Live-Cell Imaging of Sterculic Acid-a Naturally Occurring 1,2-Cyclopropene Fatty Acid-by Bioorthogonal Reaction with Turn-On Tetrazine-Fluorophore Conjugates

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Automated summary

In the field of lipid research, bioorthogonal chemistry has enabled the study of lipid uptake and processing in living systems with minimal interference from biological properties. This study introduces a new bioorthogonal probe for investigating unsaturated free fatty acids in live cells. The probe is taken up by dendritic cells without toxicity and can be visualized using a specific reaction. It can also be used to identify changes in protein oleoylation after immune cell activation. Moreover, it can be integrated into a multiplexed bioorthogonal reaction workflow for simultaneous imaging of multiple biomolecules in the cell using confocal microscopy.