Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer–small molecule interaction

An, Chung Il; Trinh, Vu B.; Yokobayashi, Yohei

doi:10.1261/rna.2299306

Cited by 134 publications

(104 citation statements)

References 35 publications

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“…A striking example of a biological communication and control system is the class of RNA-regulatory elements called riboswitches, comprised of distinct sensor and actuation (gene-regulatory) functions, that control gene expression in response to specific ligand concentrations (6). Building on these natural examples, engineered riboswitch elements have been developed for use as synthetic ligand-controlled gene-regulatory systems (7)(8)(9)(10). However, these early examples of riboswitch engineering do not address the challenges posed above because they lack portability across organisms and systems, and their designs and construction do not support modularity and component reuse.…”

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confidence: 99%

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Win

Smolke

2007

Proc. Natl. Acad. Sci. U.S.A.

358

369

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Engineered biological systems hold promise in addressing pressing human needs in chemical processing, energy production, materials construction, and maintenance and enhancement of human health and the environment. However, significant advancements in our ability to engineer biological systems have been limited by the foundational tools available for reporting on, responding to, and controlling intracellular components in living systems. Portable and scalable platforms are needed for the reliable construction of such communication and control systems across diverse organisms. We report an extensible RNA-based framework for engineering ligand-controlled gene-regulatory systems, called ribozyme switches, that exhibits tunable regulation, design modularity, and target specificity. These switch platforms contain a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of a hammerhead ribozyme sequence. We examined two modes of standardized information transmission between these domains and demonstrate a mechanism that allows for the reliable and modular assembly of functioning synthetic RNA switches and regulation of ribozyme activity in response to various effectors. In addition to demonstrating examples of small molecule-responsive, in vivo functional, allosteric hammerhead ribozymes, this work describes a general approach for the construction of portable and scalable gene-regulatory systems. We demonstrate the versatility of the platform in implementing application-specific control systems for small molecule-mediated regulation of cell growth and noninvasive in vivo sensing of metabolite production.

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mentioning

confidence: 99%

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Win

Smolke

2007

Proc. Natl. Acad. Sci. U.S.A.

358

369

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“…With the adjuvant siRNA, the aptamer was thus transformed into a promising tool to post-transcriptionally regulate expression in mammalian cells. 63 Intracellularly expressed aptamers were also shown to be viable alternatives to siRNA knockdown for direct regulation of gene expression. 64 Thus, it is possible that a combination of an aptamer and siRNA against a gene target may be the method of choice to maximize knockdown of protein activity.…”

Section: Aptamers For Intracellular Targets Are Being Developedmentioning

confidence: 99%

Gene therapy progress and prospects: RNA aptamers

2007

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Aptamers are oligonucleotides evolved in vitro or in nature to bind target ligands with high affinity and specificity. They are emerging as powerful tools in the fields of therapeutics, drug development, target validation and diagnostics. Aptamers are attractive alternatives to antibody-and small-moleculebased therapeutics owing to their stability, low toxicity, low immunogenicity and improved safety. With the recent approval of the first aptamer drug Macugen by the US FDA, there is great impetus to develop therapeutic aptamers that can target a wide array of disease states. The recent demonstration that aptamer activity can be reversed by the administration of a simple antidote greatly enhances the potential value of aptamers as therapeutic agents.

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“…RNA aptamers that bind to small-molecule ligands (e.g., theophylline) have been instrumental in the development of novel systems that allow for ligand-dependent regulation of mRNA translational activity (3)(4)(5)(6)(7)(8)(9)(10), ribozyme activity (10,11), or other cellular processes (3,9,12,13). In these systems, the aptamers are able to directly or indirectly influence their targets through ligand-binding-induced changes in RNA structure (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). This ability to modulate RNA conformation makes aptamers amenable for use in the development of other biologically based regulatory systems.…”

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confidence: 99%

Riboswitching on RNA virus replication

Wang

White

2007

Proc. Natl. Acad. Sci. U.S.A.

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Positive-strand RNA viruses direct different virus-specific processes during their infection of host cells. Fundamental events such as viral RNA genome replication are controlled by viral regulatory RNA elements (REs). Here, we have investigated the possibility of specifically modulating the action of a viral RE using RNA aptamer technology. Through rational design, a tombusvirus RE, which has the structure of a perfect RNA stem loop in the plus-strand RNA genome, was replaced with a theophylline-binding RNA aptamer sequence, an imperfect stem loop. The aptamer-RE hybrid was designed so that, upon binding theophylline, it would become more stable and structurally mimic the functional RE (i.e., represent a ligand-inducible RE riboswitch). Initial experiments were conducted with a small noncoding virus genome-derived RNA replicon, and the results showed that replication was inducible, up to Ϸ10-fold, in a theophylline-specific and dose-dependent manner. A similar level of theophylline-dependent induction was also observed when a full-length viral genome containing an RE riboswitch was tested. Analysis of this engineered viral genome revealed that this RE, located in the 5 untranslated region, specifically mediates efficient accumulation of plus-strands of the virus genome. Therefore, in addition to allowing for modulation of virus reproduction, the RE riboswitch system also provided insight into RE function. The ability to chemically induce a viral process via modulation of virus genome structure could be useful for basic and applied aspects of research.aptamer ͉ gene regulation ͉ riboswitch ͉ RNA structure ͉ tombusvirus R NA aptamers are RNA molecules that are capable of specifically binding to cognate ligands. Small moleculebinding RNA aptamers were first identified through artificial selection by using systematic evolution of ligands by exponential enrichment (i.e., SELEX) (1). Surprisingly, some of these RNA aptamers showed favorable binding affinities and high levels of specificity (1). These observations suggested that natural RNA aptamers might exist and function in nature. Indeed, naturally occurring RNA aptamers, present as components of riboswitches, were subsequently identified in bacteria and shown to be involved in regulating translation or transcription of cellular mRNA (2). These riboswitches contain two mutually exclusive structures, one functional and the other nonfunctional, and ligand binding to the aptamer domain within the riboswitch acts to stabilize one of the two structures (2). In this way, the presence or absence of the ligand dictates which RNA structure dominates and, thus, the activity of the functional RNA module.Engineered RNA-based regulation of gene expression in living cells is now a rapidly expanding area of biotechnology. RNA aptamers that bind to small-molecule ligands (e.g., theophylline) have been instrumental in the development of novel systems that allow for ligand-dependent regulation of mRNA translational activity (3-10), ribozyme activity (10, 11), or other cellular proc...

show abstract

Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer–small molecule interaction

Cited by 134 publications

References 35 publications

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Gene therapy progress and prospects: RNA aptamers

Riboswitching on RNA virus replication

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