate constants (kobs) are reported as the mean ± SD from fit of cleavage kinetics (Supplementary Figure S10) to one phase exponential (R2 > 0.99) of at least three independent assays for each device. (F) Correlation analysis of RNA device in vivo basal level gene-regulatory activities in mammalian cells (Figure 5; ‘No MS2’ condition) and in vitro cleavage activity (cleavage time constant; kobs−1). Pearson correlation coefficient (r): 0.97, slope = 0.008 ± 0.001.
Mentions: The gel-based cleavage assays were performed on radiolabeled transcripts at physiologically relevant reaction conditions at 37°C in the presence and absence of 2 μM MS2 protein (Figure 6A, Supplementary Figure S6). The cleavage rate constants for the theophylline-responsive control device (L2b8) were comparable in the absence and presence of MS2 protein, indicating that MS2 protein has no non-specific effect on the cleavage activity. In contrast, for almost all the MS2-responsive ON switches, kobs values measured in the presence of MS2 protein were lower than those measured in the absence of MS2 protein, indicating that MS2 protein binding to the aptamer shifts the distribution between the two functional conformations to the ribozyme-inactive state, resulting in slower cleavage activity as expected. For the OFF switch (MS2-B1), kobs in the presence of MS2 protein is higher, indicating that MS2 protein binding shifts the conformational distribution toward the ribozyme-active state, resulting in faster cleavage activity as expected. The MS2-A8 device cleavage activity (kobs) slightly increases, from 0.08 to 0.10 min−1 in the presence of MS2 protein, supporting the observed OFF-switch behavior in yeast (Figure 4B). We compared the corresponding cleavage time constants (kobs−1) to the yeast gene-regulatory activities (Figure 4B; ‘No MS2’ and ‘+MS2’ condition). The cleavage time constants and in vivo yeast gene-regulatory activities exhibit a strong linear correlation (Figure 6B; Pearson product-moment correlation coefficient (Pearson r): 0.89). The in vivo mammalian gene-regulatory activities (Figure 5C) also correlated to the cleavage time constants, but this correlation was not significant at a P-value of 0.01 (Supplementary Figure S7; Pearson r: 0.58). These results suggest that the in vitro gel-based cleavage assay conditions are more reflective of in vivo RNA device cleavage in yeast cells than mammalian 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.

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