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Project

Chemoenzymatic labelling of hybridization probes for spatially super-evolved omics

Microscopy and more specifically fluorescence microscopy is a beloved tool of many scientists to study small biological structures that cannot be resolved by the human eye. Still, even regular fluorescence microscopy is limited in resolution and detection of biomolecules that reside at the nanometer level require an improvement in resolving power. As a solution, super resolution microscopy has been developed over the years, realizing visualization of these ultra-small structures in different ways. In 2015, the lab of Edward Boyden at MIT established a novel and straightforward method to accomplish nanoscale resolution on a conventional diffraction limited microscope, called expansion microscopy. By infusing biological samples with suitable monomers, a super-absorbent polymer can be formed throughout the sample, which can be expanded and produce a perfectly transparent matrix. In order to preserve their original geometry, the biomolecules of interest are cross-linked inside this polymer, allowing not only to image the sample with an increased resolution but also retain spatial information to study these molecules within their cellular context.

In this research, we will make use of expansion microscopy to investigate biological questions in various scientific fields ranging from membrane studies and epigenetics to virology studies. We demonstrate the development of trifunctional labels to target, anchor and detect biomolecules simultaneously inside the hydrogel network. This allowed expansion of membrane structures or the use of small labeling molecules like phalloidin, which was not possible before in expansion microscopy. Interestingly, we also show how different lipid bilayers inside the cells are targeted when another reporter, in this case a fluorescent dye, is used. Next, we move away from the more structural studies performed and dive deeper into cell nuclei to study the epigenome. We aimed to develop a method to gain information on interactions happening between an epigenetic reader and several epigenetic histone modifications. This was achieved via a combined technique of immunostaining, expansion microscopy and co-localization. We call this method expansion microscopy for epigenetics or ExEpi. Two different epigenetic readers, BRD4 and LEDGF, are studied with more detail to validate the technique and their preferred epigenetic modifications, H3K9/14ac and H3K36me3 respectively, are confirmed by ExEpi. When an increased concentration of the BET-inhibitor JQ1 was added to the assay, we also illustrated a decrease in co-localization between BRD4 and H3K9/14ac. Therefore we hypnotize this method could be used in the future as an epigenetic drug discovery assay. Finally, expansion microscopy is used as a tool to investigate two different retroviruses, MLV and HIV-1. More specifically, we look into interactions between MLV viruses and histone acetylation by adapting our ExEpi technique for localization of fluorescent MLV pre-integration complexes instead of epigenetic readers. As such, a comparative study between MLV wild-type vectors and MLV BET-independent vectors was executed to analyze potential distribution differences inside the nucleus. In the end, we implemented expansion microscopy in single-virus analysis of HIV-1 and investigate nuclear entry. For this, different structures inside the cell were targeted such as the nuclear pore complex and nuclear lamin but also the nuclear membrane was stained with our own trifunctional labels. In addition, a new variation of expansion microscopy was tested to increase our expansion factor and thus final resolution even more.

In conclusion, expansion microscopy was used as a clever tool to easily investigate different biomolecules inside single-cells. This allowed us to study an array of biological questions at the nanoscale, all performed on regular diffraction limited microscopes and with relatively simple adaptations to the original expansion protocol.

 

Date:31 Aug 2017 →  3 Feb 2022
Keywords:Chemoenzymatic labelling, 3D microscopy
Disciplines:Sustainable chemistry, Inorganic chemistry, Organic chemistry, Theoretical and computational chemistry, Other chemical sciences, Physical chemistry
Project type:PhD project