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A minimal Cys-cyclobutene non-canonical amino acid for bioorthogonal imaging of proteins in live cells

Objective

Insight into dynamic processes such as uptake and intracellular trafficking and signalling of small molecules and proteins in cells is key in basic biology and drug development. The use of bioorthogonal chemical reactions for labelling and probing specific biomolecules is an exciting option but a very challenging task as these transformations should proceed very rapidly in physiological conditions with complete chemoselectivity. So, in this application we propose to achieve site-selective protein labelling by the introduction of a non-canonical Cys-cyclobutene aminoacid and a very fast alkene chemoselective ligation reaction. The small size of the proposed alkene-tagged aminoacid should not interfere with the protein structure, function, activity or localisation in contrast with other fluorescent tags such as the use of fusion proteins as GFP. This project aims at 1) developing a new photo triggered [2+2] cycloaddition bioorthogonal ligation between two alkene containing partners and to apply the already established IEDDA reaction between tetrazines and dienophiles. While the use of light as a trigger offers the possibility for an improved spatial and temporal resolution, the use of tetrazine fluorogenic probes allows for ‘turn-on’ fluorescence on their rapid reaction with dienophiles 2) use these optimized reaction to study apoptosis in cells and tissues by introducing the proposed Cys-cyclobutene non-canonical amino acid into C2A Domain of Synaptotagmin-I followed by the ligation reaction ex vivo and in vivo

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Topic(s)

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Funding Scheme

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MSCA-IF-EF-ST - Standard EF

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Call for proposal

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(opens in new window) H2020-MSCA-IF-2015

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Coordinator

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 183 454,80
Address
TRINITY LANE THE OLD SCHOOLS
CB2 1TN CAMBRIDGE
United Kingdom

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Region
East of England East Anglia Cambridgeshire CC
Activity type
Higher or Secondary Education Establishments
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 183 454,80
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