Directed evolution of orthogonal RNA–RBP pairs through library-vs-library <i>in vitro</i> selection

Fukunaga, Kimitoshi; Yokobayashi, Yohei

doi:10.1093/nar/gkab527

Cited by 10 publications

(5 citation statements)

References 74 publications

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“…For RNA aptamers, given their generally small size (tens of nucleotides), it is possible to create fully randomized 20- to 40-nucleotide (nt) libraries using commercial DNA synthesis. These can then be transcribed and screened in vitro by systematic evolution of ligands using exponential enrichment (SELEX) [ 116 , 117 ]. Randomized 20-nt regions can also be fused to a catalytic RNA domains (ribozymes) [ 118 ] to create synthetic aptamers with novel functions (e.g., inducible self-cleaving ribozymes) [ 116 , 119 , 120 ].…”

Section: Discovery Of New Genetic Sensorsmentioning

confidence: 99%

Accelerating Genetic Sensor Development, Scale-up, and Deployment Using Synthetic Biology

Joshi,

Jenkins,

Ulaeto

et al. 2024

BioDesign Res

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Living cells are exquisitely tuned to sense and respond to changes in their environment. Repurposing these systems to create engineered biosensors has seen growing interest in the field of synthetic biology and provides a foundation for many innovative applications spanning environmental monitoring to improved biobased production. In this review, we present a detailed overview of currently available biosensors and the methods that have supported their development, scale-up, and deployment. We focus on genetic sensors in living cells whose outputs affect gene expression. We find that emerging high-throughput experimental assays and evolutionary approaches combined with advanced bioinformatics and machine learning are establishing pipelines to produce genetic sensors for virtually any small molecule, protein, or nucleic acid. However, more complex sensing tasks based on classifying compositions of many stimuli and the reliable deployment of these systems into real-world settings remain challenges. We suggest that recent advances in our ability to precisely modify nonmodel organisms and the integration of proven control engineering principles (e.g., feedback) into the broader design of genetic sensing systems will be necessary to overcome these hurdles and realize the immense potential of the field.

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Section: Discovery Of New Genetic Sensorsmentioning

confidence: 99%

Accelerating Genetic Sensor Development, Scale-up, and Deployment Using Synthetic Biology

Joshi,

Jenkins,

Ulaeto

et al. 2024

BioDesign Res

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“…To overcome this limitation, creating novel RNA – RBP pairs by redesigning existing pairs was thought to be a reasonable strategy. One recently established method by Fukunaga and Yokobayashi, PD-SELEX, combined SELEX with phage display (PD), an in vitro screening method to identify ligands for proteins displayed on phage particles [ 66–68 ], for the systematic co-evolution of RNA – RBP pairs ( Figure 3B ) [ 69 ]. In addition to a random RNA library, they generated a library of randomly mutated L7Ae ( Figure 1 ).…”

Section: Protein Sensorsmentioning

confidence: 99%

Sensing intracellular signatures with synthetic mRNAs

Ono,

Saito

2023

RNA Biology

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The bottom-up assembly of biological components in synthetic biology has contributed to a better understanding of natural phenomena and the development of new technologies for practical applications. Over the past few decades, basic RNA research has unveiled the regulatory roles of RNAs underlying gene regulatory networks; while advances in RNA biology, in turn, have highlighted the potential of a wide variety of RNA elements as building blocks to construct artificial systems. In particular, synthetic mRNA-based translational regulators, which respond to signals in cells and regulate the production of encoded output proteins, are gaining attention with the recent rise of mRNA therapeutics. In this Review, we discuss recent progress in RNA synthetic biology, mainly focusing on emerging technologies for sensing intracellular protein and RNA molecules and controlling translation.

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“…For example, Stapleton et al developed several L7Ae mutants with different affinities, although none of them showed higher translational repression than the wild type [22]. Recently, Fukunaga and Yokobayashi reported L7Ae mutants with different sequence selectivity, but their translational repression efficiency remains unknown [57].…”

Section: Protein Module Optimizationmentioning

confidence: 99%

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

Nakanishi

2021

Life

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Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits.

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Directed evolution of orthogonal RNA–RBP pairs through library-vs-library in vitro selection

Cited by 10 publications

References 74 publications

Accelerating Genetic Sensor Development, Scale-up, and Deployment Using Synthetic Biology

Accelerating Genetic Sensor Development, Scale-up, and Deployment Using Synthetic Biology

Sensing intracellular signatures with synthetic mRNAs

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

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