Final Report Summary - NUPTAG (Protein-engineering based approach for super-resolution imaging of nucleoporin-chromatin interactions)
Recent developments of super-resolution microscopy (SRM) techniques allow us to follow biological processes with nanometer resolution. The impact of such techniques on biological studies is enormous as we can now look at single fluorescently labeled molecules inside the cells. This has also been recognized by the 2014 Nobel prize in chemistry which was awarded for the development of super-resolved fluorescence microscopy techniques. Continuous developments of optics, instrumentation, and software, lead to further improvements of these techniques, pushing the limits of light microscopy based studies. However, all fluorescence based techniques come with a demand for an optimal labeling method to make the underlying biological process visible under the light microscope. Labeling tags with suitable properties for the respective SRM method should be used and the tag should be small such that labeling density can be as high as possible. This makes small synthetic dyes very attractive labeling tags but their direct attachment to proteins in a specific way is not straight-forward.
Goal of this project was to develop new protein labeling methods for SRM with small dyes. I combined state-of-the-art tools of chemical biology and protein engineering to achieve this. By using genetic code expansion and very fast chemical reactions, known as click-chemistry, I developed a method for live mammalian cell protein multi-color labeling for SRM. Genetic code expansion is used to introduce functional groups into proteins of interest. In a subsequent step, these functional groups are selectively labeled with a dye of choice via nontoxic click-chemistry reactions.
To the best of my knowledge, this work represents the first evidence of SRM based on click-chemistry protein labeling by means of genetic code expansion. To make it more versatile, I achieved dual-color labeling by combining two very fast click-reactions in a way that was not described before. The methodology was published in several journal articles and a review and was also used as a basis for our patent application. It opens many possibilities for labeling of cells in a non-invasive way. We showed the potential of the technique by labeling distinct populations of membrane proteins, such as insulin receptors, and viral like particles. In the meantime, we are working on the improvement of the method, and we can also label intracellular proteins such as cytoskeleton, etc.
This methodology has raised a lot of interest in the scientific community as it represents the smallest available protein labeling tag. Furthermore, labeling is done in living cells on a rapid timescale, which is crucial for studies of dynamic biological processes. Material necessary for implementation of this method has been shipped to many labs world-wide and we started many collaborations. We are continuously working on the improvement of the method in order to make it more optimal for very demanding biological applications. We also wrote a protocol paper to make it easier for non-specialized laboratories to adapt the labeling for their studies.
Goal of this project was to develop new protein labeling methods for SRM with small dyes. I combined state-of-the-art tools of chemical biology and protein engineering to achieve this. By using genetic code expansion and very fast chemical reactions, known as click-chemistry, I developed a method for live mammalian cell protein multi-color labeling for SRM. Genetic code expansion is used to introduce functional groups into proteins of interest. In a subsequent step, these functional groups are selectively labeled with a dye of choice via nontoxic click-chemistry reactions.
To the best of my knowledge, this work represents the first evidence of SRM based on click-chemistry protein labeling by means of genetic code expansion. To make it more versatile, I achieved dual-color labeling by combining two very fast click-reactions in a way that was not described before. The methodology was published in several journal articles and a review and was also used as a basis for our patent application. It opens many possibilities for labeling of cells in a non-invasive way. We showed the potential of the technique by labeling distinct populations of membrane proteins, such as insulin receptors, and viral like particles. In the meantime, we are working on the improvement of the method, and we can also label intracellular proteins such as cytoskeleton, etc.
This methodology has raised a lot of interest in the scientific community as it represents the smallest available protein labeling tag. Furthermore, labeling is done in living cells on a rapid timescale, which is crucial for studies of dynamic biological processes. Material necessary for implementation of this method has been shipped to many labs world-wide and we started many collaborations. We are continuously working on the improvement of the method in order to make it more optimal for very demanding biological applications. We also wrote a protocol paper to make it easier for non-specialized laboratories to adapt the labeling for their studies.