Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes

Stifel, Julia; Spöring, Maike; Hartig, Jörg S.

doi:10.1093/synbio/ysy022

Cited by 27 publications

(37 citation statements)

References 32 publications

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“…The most interesting switches in terms of fold change and dynamic range were GuaB4HHR (5.0-34.2% of the expression levels of a conventional, riboswitch-free construct, 6.9-fold change) and GuaB6HHR (19.6-84.1%, 4.3-fold change). Given that the previous rationally designed variants only showed up to 2-fold expression induction 13 , these results nicely demonstrate the power of our screen to identify higher-potency switches from large sequence space.…”

Section: Resultssupporting

confidence: 57%

“…4a). In a previous study, we used a rational approach to construct Gua-dependent ON-switches using this design 13 . Now, we aimed to utilize amplicon-seq to explore a larger sequence space by randomizing the communication module at seven positions ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To establish this method and to demonstrate its ability to identify functional riboswitch constructs, we first screened libraries based on previously described tetracycline (Tet)-hammerhead (HHR), guanine (Gua)-hepatitis-delta-virus (HDV) and Guahammerhead ribozyme designs 4,12,13 . Using this approach, we recovered known and identified new Tet-responsive ON-as well as Gua-responsive OFF-and ON-switch sequences, respectively.…”

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confidence: 99%

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High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

et al. 2020

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Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including nextgeneration gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires largescale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 10 4 constructs) by cDNAamplicon-sequencing in transiently transfected and stimulated human cells. The selfbarcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline-and guanine-responsive ON-and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

et al. 2020

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“…Bacteria and yeast are robust and easily-handled, but library sizes are limited by the transfection bottleneck [167] and riboswitches selected within them may also show reduced performance in mammalian cells. This difficulty is reflected in efforts to develop guanine-regulated aptazymes by Stifel et al, who enriched aptazymes using in vitro selection in E. coli but used rational design to develop less effective aptazymes for use in human cells [168]. Nonetheless, some aptazymes selected in prokaryotes or yeast can regulate transgene expression in mammals.…”

Section: Improving the Function Of Aptazyme Riboswitchesmentioning

confidence: 99%

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

Tickner

Farzan

2021

Pharmaceuticals

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Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

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“…RNA-mediated cis-regulation of expression at the transcriptional and translational level using natural, engineered, and synthetic riboswitches [89][90][91][92][93][94][95][96][97] and ribozymes [98][99][100][101] has been shown to provide several of the desirable characteristics of a gene expression system. These include tight regulation of basal expression, large dynamic range, and high signal:noise ratio.…”

Section: Cis-mediated Riboregulationmentioning

confidence: 99%

Contemporary Tools for Regulating Gene Expression in Bacteria

Kent

Dixon

2020

Trends in Biotechnology

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Insights from novel mechanistic paradigms in gene expression control have led to the development of new gene expression systems for bioproduction, control, and sensing applications. Coupled with a greater understanding of synthetic burden and modern creative biodesign approaches, contemporary bacterial gene expression tools and systems are emerging that permit fine-tuning of expression, enabling greater predictability and maximisation of specific productivity, while minimising deleterious effects upon cell viability. These advances have been achieved by using a plethora of regulatory tools, operating at all levels of the so-called 'central dogma' of molecular biology. In this review, we discuss these gene regulation tools in the context of their design, prototyping, integration into expression systems, and biotechnological application. From Overexpression to Precise Regulation: Engineering Gene Expression Control Historically, researchers have relied on a relatively small number of mechanisms to regulate heterologous gene expression [1]. These traditional systems have focussed on massive overexpression of genes in an effort to maximise product yield (see Glossary). Often systems developed for overexpression are adapted ad hoc for integration into genetic circuits. While suited for the rapid accumulation of high levels of protein, these tools are often insufficient for developing the more precise systems required for many modern applications in sensing, computation and production. Recent years have seen improvements in the functionality of gene regulation tools, enhancing utility for dynamic, tuneable regulation. With any bioproduction effort, there is often an important tradeoff between yield, biomass accumulation, and titre that must be considered (Figure 1A). This balance is important for an array of biotechnological applications to ensure process viability and stability. As our ability to engineer more complex systems increases, the importance of harmonising gene expression with the metabolic capacity of the host organism, or chassis, by reducing the burden associated with the engineered function only increases. In the case of recombinant protein production, aberrant protein synthesis can lead to an overload in cellular capacity, such as synthesis or secretion. Both resource limitations, such as limited amino acid and ribosome availability [2], and symptoms of cellular capacity overload, such as impaired protein folding and secretion capacity [3-5], can impact cellular viability. Resource demand and overload also present a challenge to metabolic engineering efforts where substrate, intermediate, and product concentration can all have deleterious effects on cell viability [6]. This issue is exacerbated when central metabolites and/or cofactors are consumed, creating further competition for cellular resources.

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Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes

Cited by 27 publications

References 32 publications

High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

Contemporary Tools for Regulating Gene Expression in Bacteria

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