A quantitative framework for the forward design of synthetic miRNA circuits

Bloom, Ryan; Winkler, Sally M.; Smolke, Christina D.

doi:10.1038/nmeth.3100

Cited by 39 publications

(46 citation statements)

References 46 publications

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“…Examples include the regulation of mRNA translation (24,25,27), shRNA/miRNA processing (14,45), alternative splicing (43), nonsense-mediated mRNA decay (46) and ribozyme activity. However, these devices were exclusively delivered by plasmid DNA, which may raise the risk of random genomic integration.…”

Section: Discussionmentioning

confidence: 99%

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

Kawasaki

Fujita

Nagaike

et al. 2017

Nucleic Acids Research

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Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information.

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Section: Discussionmentioning

confidence: 99%

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

Kawasaki

Fujita

Nagaike

et al. 2017

Nucleic Acids Research

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“…Advances in generating synthetic riboswitches have accelerated their application as genetic controllers in the areas of molecular sensors, metabolic pathway optimization (Cress et al, ; Dietrich et al, ; Liu et al, ; McKeague et al, ; Rogers et al, ) and therapeutic applications (Davydova et al, ; Lee et al, ). In the past decade, several natural and artificial RNA‐based sensors have been engineered to respond to a wide range of metabolites, including folinic acid (Trausch et al, ), theophylline (Beisel et al, ; Lynch et al, ; Michener and Smolke, ; Topp et al, ; Wachsmuth et al, ), xanthine (Beisel et al, ), tetracycline (Beisel et al, ; Weigand and Suess, ), ammeline (Dixon et al, ), cyclic‐di‐GMP (Kellenberger et al, ; Lynch et al, ), cyclic‐di‐AMP (Kellenberger et al, ), β‐catenin (Bloom et al, ), thiamine 5′‐pyrophosphate (TPP) (You et al, ), guanine (Paige et al, ; You et al, ), adenine (You et al, ), S‐adenosyl‐methionine (SAM) (Paige et al, ; You et al, ), adenosine 5‐diphosphate (ADP) (Paige et al, ), guanosine 5‐triphosphate (GTP) (Paige et al, ), flavin mononucleotide (FMN) (Meyer et al, ), lysine (Yang et al, ; Zhou and Zeng, ), glucosamine 6‐phosphate (Lee and Oh, ) in prokaryotic, eukaryotic, and mammalian cells through various mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Naringenin‐responsive riboswitch‐based fluorescent biosensor module for Escherichia coli co‐cultures

Xiu

Jang

Jones

et al. 2017

Biotech & Bioengineering

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The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. © 2017 Wiley Periodicals, Inc.

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“…The difference in time scales between transcription and RNA binding and cofactor-assisted activation would make it possible to implement a system with the required separations of time scales. A growing set of tools for engineering in vivo RNA-based logic could be used to design these mechanisms [30][31][32][33]. In vitro, transcriptional circuits [13,34,35] or strand displacement circuits [9,36] could potentially also implement the mechanisms we describe.…”

Section: Discussionmentioning

confidence: 99%

A self-regulating biomolecular comparator for processing oscillatory signals

Agrawal

Franco

Schulman

2015

J. R. Soc. Interface.

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While many cellular processes are driven by biomolecular oscillators, precise control of a downstream on/off process by a biochemical oscillator signal can be difficult: over an oscillator's period, its output signal varies continuously between its amplitude limits and spends a significant fraction of the time at intermediate values between these limits. Further, the oscillator's output is often noisy, with particularly large variations in the amplitude. In electronic systems, an oscillating signal is generally processed by a downstream device such as a comparator that converts a potentially noisy oscillatory input into a square wave output that is predominantly in one of two well-defined on and off states. The comparator's output then controls downstream processes. We describe a method for constructing a synthetic biochemical device that likewise produces a square-wave-type biomolecular output for a variety of oscillatory inputs. The method relies on a separation of time scales between the slow rate of production of an oscillatory signal molecule and the fast rates of intermolecular binding and conformational changes. We show how to control the characteristics of the output by varying the concentrations of the species and the reaction rates. We then use this control to show how our approach could be applied to process different in vitro and in vivo biomolecular oscillators, including the p53-Mdm2 transcriptional oscillator and two types of in vitro transcriptional oscillators. These results demonstrate how modular biomolecular circuits could, in principle, be combined to build complex dynamical systems. The simplicity of our approach also suggests that natural molecular circuits may process some biomolecular oscillator outputs before they are applied downstream.

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A quantitative framework for the forward design of synthetic miRNA circuits

Cited by 39 publications

References 46 publications

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

Naringenin‐responsive riboswitch‐based fluorescent biosensor module for Escherichia coli co‐cultures

A self-regulating biomolecular comparator for processing oscillatory signals

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