Synthetic riboswitches — A tool comes of age

Groher, Florian; Suess, Beatrix

doi:10.1016/j.bbagrm.2014.05.005

Cited by 90 publications

(69 citation statements)

References 92 publications

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“…However, a 1.5-fold higher AAV dose had to be used in order to fully reach the functional outcome observed with the riboswitch-free construct. Reasons for the slightly lower initial expression might include ligandindependent basal activity of the riboswitch, which might be more pronounced in vivo as compared to the only slight effects observed in vitro, and minor riboswitch activation by endogenously present guanine or cellular disturbing factors such as RNA binding proteins 35 . Nevertheless, even a 1.5-fold higher AAV dose can be easily tolerated given that the average riboswitch-mediated increase in viral vector yield of 12 test-productions (see Besides the use of an inducible expression system, transgene expression during viral vector production could be avoided by using tissue-specific promoters that are inactive in HEK-293 producer cells.…”

Section: Discussionmentioning

confidence: 99%

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

et al. 2015

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Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.

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Section: Discussionmentioning

confidence: 99%

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

et al. 2015

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“…As riboswitches are promising modular tools for RNAbased gene regulation (Wittmann & Suess, 2012), artificial riboswitches have been developed through either rational design or library screening (Berens, Groher, & Suess, 2015;Sharma, Nomura, & Yokobayashi, 2008;Werstuck & Green, 1998). In addition to the generation of fully artificial riboswitches, semi-artificial riboswitches constructed through redesign of naturally occurring riboswitches are also attractive, in which the repertoire of riboswitches can be expanded as artificial tools for genetic circuit design (Berens et al, 2015;Groher & Suess, 2014). In the modular organization of the full-length Vc2 riboswitch, the P1 element plays key roles in determining the turn-ON or turn-OFF of translation.…”

Section: Discussionmentioning

confidence: 99%

Recognition of cyclic‐di‐GMP by a riboswitch conducts translational repression through masking the ribosome‐binding site distant from the aptamer domain

et al. 2018

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The riboswitch is a class of RNA-based gene regulatory machinery that is dependent on recognition of its target ligand by RNA tertiary structures. Ligand recognition is achieved by the aptamer domain, and ligand-dependent structural changes of the expression platform then usually mediate termination of transcription or translational initiation. Ligand-dependent structural changes of the aptamer domain and expression platform have been reported for several riboswitches with short (<40 nucleotides) expression platforms. In this study, we characterized structural changes of the Vc2 c-di-GMP riboswitch that represses translation of downstream open reading frames in a ligand-dependent manner. The Vc2 riboswitch has a long (97 nucleotides) expression platform, but its structure and function are largely unknown. Through mutational analysis and chemical probing, we identified its secondary structures that are possibly responsible for switch-OFF and switch-ON states of translational initiation.

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“…The combination of in vitro generated aptamer domains specific for a whole plethora of target molecules with suited regulatory domains opens the principle possibility to generate custom-designed orthogonal devices regulating gene expression in pro- and eukaryotes (57,55,10,58,59). However, the application of such aptamers as synthetic riboswitches is a rather difficult task.…”

Section: Discussionmentioning

confidence: 99%

“…This modular nature of riboswitches holds the potential for constructing synthetic riboswitches and reprogramming cells by replacing the aptamer domain with another aptamer–ligand-pair (9). In vitro selection procedures like SELEX enable researchers to isolate aptamer sequences that in theory can target any desired ligand molecule, rendering synthetic riboswitches an ideal tool to manipulate gene expression at the level of transcription or translation (10–12). …”

Section: Introductionmentioning

confidence: 99%

Applicability of a computational design approach for synthetic riboswitches

et al. 2016

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Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand-dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.

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Synthetic riboswitches — A tool comes of age

Cited by 90 publications

References 92 publications

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

Recognition of cyclic‐di‐GMP by a riboswitch conducts translational repression through masking the ribosome‐binding site distant from the aptamer domain

Applicability of a computational design approach for synthetic riboswitches

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