University of Pittsburgh researchers have developed a novel method to rapidly generate single-molecule information for fluorescently tagged proteins isolated from nuclear extracts of human cells.
Description
Understanding how proteins detect and bind to targets in real-time can allow for unprecedented insight into biological mechanisms. This method uses multiple fluorescent color tagged proteins where interactions are simultaneously measured to characterize the assembly and disassembly kinetics of multi-protein complexes on DNA.
Applications
1. Analysis of interaction of DNA-binding proteins with DNA substrates in real time.
2. Gaining mechanistic insight into diverse protein-DNA and protein-protein interactions.
3. Rapid screening for structural variants of proteins linked to tumors.
Advantages
Current single-molecule fluorescence studies of DNA-binding proteins require overexpression, purification and labeling with a fluorescent tag of a targeted protein. It is time consuming and can prove difficult for some proteins due to loss of activity and may not fully represent how these proteins interact in a complex cell. In vivo imaging of DNA-binding is technically possibly but has proven challenging, particularly watching DNA-binding proteins find their specific binding sites.
Single-Molecule Analysis of DNA-Binding Proteins from Nuclear Extracts (SMADNE) combines both approaches, providing the specificity of purified proteins without the need for costly purification techniques. SMADNE utilizes readily available fluorescent tags and multiple targets can be labelled to better study interacting proteins.
Invention Readiness
Laboratory experiments have shown SMADME can provide new insights about DNA damage recognition and is a universal technique that can be used to rapidly characterize numerous protein-DNA interactions. A workflow has been developed with scanning rates as fast as 6 msec per scan along the central DNA position.
Laboratory experiments used three forms of DNA damage to demonstrate how this approach can be successfully used to determine binding lifetimes, events per second, positional dependance (specificity) and characterization of 1D diffusion along DNA.
IP Status
https://patents.google.com/patent/WO2024035721A1
