Design and Application of Probes to Understand Peroxisomal Localisation of BODIPYs

Abstract

BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) constitute an important class of fluorophores. The excellent qualities of BODIPY comprise relatively high fluorescence quantum yields and molar absorption coefficients, narrow emission bandwidths, high elevated stability towards chemicals and light, and the extra feature of excitation/emission wavelengths in the visible region.
Previous work in our group generated a small set of BODIPY fluorophores, including one, nitro-BODIPY, 1,3,5,7‐tetramethyl-8‐(4‐nitrophenyl)‐BODIPY, that is intrinsically non-fluorescent but upon incubation of cells with this compound, fluorescence is observed at the target site. The target site in plant cells was identified as peroxisomes, as verified by co-localization with an SKL-FP construct (peroxisome-specific carboxyl-terminal targeting sequence fluorescent proteins). It is key to future applications to be able to understand how this is occurring at a molecular level.
These objectives are challenged by a lack of knowledge of this protein target and the physicochemical profile of nitro-BODIPY. This leads to the specific aims of this project which are: 1. To develop a ‘toolbox’ of analogues that enable multicolour visualisation. 2. To confirm the nature of the binding process between nitro-BODIPY and the target protein and to determine its reversibility. 3. To identify the target protein of nitro-BODIPY in plant peroxisomes.
Therefore, a family of red-shifted nitro-BODIPY probes were synthesised. Most of them showed selectivity towards plant peroxisomes and their emission wavelength could reach to a maximum of around 700 nm. Water-soluble probes were prepared and proved to exhibit selective labelling towards plant peroxisomes as well. In addition, a group of dual-fluorophore probes (nitro-BODIPY probes with a second fluorophore including Rhodamine B, Cy5, dansyl and NBD) were developed to determine whether nitro-BODIPY only localizes at peroxisome or somewhere else. Although several dual-fluorophore probes showed selective labelling of the peroxisome, the reporter function of the second fluorophore in all probes was challenged by various degrees of FRET. Finally, a photoaffinity labelling probe (nitro-BODIPY core attatched with a benzophenone photoaffinity group) was prepared to determine the target proteins of nitro-BODIPY in plant peroxisomes. Several potential protein targets of nitro-BODIPY in plant peroxisomes were detected by this photoaffinity labelling probe via SDS-PAGE technique.