Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans.

Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5'-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.

Neomycin-dependent hammerhead ribozymes for the direct control of gene expression in Saccharomyces cerevisiae.

Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast.

Synthesis of All Possible Canonical (3'-5'-Linked) Cyclic Dinucleotides and Evaluation of Riboswitch Interactions and Immune-Stimulatory Effects.

The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.

Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes.

Artificial riboswitches based on ribozymes serve as versatile tools for ligand-dependent gene expression regulation. Advantages of these so-called aptazymes are their modular architecture and the comparably little coding space they require. A variety of aptamer-ribozyme combinations were constructed in the past 20 years and the resulting aptazymes were applied in diverse contexts in prokaryotic and eukaryotic systems. Most in vivo functional aptazymes are OFF-switches, while ON-switches are more advantageous regarding potential applications in e.g. gene therapy vectors. We developed new ON-switching aptazymes in the model organism Escherichia coli and in mammalian cell culture using the intensely studied guanine-sensing xpt aptamer. Utilizing a high-throughput screening based on fluorescence-activated cell sorting in bacteria we identified up to 9.2-fold ON-switches and OFF-switches with a dynamic range up to 32.7-fold. For constructing ON-switches in HeLa cells, we used a rational design approach based on existing tetracycline-sensitive ON-switches. We discovered that communication modules responding to tetracycline are also functional in the context of guanine aptazymes, demonstrating a high degree of modularity. Here, guanine-responsive ON-switches with a four-fold dynamic range were designed. Summarizing, we introduce a series of novel guanine-dependent ribozyme switches operative in bacteria and human cell culture that significantly broaden the existing toolbox.

Twister ribozymes as highly versatile expression platforms for artificial riboswitches.

The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class.

Synthesis of mannoheptose derivatives and their evaluation as inhibitors of the lectin LecB from the opportunistic pathogen Pseudomonas aeruginosa.

Biofilm formation and chronic infections with Pseudomonas aeruginosa depend on lectins produced by the bacterium. The bacterial C-type lectin LecB binds to the two monosaccharides l-fucose and d-mannose and conjugates thereof. Previously, d-mannose derivatives with amide and sulfonamide substituents at C6 were reported as potent inhibitors of the bacterial lectin LecB and LecB-mediated bacterial surface adhesion. Because d-mannose establishes a hydrogen bond via its 6-OH group with Ser23 of LecB in the crystal structure and may be beneficial for binding affinity, we extended d-mannose and synthesized mannoheptoses bearing the free 6-OH group as well as amido and sulfonamido-substituents at C7. Two series of diastereomeric mannoheptoses were synthesized and the stereochemistry was determined by X-ray crystallography. The potency of the mannoheptoses as LecB inhibitors was assessed in a competitive binding assay. The data reveal a diastereoselectivity of LecB for (6S)-mannoheptose derivatives with increased activity over methyl α-d-mannoside.