Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies

Meyer, Sarai; Chappell, James; Sankar, Sitara B.; Chew, Rebecca; Lucks, Julius B.

doi:10.1002/bit.25693

Cited by 39 publications

(32 citation statements)

References 48 publications

(98 reference statements)

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“…All tested constructs in this study are engineered combining known RNA sequences, e.g., attenuator and terminator sequences, and building their complements. In a followup publication the Lucks lab investigated the possibility to further increase the effect of the worst performing pbuE design [ 126 ]. Here again known parts such as promoter sequences of varying strength, stabilizing 5

stems and sequence scaffolds taken from naturally occurring sRNAs were used in a plug and play manner.…”

Section: Practical Designsmentioning

confidence: 99%

Design of Artificial Riboswitches as Biosensors

Findeiß

Etzel

Will

et al. 2017

Sensors

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RNA aptamers readily recognize small organic molecules, polypeptides, as well as other nucleic acids in a highly specific manner. Many such aptamers have evolved as parts of regulatory systems in nature. Experimental selection techniques such as SELEX have been very successful in finding artificial aptamers for a wide variety of natural and synthetic ligands. Changes in structure and/or stability of aptamers upon ligand binding can propagate through larger RNA constructs and cause specific structural changes at distal positions. In turn, these may affect transcription, translation, splicing, or binding events. The RNA secondary structure model realistically describes both thermodynamic and kinetic aspects of RNA structure formation and refolding at a single, consistent level of modelling. Thus, this framework allows studying the function of natural riboswitches in silico. Moreover, it enables rationally designing artificial switches, combining essentially arbitrary sensors with a broad choice of read-out systems. Eventually, this approach sets the stage for constructing versatile biosensors.

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stems and sequence scaffolds taken from naturally occurring sRNAs were used in a plug and play manner.…”

Section: Practical Designsmentioning

confidence: 99%

Design of Artificial Riboswitches as Biosensors

Findeiß

Etzel

Will

et al. 2017

Sensors

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“…While directly engineering natural regulators for improved function within these applications is showing promise, our incomplete understanding of the complex mechanisms underlying many natural RNA regulators hinders our ability to quickly design them de novo. The puzzling nature of these mechanisms has sparked an increased interest in uncovering RNA structure-function design principles that can be used to efficiently engineer large libraries of RNA regulators with optimized ) and sometimes expanded function (Chappell et al 2015a,b;Meyer et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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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“…[23a,d] Similarly,s ynthetic transacting RNAs,s uch as small RNAs (sRNAs), have been engineered to control bacterial transcription by preventing the formation of integrated terminator hairpins within the mRNAm olecule of interest. [39] Post-translational switches act on the protein product itself and regulate its function by influencing protein stability, localization, and splicing. [40] Several ligand-responsive protein-degradation technologies have been developed that are based on tagging the protein of interest with adestabilization domain called adegron.…”

Section: Gene Switchesmentioning

confidence: 99%

Synthetic Biology—The Synthesis of Biology

2017

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Synthetic biology concerns the engineering of man-made living biomachines from standardized components that can perform predefined functions in a (self-)controlled manner. Different research strategies and interdisciplinary efforts are pursued to implement engineering principles to biology. The "top-down" strategy exploits nature's incredible diversity of existing, natural parts to construct synthetic compositions of genetic, metabolic, or signaling networks with predictable and controllable properties. This mainly application-driven approach results in living factories that produce drugs, biofuels, biomaterials, and fine chemicals, and results in living pills that are based on engineered cells with the capacity to autonomously detect and treat disease states in vivo. In contrast, the "bottom-up" strategy seeks to be independent of existing living systems by designing biological systems from scratch and synthesizing artificial biological entities not found in nature. This more knowledge-driven approach investigates the reconstruction of minimal biological systems that are capable of performing basic biological phenomena, such as self-organization, self-replication, and self-sustainability. Moreover, the syntheses of artificial biological units, such as synthetic nucleotides or amino acids, and their implementation into polymers inside living cells currently set the boundaries between natural and artificial biological systems. In particular, the in vitro design, synthesis, and transfer of complete genomes into host cells point to the future of synthetic biology: the creation of designer cells with tailored desirable properties for biomedicine and biotechnology.

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Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies

Cited by 39 publications

References 48 publications

Design of Artificial Riboswitches as Biosensors

Design of Artificial Riboswitches as Biosensors

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

Synthetic Biology—The Synthesis of Biology

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