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
Mentions: We investigated different RNA device architectures for physically and functionally coupling the sTRSV HHRz actuator (Figure 1A) and MS2 coat protein aptamer sensor (Figure 1B) domains. The MS2 coat protein (35–38) was selected as a ligand input because it has a well-characterized binding interaction with RNA aptamers and due to its relatively small size is expected to be present in both the nucleus and the cytoplasm (39). Our designs leverage the fact that the lower stem of the aptamer (Figure 1B; black backbone bases) is only structurally conserved (35), which allows sequence substitutions in this region without a loss in binding affinity.
Affiliation: Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA.