Predictable control of RNA lifetime using engineered degradation-tuning RNAs

Zhang, Qi; Ma, Duo; Wu, Fuqing; Standage-Beier, Kylie; Chen, Xingwen; Wu, Kaiyue; Green, Alexander A.; Wang, Xiao

doi:10.1038/s41589-021-00816-4

Cited by 26 publications

(19 citation statements)

References 59 publications

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“…Adopting ctRSD circuits for these diverse applications will require overcoming challenges in controlling expression, degradation, and cleavage rates−especially in vivo. These issues could be addressed by optimizing 5′ hairpins to tune expression levels ( 30 ) or increase RNA stability ( 48 ), as well as exploring HDV ribozyme variants ( 49 ). Ultimately, ctRSD circuits are poised to be a versatile, enabling technology across many synthetic biology platforms.…”

Section: Discussionmentioning

confidence: 99%

Cotranscriptionally encoded RNA strand displacement circuits

Schaffter

Strychalski

2022

Sci. Adv.

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Engineered molecular circuits that process information in biological systems could address emerging human health and biomanufacturing needs. However, such circuits can be difficult to rationally design and scale. DNA-based strand displacement reactions have demonstrated the largest and most computationally powerful molecular circuits to date but are limited in biological systems due to the difficulty in genetically encoding components. Here, we develop scalable cotranscriptionally encoded RNA strand displacement (ctRSD) circuits that are rationally programmed via base pairing interactions. ctRSD circuits address the limitations of DNA-based strand displacement circuits by isothermally producing circuit components via transcription. We demonstrate circuit programmability in vitro by implementing logic and amplification elements, as well as multilayer cascades. Furthermore, we show that circuit kinetics are accurately predicted by a simple model of coupled transcription and strand displacement, enabling model-driven design. We envision ctRSD circuits will enable the rational design of powerful molecular circuits that operate in biological systems, including living cells.

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Section: Discussionmentioning

confidence: 99%

Cotranscriptionally encoded RNA strand displacement circuits

Schaffter

Strychalski

2022

Sci. Adv.

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“…Despite the benefit of the TPRS and TPAS systems for regulating metabolic pathways, as discussed in the present work, these systems depend on multiple proteins built from cellular resources and may cause a metabolic burden to cells. As a result, novel and adaptive strategies can be applied to creating robust and much simpler systems that may reduce cellular burdens [ 57 , 58 ]. Among those strategies, orthogonal DNA binding systems such as bZip, RNAi, or other synthetic DNA binding strategies to avoid non-specific recombination or protein aggregation before activation.…”

Section: Discussionmentioning

confidence: 99%

Bifunctional optogenetic switch for improving shikimic acid production in E. coli

Komera

Gao

Guo

et al. 2022

Biotechnol Biofuels

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Background Biomass formation and product synthesis decoupling have been proven to be promising to increase the titer of desired value add products. Optogenetics provides a potential strategy to develop light-induced circuits that conditionally control metabolic flux redistribution for enhanced microbial production. However, the limited number of light-sensitive proteins available to date hinders the progress of light-controlled tools. Results To address these issues, two optogenetic systems (TPRS and TPAS) were constructed by reprogramming the widely used repressor TetR and protease TEVp to expand the current optogenetic toolkit. By merging the two systems, a bifunctional optogenetic switch was constructed to enable orthogonally regulated gene transcription and protein accumulation. Application of this bifunctional switch to decouple biomass formation and shikimic acid biosynthesis allowed 35 g/L of shikimic acid production in a minimal medium from glucose, representing the highest titer reported to date by E. coli without the addition of any chemical inducers and expensive aromatic amino acids. This titer was further boosted to 76 g/L when using rich medium fermentation. Conclusion The cost effective and light-controlled switch reported here provides important insights into environmentally friendly tools for metabolic pathway regulation and should be applicable to the production of other value-add chemicals.

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“…Lastly, the system inherits the general advantages of RNA-based operations, including a fast response time, reduced resource usage, and multiplexing [46,91,92]. Recent developments on degradation-tunable RNAs in combination with toehold switches may provide further design flexibility [93]. Notably, a variety of ALU circuits using DNA strand displacement reactions [24] showcases the power of nucleic-acid-based molecular computations.…”

Section: Discussionmentioning

confidence: 99%

Cellular Computational Logic Using Toehold Switches

Choi

Lee

Kim

2022

IJMS

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The development of computational logic that carries programmable and predictable features is one of the key requirements for next-generation synthetic biological devices. Despite considerable progress, the construction of synthetic biological arithmetic logic units presents numerous challenges. In this paper, utilizing the unique advantages of RNA molecules in building complex logic circuits in the cellular environment, we demonstrate the RNA-only bitwise logical operation of XOR gates and basic arithmetic operations, including a half adder, a half subtractor, and a Feynman gate, in Escherichia coli. Specifically, de-novo-designed riboregulators, known as toehold switches, were concatenated to enhance the functionality of an OR gate, and a previously utilized antisense RNA strategy was further optimized to construct orthogonal NIMPLY gates. These optimized synthetic logic gates were able to be seamlessly integrated to achieve final arithmetic operations on small molecule inputs in cells. Toehold-switch-based ribocomputing devices may provide a fundamental basis for synthetic RNA-based arithmetic logic units or higher-order systems in cells.

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Predictable control of RNA lifetime using engineered degradation-tuning RNAs

Cited by 26 publications

References 59 publications

Cotranscriptionally encoded RNA strand displacement circuits

Cotranscriptionally encoded RNA strand displacement circuits

Bifunctional optogenetic switch for improving shikimic acid production in E. coli

Cellular Computational Logic Using Toehold Switches

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