MinBioTag · A minimal Cys-cyclobutene non-canonical amino acid for bioorthogonal imaging of proteins in live cells
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2016-04-01 → 2018-03-31
- EU contribution
- €183,455
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
A minimal Cys-cyclobutene non-canonical amino acid for bioorthogonal imaging of proteins in live cells
Nature has produced an intricate machinery to diversify the structure of proteins after their synthesis in the ribosome. Recently and in an attempt to mimic nature, reactions that allow for post-expression modification of proteins at selected sites had been developed. These reactions are used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they can be used to study and monitor disease-associated proteins or to conjugate cytotoxic molecules to antibodies to improve efficacy and reduce side effects of cancer treatments. The aim of my research is to develop a new non-natural small amino acid, tagged with a cyclobutene moiety, that can be introduced in proteins followed a very fast and selective chemical reaction. Establishment of this new chemical site-selective bioorthogonal protein labelling strategy will allow us, as a proof of principle, to modify the C2A Domain of Synaptotagmin-I in a chemoselective fashion with no toxicity for living cells in order to image apoptotic cells without interfere with the protein’s innate structure, function, activity and localisation as well as cellular functions.. We expect this project find wide application as the strategy here outline will allow to label and monitor proteins with a minimal interference on their function and localisation, creating a valuable future tool for chemists and biologists.
Data: CORDIS, © European Union
Project 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
Original text from CORDIS.
Participants
- THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE · CAMBRIDGECoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union