Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells

Zhong, Guocai; Wang, Haimin; Bailey, Charles C.; Gao, Guangping; Farzan, Michael

doi:10.7554/elife.18858

Cited by 83 publications

(104 citation statements)

References 39 publications

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“…The Farzan group demonstrated an intriguing strategy that combines empirical experimental data with quantitative modeling [18]. They first synthesized a panel of 32 aptazymes and measured their riboswitch performance in cultured mammalian cells, and attempted to correlate the experimental results with various design parameters such as calculated annealing energy and the number of hydrogen bonds in the communication module.…”

Section: Controlling Mrna Cleavage By Aptazymesmentioning

confidence: 99%

“…The researchers concluded that the proximity of base pairing (or lack thereof) within the communication module to the ribozyme affects the ribozyme activity, and devised 'Weighted Hydrogen Bond Score (WHBS)' as a calculable parameter that correlates with the ribozyme performance. They used WHBS as a guide to design aptazyme-based riboswitches using three aptamers with good switching characteristics [18]. It remains to be seen if the strategy can be extrapolated to different aptamers, ribozymes, and aptazyme architectures.…”

Section: Controlling Mrna Cleavage By Aptazymesmentioning

confidence: 99%

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Aptamer-based and aptazyme-based riboswitches in mammalian cells

Yokobayashi

2019

Current Opinion in Chemical Biology

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Molecular recognition by RNA aptamers has been exploited to control gene expression in response to small molecules in mammalian cells. These mammalian synthetic riboswitches offer attractive features such as small genetic size and lower risk of immunological complications compared to proteinbased transcriptional gene switches. The diversity of gene regulatory mechanisms that involve RNA has also inspired the development of mammalian riboswitches that harness various regulatory mechanisms. In this report, recent advances in synthetic riboswitches that function in mammalian cells are reviewed focusing on the regulatory mechanisms they exploit such as mRNA degradation, microRNA processing, and programmed ribosomal frameshifting.

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Section: Controlling Mrna Cleavage By Aptazymesmentioning

confidence: 99%

Section: Controlling Mrna Cleavage By Aptazymesmentioning

confidence: 99%

Aptamer-based and aptazyme-based riboswitches in mammalian cells

Yokobayashi

2019

Current Opinion in Chemical Biology

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Section: Molecular Engineering Of Aptazyme‐based Nanomaterials Towardmentioning

confidence: 99%

“…Later, aptazymes have been demonstrated for diverse applications, including construction of synthetic gene networks in yeast, and regulation of transgene expression, or virus replication in mammalian cell culture . Recently, rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells was developed by Zhong et al (Figure C) . These aptazymes efficiently regulated adeno‐associated virus (AAV)‐vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches.…”

Section: Molecular Engineering Of Aptazyme‐based Nanomaterials Towardmentioning

confidence: 99%

Molecular Engineering of Functional Nucleic Acid Nanomaterials toward In Vivo Applications

Zhang

Lan

2019

Adv Healthcare Materials

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equally exciting and perhaps more challenging field of application is toward their in vivo applications, including living animals and human bodies, in diverse areas, [2] such as sensing, drug delivery, medical imaging, and cancer therapy. However, most of these nanomaterials lack selectivity toward targets of interests. Therefore, to employ these nanomaterials for a wider range of in vivo applications, various molecular engineering approaches have been employed to obtained nanomaterials functionalized with biomolecules to enable selective targeting. [3][4][5] Among these biomolecules, functional nucleic acids, or FNAs, have shown the most promise.FNAs are DNA or RNA molecules that can interact with or bind to a specific analyte, resulting in a conformational change and/or a catalytic reaction. [6] As their names suggested, ribozymes and deoxyribozymes (called DNAzyme hereafter) can act as enzymes in the presence of cofactors to catalyze various reactions, including cleavage and ligation of RNA/DNA, and phosphorylation and peroxidation of DNA. On the other hand, riboswitches and DNA aptamers are antibody-like receptors that can bind target molecules with high affinity and selectivity. Furthermore, the binding abilities of riboswitches and aptamers can be combined with the enzymatic function of ribozymes or DNAzymes to form aptazymes so that the binding of targets can inhibit or enhance the catalytic activities. These nucleic acids, including DNAzymes, aptamers, and aptazymes, are collectively known as FNAs to emphasize their functions beyond the traditional genetic storage and transformations roles for DNA and RNA. Unlike DNA and RNA inside biological systems, FNAs are isolated via a combinatorial technique known as in vitro selection, or a process also known as systematic evolution of ligands by exponential enrichment (SELEX). The discovery of new functions of nucleic acids has not only resulted in major advances in the fields of molecular biology and biochemistry, but also revolutionized sensors designs for both in vitro and in vivo applications. [7,8] Over the past decade, numerous FNA-based nanomaterials have been developed. [9][10][11][12] Among these FNA-based nanosystems, the functions of FNAs can be categorized into two types: diagnostic function and therapeutic function. For the diagnostic function, the FNAs serve either as the biorecognition ligands for direct sensing and imaging of the Recent advances in nanotechnology and engineering have generated many nanomaterials with unique physical and chemical properties. Over the past decade, numerous nanomaterials are introduced into many research areas, such as sensors for environmental monitoring, food safety, point-ofcare diagnostics, and as transducers for solar energy transfer. Meanwhile, functional nucleic acids (FNAs), including nucleic acid enzymes, aptamers, and aptazymes, have attracted major attention from the biomedical community due to their unique target recognition and catalytic properties. Benefiting from the recent progress of molecular engine...

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“…To design synthetic riboswitches, mostly self-cleaving ribozymes are used as expression platforms that are linked to an aptamer domain via a communication sequence, yielding so-called aptazymes (ribozyme plus aptamer). Development of synthetic RNA aptamers via SELEX (Systematic Evolution of Ligands by Exponential enrichment) along with rational design of the communication sequences brought the possibility to generate aptazymes that sense a broad variety of molecular inputs, such as proteins, RNAs, metabolites and co-factors (Townshend et al, 2015; McKeague et al, 2016; Zhong et al, 2016). The option to induce conformational changes or destabilize mRNAs by a ligand-responsive aptazyme makes them versatile tools for genetic control in diverse biological systems.…”

Section: Introductionmentioning

confidence: 99%

A theophylline-responsive riboswitch regulates expression of nuclear-encoded genes in Arabidopsis

Shanidze

Lenkeit

Hartig

et al. 2019

Preprint

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Ligand-responsive synthetic riboswitches are versatile and innovative tools for external gene regulation in pro- and eukaryotes. Riboswitches are small cis-regulatory RNA elements that regulate gene expression by conformational changes in response to ligand binding. In plants, synthetic riboswitches were used to regulate gene expression in plastids, but the application of synthetic riboswitches for the regulation of nuclear-encoded genes in planta has not been reported so far. Here we characterize the properties of a theophylline-responsive synthetic aptazyme for control of nuclear-encoded transgenes in Arabidopsis (Arabidopsis thaliana). Activation of the aptazyme, inserted in the 3-UTR of the target gene, resulted in rapid self-cleavage and subsequent decay of the mRNA. This riboswitch allowed reversible, theophylline-dependent downregulation of the Green Fluorescent Protein (GFP) reporter gene in a dose- and time- dependent manner. Insertion of the riboswitch into the One Helix Protein 1 (OHP1) gene allowed complementation of ohp1 mutants and induction of the mutant phenotype by theophylline. GFP or OHP1 transcript levels were downregulated by maximally 90%, and GFP protein levels by 95%. These results establish artificial riboswitches as tools for externally controlled gene expression in synthetic biology in plants or functional crop design.One sentence summaryArtificial, ligand-responsive RNA aptazymes are an efficient tool for dose- and time-dependent external control of nuclear gene expression in plants.

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Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells

Cited by 83 publications

References 39 publications

Aptamer-based and aptazyme-based riboswitches in mammalian cells

Aptamer-based and aptazyme-based riboswitches in mammalian cells

Molecular Engineering of Functional Nucleic Acid Nanomaterials toward In Vivo Applications

A theophylline-responsive riboswitch regulates expression of nuclear-encoded genes in Arabidopsis

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