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