Fluorogenic protein labeling using a genetically encoded unstrained alkene

Shang, Xin; Song, Xinyu; Faller, Christina E.; Lai, Rui; Li, H.; Cerny, Ronald L.; Niu, Wei; Guo, Jiantao

doi:10.1039/c6sc03635j

Cited by 54 publications

(44 citation statements)

References 41 publications

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“…In our previously report, an inverse electron-demand Diels-Alder (iEDDA) reaction between KStyr and tetrazine was examined for fluorogenic protein labeling. 10 It was intriguing that styrene could serve as a versatile reagent for two different fluorophore-forming bioorthogonal reactions. By employing our previously identified pyrrolysyl-tRNA synthetase (PylRS) mutant-tRNA Pyl pair, 10 we were able to obtained 25 mg/L of sfGFP mutant (sfGFP-Asn149KStyr) that contains KStyr at position Asn149.…”

Section: Resultsmentioning

confidence: 99%

“…10 It was intriguing that styrene could serve as a versatile reagent for two different fluorophore-forming bioorthogonal reactions. By employing our previously identified pyrrolysyl-tRNA synthetase (PylRS) mutant-tRNA Pyl pair, 10 we were able to obtained 25 mg/L of sfGFP mutant (sfGFP-Asn149KStyr) that contains KStyr at position Asn149. In a typical labeling experiment, sfGFP-Asn149KStyr was incubated with 500 μM tetrazole in PBS buffer under UV irradiation at 302 nm.…”

Section: Resultsmentioning

confidence: 99%

“…10 Cells were grown on a LB agar plate containing 100 mg/L ampicillin and 34 mg/L chloramphenicol at 37 °C overnight. A single colony from the plate was inoculated into 5 mL LB media supplemented with 100 mg/L ampicillin and 34 mg/L chloramphenicol.…”

Section: Methodsmentioning

confidence: 99%

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Improved Photoinduced Fluorogenic Alkene–Tetrazole Reaction for Protein Labeling

et al. 2017

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The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene-tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene-tetrazole reaction product. This finding provides a useful guidance to the future design of alkene-tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene-tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene-tetrazole chemistry in real-time imaging in live cells.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Improved Photoinduced Fluorogenic Alkene–Tetrazole Reaction for Protein Labeling

et al. 2017

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“…40 Protein expression was carried out in LB medium supplemented with and without 1 mM CbK. Fluorescence analysis of E. coli cultures showed that full-length sfGFP protein was produced only in the presence of CbK (Fig.…”

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confidence: 99%

“…With the purified sfGFP-N149CbK in hand, we explored the inverse Diels-Alder reaction between the recombinant sfGFP-N149CbK and a commonly used tetrazine scaffold 40–41 containing a fluorescein conjugate (Fl-Tet; Fig. 1).…”

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A genetically encoded cyclobutene probe for labelling of live cells

et al. 2017

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Mechanism‐Based Fluorogenic trans‐Cyclooctene–Tetrazine Cycloaddition

et al. 2016

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The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable,b ut challenging.R eported herein is an ew concept of fluorescent product formation upon the inverse electron-demand Diels-Alder reaction of 1,2,4,5-tetrazines with particular trans-cyclooctene (TCO) isomers.Insharp contrast to knownfluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4-dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5-tetrazine.W es upport the claim with NMR measurements and rationalizethe data by computational study. Cell-labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.

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Fluorogenic protein labeling using a genetically encoded unstrained alkene

Abstract: A new fluorogenic bioorthogonal reaction between styrene (an unstrained alkene) and a tetrazine was developed.

Cited by 54 publications

References 41 publications

Improved Photoinduced Fluorogenic Alkene–Tetrazole Reaction for Protein Labeling

Improved Photoinduced Fluorogenic Alkene–Tetrazole Reaction for Protein Labeling

A genetically encoded cyclobutene probe for labelling of live cells

Mechanism‐Based Fluorogenic trans‐Cyclooctene–Tetrazine Cycloaddition

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