Detection and study of protein–DNA interactions are of great importance for they govern many cellular processes. Interactions might be stable or transient, strong or weak and depend on several conditions which can influence our experimental results. There are two major limitations of classical methods to detect protein interactions partners of a known DNA. First, they do not allow us to study interactions under in vivo conditions, and second, they are not suitable for detection of transient and weak ones. With the aim of making those two things possible we set on to develop a novel approach for DNA-protein partners discovery, which is based on a three-component system that enables enzyme-catalysed proximity labeling of proteins with biotin and also serves as a practical tool to perform chromatin immunoprecipitation. The system consists of a nucleotide fragment F-DNA and two chimeric proteins, namely Tus-link72-TurboID and GAL4(1–147)-link45-sfGFP. F-DNA represents the core element of the system, which contains DNA sequence or secondary structure of interest and enables colocalization of its interaction partners with chimeric proteins. Tus-link72-TurboID contains a mutant biotin ligase which biotinylates colocalised proteins on F-DNA, whereas GAL4(1–147)-link45-sfGFP represents the internal control of the system. The system can be used either in vivo or in vitro. To test its functionality, we constructed a proof-of-concept in vitro system involving a known interaction partner. However, we were not able to detect target proteins after their isolation. Our hypothesis is that the recombinant biotin ligase in Tus-link72-TurboID has slower kinetics as we expected based on its activity in vivo. Before proceeding with optimization of the system, we should therefore first test activity of the enzyme and determine its kinetics. In order to make the system useful and commercially available, we should also optimize the preparation of its components.
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