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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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Sequence and secondary structures of the actuator (sTRSV HHRz) and sensor (MS2 aptamer) domains. (A) The sTRSV HHRz consists of a catalytic core flanked by three helices. The HHRz is integrated into the transcript through stem III and the loop sequences auxilliary to stems I and II are involved in mediating tertiary contacts required for catalytic activity at physiological Mg2+ concentrations. The Hoogsteen U-A-U base triple and GG base pair HHRz tertiary interactions (40) are denoted with boxed green and gray nucleotides, respectively. The site-specific phosphodiester bond isomerization that results in cleavage of the RNA is marked with a red arrow. (B) The high affinity variant of the MS2 bacteriophage translational operator RNA (38) used as the MS2 aptamer for all devices in this study. Bases involved directly in MS2 protein binding are indicated in orange, while all other bases are only structurally conserved (35). The MS2 F5 aptamer variant has a non-canonical GA pair (brown dotted boxes) below the bulged adenine (36). In device construction, the aptamer is truncated to the subsequence above the bulged adenine (yellow backbone) and grafted onto functional sequences (e.g. transmitter, HHRz stem) to reconstitute the aptamer lower stem (black backbone). All secondary structures were predicted by RNAstructure folding software (41).
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Figure 1: Sequence and secondary structures of the actuator (sTRSV HHRz) and sensor (MS2 aptamer) domains. (A) The sTRSV HHRz consists of a catalytic core flanked by three helices. The HHRz is integrated into the transcript through stem III and the loop sequences auxilliary to stems I and II are involved in mediating tertiary contacts required for catalytic activity at physiological Mg2+ concentrations. The Hoogsteen U-A-U base triple and GG base pair HHRz tertiary interactions (40) are denoted with boxed green and gray nucleotides, respectively. The site-specific phosphodiester bond isomerization that results in cleavage of the RNA is marked with a red arrow. (B) The high affinity variant of the MS2 bacteriophage translational operator RNA (38) used as the MS2 aptamer for all devices in this study. Bases involved directly in MS2 protein binding are indicated in orange, while all other bases are only structurally conserved (35). The MS2 F5 aptamer variant has a non-canonical GA pair (brown dotted boxes) below the bulged adenine (36). In device construction, the aptamer is truncated to the subsequence above the bulged adenine (yellow backbone) and grafted onto functional sequences (e.g. transmitter, HHRz stem) to reconstitute the aptamer lower stem (black backbone). All secondary structures were predicted by RNAstructure folding software (41).

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.


Protein-responsive ribozyme switches in eukaryotic cells.

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

Sequence and secondary structures of the actuator (sTRSV HHRz) and sensor (MS2 aptamer) domains. (A) The sTRSV HHRz consists of a catalytic core flanked by three helices. The HHRz is integrated into the transcript through stem III and the loop sequences auxilliary to stems I and II are involved in mediating tertiary contacts required for catalytic activity at physiological Mg2+ concentrations. The Hoogsteen U-A-U base triple and GG base pair HHRz tertiary interactions (40) are denoted with boxed green and gray nucleotides, respectively. The site-specific phosphodiester bond isomerization that results in cleavage of the RNA is marked with a red arrow. (B) The high affinity variant of the MS2 bacteriophage translational operator RNA (38) used as the MS2 aptamer for all devices in this study. Bases involved directly in MS2 protein binding are indicated in orange, while all other bases are only structurally conserved (35). The MS2 F5 aptamer variant has a non-canonical GA pair (brown dotted boxes) below the bulged adenine (36). In device construction, the aptamer is truncated to the subsequence above the bulged adenine (yellow backbone) and grafted onto functional sequences (e.g. transmitter, HHRz stem) to reconstitute the aptamer lower stem (black backbone). All secondary structures were predicted by RNAstructure folding software (41).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4231745&req=5

Figure 1: Sequence and secondary structures of the actuator (sTRSV HHRz) and sensor (MS2 aptamer) domains. (A) The sTRSV HHRz consists of a catalytic core flanked by three helices. The HHRz is integrated into the transcript through stem III and the loop sequences auxilliary to stems I and II are involved in mediating tertiary contacts required for catalytic activity at physiological Mg2+ concentrations. The Hoogsteen U-A-U base triple and GG base pair HHRz tertiary interactions (40) are denoted with boxed green and gray nucleotides, respectively. The site-specific phosphodiester bond isomerization that results in cleavage of the RNA is marked with a red arrow. (B) The high affinity variant of the MS2 bacteriophage translational operator RNA (38) used as the MS2 aptamer for all devices in this study. Bases involved directly in MS2 protein binding are indicated in orange, while all other bases are only structurally conserved (35). The MS2 F5 aptamer variant has a non-canonical GA pair (brown dotted boxes) below the bulged adenine (36). In device construction, the aptamer is truncated to the subsequence above the bulged adenine (yellow backbone) and grafted onto functional sequences (e.g. transmitter, HHRz stem) to reconstitute the aptamer lower stem (black backbone). All secondary structures were predicted by RNAstructure folding software (41).
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.

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