Researchers at Colorado State University have developed a novel live-cell sensor system called Reader4 for detecting protein ubiquitylation in real time. This innovative two-part, split-protein system generates a fluorescent signal when a target protein (like a histone) is ubiquitinated. By using a reversible “splitFAST” approach, the sensor can monitor the dynamic changes of ubiquitylation within an intact cell. This advancement provides a powerful new tool for research into cellular processes and drug screening.
Ubiquitylation is a crucial post-translational modification involved in numerous cellular processes, including DNA repair and transcriptional regulation. Traditional detection methods typically require cell lysis, which prevents real-time monitoring of spatial and temporal dynamics within living cells. A significant challenge in the field has been the lack of specific antibodies or reagents for detecting ubiquitylation at specific residues, such as H2AK127. This new technology addresses this gap, enabling specific, real-time analysis of these modifications.
The system is an avidity-based biosensor that utilizes a split version of the Fluorescence-Activating and absorption-Shifting Tag (FAST) protein. One fragment of FAST (NFAST) is fused to a ubiquitin binding domain (UBD), and the other (CFAST) is fused to the target protein, such as histone H2A. When ubiquitylation occurs on the target protein, the UBD binds to the attached ubiquitin, bringing the two FAST fragments into close proximity. In the presence of a non-fluorescent fluorogen, like HBR-DOM2, the reconstituted FAST protein becomes fluorescent, providing a detectable signal indicative of the modification).
Optimization efforts, including using an RspA-NFAST variant, significantly improved the sensor’s performance by reducing the background signal from self-complementation, increasing the dynamic range from ~5-fold to over 7-fold. This allows for quantitative measurement of BRCA1-BARD1 ligase activity and detection of low-abundance endogenous ubiquitylation events. The system’s reversibility is a key advantage, allowing for the study of the dynamic equilibrium of ubiquitylation and deubiquitylation.
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TRL: 5
US Provisional Patent
Tingting Yao
Bob Cohen
Carolina dos Santos Passos
Steve Foster
Steve.Foster@colostate.edu
970-491-7100