I am especially indebted to Maung Win, whose initial development of the ribosome switching platform helped to put the lab at the forefront of synthetic biology and gave me a starting point for my project. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to gene regulation and thus cellular function. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct the stability, structure, and localization of the device.
This genetically encoded technology could find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.
Introduction
Many RNA-based devices use architectures that also include a transmitter component, which connects the sensor and actuator components and transmits information between them by modulating the actuator's activity based on the ligand-bound state of the sensor. Ligand binding to an aptamer integrated into the base of the miRNA stem prevented processing of the primary miRNA (pri-miRNA) by Drosha, increasing target gene expression levels as a function of increasing ligand concentrations. The RNA machinery is linked to the 3' UTR of the target gene, where ribozyme self-cleavage inactivates the transcript and thereby reduces gene expression, independently of cell-specific machinery.
The precise design of the transmitter component determines whether the ribozyme switch will suppress or enhance gene expression, unlike many of the switches described above, which are only capable of regulating gene expression in one direction. Other investigators have demonstrated switching activity of a theophylline-responsive ribozyme switch coupled to the 5' UTR45,46, but this strategy may lead to nonspecific reduction of translation initiation due to the high degree of secondary structure upstream of the start codon. In another example, ribozyme switches responsive to small molecule drugs were used to regulate the expression of the cytokines IL-2 and IL-15 in engineered T cells, thereby imparting drug-modulated control of T cell proliferation and survival in vitro and in vivo43.
However, in vivo activity of trans-ribozymes was not established, probably due to the inability of the two RNA strands to hybridize correctly inside the cell. We developed a genetic system for quantitative characterization of the activity of these units in human cells.
Development of an RNA device framework that targets endogenous genes in human cells
41 Supplementary Tables
42 ancillary cis-
Development of an RNA device framework that responds to proteins in human cells
We also investigated the mechanism of action and ligand localization requirements of the ribozyme switch by localizing the protein ligand in different cellular compartments. Binding of L7Ae to its aptamer in the regulator RNA prevented binding to the mRNA, abrogating expression of the target gene. MS2-C: The MS2 aptamer is linked via a transmitter that affects the secondary structure of the loop.
MS2-D: The MS2 aptamer is placed directly upstream of ribosome and a transmitter that affects stem III formation. The first set of ribosome switches contains a transmitter designed to change the secondary structure of one of the catalytic stems and core (Figure 3.1, MS2-B). A tetracycline-responsive CMV-TetO2 promoter controls expression of the protein ligand, and the fluorescent reporter protein is expressed from a constitutive promoter.
We tested four of the direct-coupled designs (MS2-A1, MS2-A2, MS2-A5 and MS2-A6) in the described in vivo characterization assay. One of the loop transmitter designs (MS2-C) showed low gene knockdown and no switching activity (MS2-C2), while the other three designs responded to MS2 (Figure 3.4, MS2-C). To more accurately measure the gene regulatory activity of the protein-responsive ribozyme switches in mammalian cells, we developed and optimized an improved in vivo characterization system.
As described above, our initial characterization construct used a GFP reporter expressed from a CMV promoter to measure the gene regulatory activity of ribosome switches. Thus, we examined two alternative versions of the MS2 ligand: (i) a mutant form of MS2 (MS2mut) containing two amino acid substitutions (V75E and A81G) that is deficient in capsid formation but retains RNA binding affinity of wild-type protein27 and (ii) a fused dimer of the MS2 mutant (2MS2mut). The three models differ from each other in the length of the aptamer stem beyond bulge II.
The data show little gene knockdown and no switching activity for any of the PP7 designs (Figure 3.13B). Although the constructs showed a range of knockdown gene activity, none of them showed switch activity under any of the conditions tested (Figure 3.16B). The second set of designs (Bcat-B) was similar to the MS2-responsive sequence aptamer and ribozyme designs (MS2-D), with the β-catenin aptamer just upstream of the ribozyme.
Most of the loop I substitution patterns (Bcat-A) exhibited little gene knockout activity, whereas the sequential patterns (Bcat-B) exhibited high levels of gene knockout activity, similar to. The switching activity was improved by incorporating multiple copies of the ribosome switch into the 3' UTR of the target gene, resulting in 3.5-fold amplification of the ON-construct (L2b8, 2 copies and L2b9, 3 copies)17. After export to the cytoplasm, dissociation of the ligand by ribosome switching would be favored because of the extremely low local concentration of free ligand.
The coding region of the PP7 coat protein was PCR amplified from Addgene plasmid 28174 (Kathleen Collins) using primers PP7 NotIF and PP7 ApalR and inserted into pCS2595 between NotI/Apal to create pCS2847.
Conclusions
The emerging field of synthetic biology has produced a wide variety of engineered molecular devices that enable the study of cellular function and the programmed control of novel phenotypic behaviors in biological systems1-4. Synthetic molecular devices have been used to regulate gene expression in a wide variety of organisms, from prokaryotes to microbial eukaryotes to humans9-11. Some of these genetic control platforms are able to process molecular input into increases or decreases in gene expression output by combining a sensor component with an actuator component1,12.
The binding and catalytic functions of RNA strands are largely determined by their secondary structure, which can be predicted by computational models of RNA folding17-19. Furthermore, the ability of RNA to be replicated by reverse transcription and PCR enables the easy in vitro selection of RNA molecules with new functions from large libraries of different sequences20,21. The ability of RNA enzymes to cleave phosphodiester bonds is exploited in the engineering of the ribozyme switch platform, in which cleavage of an mRNA strand by a hammerhead ribozyme causes silencing of the encoded gene in response to ligand binding to an aptamer12.
Both ON and OFF switches were demonstrated to regulate gene expression in yeast and mammalian cells, and importantly, changing the aptamer component to detect an alternative ligand did not require extensive redesign of the device 12,22,23. We sought to extend the capabilities of the ribozyme switch platform to two new functions: regulation of endogenous genes and sensing of protein input. We demonstrated a higher level of ligand responsiveness than previously described small-molecule-responsive ribozyme switches in mammalian systems, and we showed that cytoplasmic and nuclear localization of ligand were each sufficient to induce switching.
We also demonstrated the versatility of our switch platform with a ribozyme switch responsive to an alternative protein ligand. We have rationally designed each device presented here, but in the future a broader sequence space could be explored using high-throughput in vivo screening methods to test large libraries of randomized devices. Our ribozyme switch is capable of responding to proteins in the nucleus or cytoplasm, while previously described gene regulatory devices in mammals required specific localization of ligand to produce a switching response.
This device and other ribozyme switches that respond to disease markers could be used to noninvasively detect diseased cellular states. Furthermore, such switches could be used to control cell fate, for example by regulating the expression of a proapoptotic transgene. As the field of synthetic biology continues to advance, we hope that the molecular device platform we have developed will be a useful tool for protein-responsive gene regulation.
