Efforts and Challenges in Engineering the Genetic Code

Lin, Xiao; Yu, Allen Chi Shing; Chan, Ting‐Fung

doi:10.3390/life7010012

Cited by 17 publications

(22 citation statements)

References 101 publications

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“…19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1. 13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1. 26…”

Section: Scheme S3unclassified

“…However, the considerably large fusion proteins (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) may perturb the functions of POIs. The challenges of UAA incorporation in mammalian cells also hindered its wide applications 13 . Most importantly, these methods can only be applied to cell types that are amenable to genetic manipulation.…”

Section: Introductionmentioning

confidence: 99%

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A Visible and Near-Infrared Light Activatable Diazocoumarin Probe for Fluorogenic Protein Labeling in Living Cells

Dai

Yang

2020

J. Am. Chem. Soc.

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Chemical modification of proteins in living cells permits valuable glimpses into the molecular interactions that underpin dynamic cellular events. While genetic engineering methods are often preferred, selective labeling of endogenous proteins in a complex intracellular milieu with chemical approaches represents a significant challenge. In this study, we report novel diazo-coumarin compounds that can be photo-activated by visible (430-490 nm) and near-infrared light (800 nm) irradiation to photo-uncage reactive carbene intermediates, which could subsequently undergo insertion reaction with concomitant fluorescence "turned-on". With these new molecules in hand, we have developed a new approach for rapid, selective and fluorogenic labeling of endogenous protein in living cells. By using CA-II and eDHFR as model proteins, we demonstrated that subcellular localization of proteins can be precisely visualized by live-cell imaging and protein levels can be reliably quantified in multiple cell types using flow cytometry. Dynamic protein regulations such as hypoxia induced CA-IX accumulation can also be detected. In addition, by two-photon excitation with an 800 nm laser, cell-selective labeling can also be achieved with spatially controlled irradiation. Our method circumvents the cytotoxicity of UV light and obviates the need for introducing external reporters with "click chemistries". We believe that this approach of fluorescence labeling of endogenous protein by bioorthogonal photo-irradiation opens up exciting opportunities for discoveries and mechanistic interrogation in chemical biology. ASSOCIATED CONTENT Supporting Information Supporting figures, tables, experimental procedures, synthesis, characterization of compounds, LC/MS/MS spectra and NMR spectra.

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Section: Scheme S3unclassified

Section: Introductionmentioning

confidence: 99%

A Visible and Near-Infrared Light Activatable Diazocoumarin Probe for Fluorogenic Protein Labeling in Living Cells

Dai

Yang

2020

J. Am. Chem. Soc.

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“…Since we are aiming to highlight standard amino acid repertoire establishment in the genetic code from the viewpoint of protein structural requirements, we apologize to all colleagues whose excellent works covering other aspects of genetic code evolution and its engineering are not mentioned here (see e.g. ).…”

Section: Code Engineering and Synthetic Life – Does It Fit Together?mentioning

confidence: 99%

Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?

Kubyshkin

Budiša

2017

Biotechnology Journal

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The main goal of synthetic biology (SB) is the creation of biodiversity applicable for biotechnological needs, while xenobiology (XB) aims to expand the framework of natural chemistries with the non-natural building blocks in living cells to accomplish artificial biodiversity. Protein and proteome engineering, which overcome limitation of the canonical amino acid repertoire of 20 (+2) prescribed by the genetic code by using non-canonic amino acids (ncAAs), is one of the main focuses of XB research. Ideally, estranging the genetic code from its current form via systematic introduction of ncAAs should enable the development of bio-containment mechanisms in synthetic cells potentially endowing them with a "genetic firewall" i.e. orthogonality which prevents genetic information transfer to natural systems. Despite rapid progress over the past two decades, it is not yet possible to completely alienate an organism that would use and maintain different genetic code associations permanently. In order to engineer robust bio-contained life forms, the chemical logic behind the amino acid repertoire establishment should be considered. Starting from recent proposal of Hartman and Smith about the genetic code establishment in the RNA world, here the authors mapped possible biotechnological invasion points for engineering of bio-contained synthetic cells equipped with non-canonical functionalities.

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“…However, the considerably large fusion proteins (19-33 kDa) may perturb the functions of POIs. The challenges of UAA incorporation in mammalian cells also hindered its wide applications 13 . Most importantly, these methods can only be applied to cell types that are amenable to genetic manipulation.…”

Section: Introductionmentioning

confidence: 99%

A Visible and Near-Infrared Light Activatable Diazo-Coumarin Probe for Fluorogenic Protein Labeling in Living Cells

Dai¹,

Yang

2020

Preprint

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Chemical modification of proteins in living cells permits valuable glimpses into the molecular interactions that underpin dynamic cellular events. While genetic engineering methods are often preferred, selective labeling of endogenous proteins in a complex intracellular milieu with chemical approaches represents a significant challenge. In this study, we report novel diazo-coumarin compounds that can be photo-activated by visible (430‒490 nm) and near-infrared light (800 nm) irradiation to photo-uncage reactive carbene intermediates, which could subsequently undergo insertion reaction with concomitant fluorescence “turned-on”. With these new molecules in hand, we have developed a new approach for rapid, selective and fluorogenic labeling of endogenous protein in living cells. By using CA-II and eDHFR as model proteins, we demonstrated that subcellular localization of proteins can be precisely visualized by live-cell imaging and protein levels can be reliably quantified in multiple cell types using flow cytometry. Dynamic protein regulations such as hypoxia induced CA-IX accumulation can also be detected. In addition, by two-photon excitation with an 800 nm laser, cell-selective labeling can also be achieved with spatially controlled irradiation. Our method circumvents the cytotoxicity of UV light and obviates the need for introducing external reporters with “click chemistries”. We believe that this approach of fluorescence labeling of endogenous protein by bioorthogonal photo-irradiation opens up exciting opportunities for discoveries and mechanistic interrogation in chemical biology.

show abstract

Efforts and Challenges in Engineering the Genetic Code

Cited by 17 publications

References 101 publications

A Visible and Near-Infrared Light Activatable Diazocoumarin Probe for Fluorogenic Protein Labeling in Living Cells

A Visible and Near-Infrared Light Activatable Diazocoumarin Probe for Fluorogenic Protein Labeling in Living Cells

Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?

A Visible and Near-Infrared Light Activatable Diazo-Coumarin Probe for Fluorogenic Protein Labeling in Living Cells

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