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In the scientific world of biochemical research there is a need for uncovering the function of proteins and RNA molecules, as well as their interactions. This can be achieved by determining their location in time and space in the cell. This can be accomplished by using photoactivation dependent proximity labeling (PDPL). PDPL encompasses techniques that use illumination to generate reactive oxygen species which will oxidize proteins and nucleotides near the area of their production. In this thesis we focused on a photosensitizer called dibromofluorescein (DBF) which after illumination produces singlet oxygen that leads to the labeling of specific amino acids and nucleotides. We examined DBF’s ability to produce singlet oxygen, as well as the effect of different probes such as 4-ethynylaniline (EA) and propargylamine (PA) in the labeling of the proteins. According to our findings, DBF was able to produce 2.67 x 10^19 molecules of singlet oxygen when illuminated for 15 minutes. When we used EA, no labeling of the BSA protein was detected. However, when PA was used, labeling could be observed. Still, having expected the signal to be more intense, we concluded that the labeling was incomplete. Moreover, we observed that the oxidized form of the protein is stable and persists after the illumination is completed. Finally, we found evidence to suggest that the buffer used can influence the labeling efficiency of the technique. Although much remains to be done, for the technique to be characterized and repeatable, we have reached a point where its main principles have been studied.

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Engineering sciences. Technology