Development of mammalian cell logic gates controlled by unnatural amino acids

Mills, Emily M.; Barlow, Victoria L.; Jones, Arwyn Tomos; Tsai, Yu‐Hsuan

doi:10.1016/j.crmeth.2021.100073

Cited by 12 publications

(5 citation statements)

References 57 publications

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“…These split o-aaRSs also serve as unique genetically encoded AND gates wherein two inputs (RAP and pIF or ABA and mIF) are required to achieve a desired output (stop codon read-through). Such genetically encoded digital logic gates are useful tools for engineering signaling pathways for synthetic biology ( 19 , 60 , 61 ).…”

Section: Resultsmentioning

confidence: 99%

“…To induce protein synthesis, an exogenous ncAA is provided—the o-aaRS attaches the ncAA onto the suppressor tRNA, which suppresses the in-frame stop codon, allowing full translation of the target gene ( 9 – 11 , 14 , 15 ). This type of extrinsic control of gene expression has been used for various applications including biocontainment of genetically modified organisms ( 16 – 18 ), to develop genetically encoded digital logic gates ( 19 , 20 ), and to control the expression of therapeutic proteins in living animals ( 7 ).…”

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confidence: 99%

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Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Jiang

Ambrose

Chung

et al. 2023

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

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Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein–protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Jiang

Ambrose

Chung

et al. 2023

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

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“…While not specifically evolved for mammalian cells, these quadruplet tRNAs were shown to be effective in incorporating a ncAA in HEK 293T cells, suggesting that anticodon arm engineering strategies are applicable to a wide range of systems ( Chen et al, 2018 ). Additional research ( Mills et al, 2021 ) screened examples of these previously-engineered quadruplet-decoding tRNA Pyl variants ( Niu et al, 2013 ; Wang et al, 2014 ; Wang et al, 2016 N. ; Chen et al, 2018 ) in HEK293 cells, and found that two of the tRNA variants (one evolved for mammalian cells, one with a simple quadruplet anticodon change) were suitable for use in ncAA implementation. Notably, engineered quadruplet tRNAs across all experiments were generally most effective only in the organism that were designed in.…”

Section: Improving Codon-anticodon Interactionsmentioning

confidence: 99%

Tuning tRNAs for improved translation

Weiss,

Decker,

Bolano

et al. 2024

Front. Genet.

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Transfer RNAs have been extensively explored as the molecules that translate the genetic code into proteins. At this interface of genetics and biochemistry, tRNAs direct the efficiency of every major step of translation by interacting with a multitude of binding partners. However, due to the variability of tRNA sequences and the abundance of diverse post-transcriptional modifications, a guidebook linking tRNA sequences to specific translational outcomes has yet to be elucidated. Here, we review substantial efforts that have collectively uncovered tRNA engineering principles that can be used as a guide for the tuning of translation fidelity. These principles have allowed for the development of basic research, expansion of the genetic code with non-canonical amino acids, and tRNA therapeutics.

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“…Metal nanoclusters (MNCs) involve basically a trivial amount of ten maximum atoms, [51,68] comprising either single/multiple elements measured commonly <2 nm unit. [46,55] They possessed and displayed striking chemical, electronic and magnetic, optical, and reactivity properties. [16,69] Such MNCs mostly exhibit an intense color instigating from their surface plasmon resonance.…”

Section: By Fluorescent Ncsmentioning

confidence: 99%

“…via β‐bromoalanine intermediate, [ 45 ] and so on. The NPAAs having similar structures with the popularly known PAAs consequently bind with AA‐tRNA synthetase at cell surfaces in abundant concentrations and erroneously form the peptide bonds [ 46 ] with no visible toxic effects (in vivo/in vitro) in the intracellular systems, thus developing an extensively misfolded polypeptide chain.…”

Section: Aas Structural and Chemical Propertiesmentioning

confidence: 99%

Fluorescent Nanomaterials for Detection and Discrimination of Amino Acids

Auwalu

Sun

et al. 2023

Advanced NanoBiomed Research

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The incessant surge and urge for amino acids (AAs) detection and discrimination has led scientists devoting extensive research efforts toward realizing an efficiently more robust methods/techniques for AAs recognition using different materials considering their crucial health impacts in the body system metabolism. Notably, fluorescent probes (FPs), as obviously known currently, are powerful noninvasive detection tools which are suitable and highly effective for biomolecules recognition displaying high‐resolution images. The last decade has witnessed exciting achievements in AAs sensing via the use of FPs and integrated probes with colorimetric sensors reviewed in 2012. However, the conducted study discovers that more advance and effective sensing along with innovative segregation methods for AAs are yet to be vastly unveiled owing to discrimination difficulties among various AAs. As a result, herein, the strategic study accounts for some recent progressive achievements made from 2013 to date for AAs recognition and discrimination via the use of fluorescent nanomaterials (FNMs) of nanoparticles, nanoclusters, and nanodots along with quantum dots (QDs) in different ways. The study concludes by outlining the sensitivity and selectivity extent of AAs, the FNMs, and QDs supramolecular chemistry studies for cancer cells, and finally their status quo based on the views and assumed perspectives.

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Development of mammalian cell logic gates controlled by unnatural amino acids

Cited by 12 publications

References 57 publications

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Tuning tRNAs for improved translation

Fluorescent Nanomaterials for Detection and Discrimination of Amino Acids

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