Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems

Takahashi, Melissa K.; Chappell, James; Hayes, Clarmyra A.; Sun, Zachary Z.; Kim, Jongmin; Singhal, Vipul; Spring, Kevin; Al‐Khabouri, Shaima; Fall, Christopher P.; Noireaux, Vincent; Murray, Richard M.; Lucks, Julius B.

doi:10.1021/sb400206c

Cited by 165 publications

(205 citation statements)

References 49 publications

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“…The data presented also provide evidence for the mechanism and importance of interior loops within antisensemediated transcriptional regulatory systems, which are being shown to be increasingly important for synthetic biology applications Chappell et al 2015a;Takahashi et al 2015). Initial in vitro structural analysis of In-cell SHAPE-Seq reactivity spectra for the upper portion of NP Fusion 1 and 2 hairpin stems.…”

Section: Discussionmentioning

confidence: 85%

“…1A; Novick et al 1989;Brantl and Wagner 2000). Because they regulate downstream RNA synthesis as a function of an RNA input, attenuators can also be used in synthetic, RNA-only transcription networks that propagate signals directly as RNA molecules Takahashi et al 2015). Attenuators also have the potential to simplify the construction of genetic networks by removing the need to express intermediate protein species.…”

Section: Introductionmentioning

confidence: 99%

“…Attenuators also have the potential to simplify the construction of genetic networks by removing the need to express intermediate protein species. In particular, variants of the Staphylococcus aureus plasmid pT181 attenuator (Novick et al 1989) have been used to construct a variety of genetic networks: In tandem they act as genetic logic gates Chappell et al 2015a), and in series they can be used to construct RNA-only transcriptional cascades ) and single input modules (Takahashi et al 2015). In addition, there is also evidence that these RNA networks propagate signals on the fast timescales governed by RNA degradation rates (Takahashi et al 2015), giving them potential kinetic advantage over their proteinmediated counterparts.…”

Section: Introductionmentioning

confidence: 99%

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Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

Takahashi

Watters

Gasper

et al. 2016

RNA

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Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators.

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Section: Discussionmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

Takahashi

Watters

Gasper

et al. 2016

RNA

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“…In this approach, commercially available cell-free transcription/translation systems (bacterial or mammalian) are freeze-dried onto paper or other porous substrates to create poised genetic regulatory networks that are stable for long-term storage at room temperature and are activated by rehydration. This work follows on other efforts in cell-free synthetic biology, which have provided important dynamic and mechanistic insight on gene regulatory networks and allowed for rapid "build-test" cycles for prototyping engineered gene circuits and biomanufacturing pathways (36)(37)(38)(39)(40)(41). The in vitro nature of these systems resolves the challenge faced by cell-based approaches of importing biomolecular components into the intracellular space, making these cell-free environments easily modified and excellent platforms for engineering.…”

Section: In Vitro Diagnosticsmentioning

confidence: 99%

Synthetic biology devices for in vitro and in vivo diagnostics

Slomovic

Pardee

Collins

2015

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

301

228

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There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.synthetic biology | diagnostics | biosensing | synthetic gene networks | nanobiotechnology

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“…These regulators are special because they can be wired together into RNA-only genetic circuits that propagate information as RNAs without the need to translate or degrade intermediate regulatory proteins 12 . Recently it has been shown that RNA transcriptional repressors, also known as attenuators 13 can be configured into NOR logic gates and transcriptional cascades 12 . They can also be used to construct more sophisticated circuits, such as single-input modules 14 that sequentially activate the expression of multiple target genes 15 . Advances in RNA engineering approaches have also greatly expanded the types of circuits that can be built out of RNA mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Generating Effective Models and Parameters for RNA Genetic Circuits

Varner

Lucks

2015

ACS Synth. Biol.

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RNA genetic circuitry is emerging as a powerful tool to control gene expression. However, little work has been done to create a theoretical foundation for RNA circuit design. A prerequisite to this is a quantitative modeling framework that accurately describes the dynamics of RNA circuits. In this work, we develop an ordinary differential equation model of transcriptional RNA genetic circuitry, using an RNA cascade as a test case. We show that parameter sensitivity analysis can be used to design a set of four simple experiments that can be performed in parallel using rapid cell-free transcription-translation (TX-TL) reactions to determine the 13 parameters of the model. The resulting model accurately recapitulates the dynamic behavior of the cascade, and can be easily extended to predict the function of new cascade variants that utilize new elements with limited additional characterization experiments. Interestingly, we show that inconsistencies between model predictions and experiments led to the model-guided discovery of a previously unknown maturation step required for RNA regulator function. We also determine circuit parameters in two different batches of TX-TL, and show that batch-to-batch variation can be attributed to differences in parameters that are directly related to the concentrations of core gene expression machinery. We anticipate the RNA circuit models developed here will inform the creation of computer aided genetic circuit design tools that can incorporate the growing number of RNA regulators, and that the parametrization method will find use in determining functional parameters of a broad array of natural and synthetic regulatory systems.

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Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems

Cited by 165 publications

References 49 publications

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

Synthetic biology devices for in vitro and in vivo diagnostics

Generating Effective Models and Parameters for RNA Genetic Circuits

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