Researchers have developed a new way to detect small decreases in proteins inside living cells, creating an amplified signal that could make it easier to discover drugs that eliminate disease-causing proteins.
The approach, called ratiometric transcriptional activation (RTA), converts the degradation of a target protein into increased gene expression. Unlike conventional methods in which protein loss produces a weaker signal, RTA generates a signal that becomes stronger as the target protein disappears. Researchers at Stanford University report the platform in Cell.
Detecting changes in protein abundance matters for both understanding cellular biology and developing targeted protein degraders, an emerging class of drugs designed to eliminate harmful proteins rather than simply block their activity. Such drugs could offer new ways to target proteins that have historically been difficult to inhibit with conventional medicines.
Existing methods, however, typically measure protein degradation through loss of signal. That can make relatively small changes difficult to detect, particularly when a protein is naturally present at low levels.
“A method that releases the constraints on protein abundance, bypasses the direct correlation of protein depletion to loss-of-signal, and provides a tunable and multiplexable signal upon target protein downregulation could enable sensitive detection of protein regulators and programmable biological outputs at scale,” the authors wrote.
RTA is designed to accomplish that by reversing the usual relationship between protein abundance and signal.
The researchers created genetic circuits in which a protein of interest is fused to an inhibitor that suppresses a transcription factor. As the target protein and its attached inhibitor are degraded, the transcription factor is freed to activate expression of a chosen gene.
Critically, the system’s sensitivity depends on the ratio between the inhibitory protein fusion and the transcription factor, rather than simply on the target protein’s abundance. By adjusting that ratio, the researchers could amplify relatively small changes in protein levels into much larger transcriptional signals.
The team demonstrated that RTA could detect protein loss caused by several different degradation mechanisms and sense changes in proteins at endogenous, or naturally occurring, levels. Protein degradation could also be connected to different outputs, including fluorescent signals, cell survival and RNA barcodes detectable by sequencing.
The researchers built three independent RTA circuits within individual cells and pooled cells carrying different reporters, allowing simultaneous monitoring of multiple protein-degradation events.
That multiplexing capability could be particularly useful for drug discovery. In high-throughput experiments, the researchers applied RTA to oncogenic targets and used the platform to identify molecular glue degraders—small molecules that induce interactions leading to the destruction of target proteins.
“RTA, with the ability to multiplex, sense fine degrees of change, and amplify signal, provides a robust and uniform platform to discover target degrader or destabilizing molecules directly from screens,” the authors wrote.
The findings suggest that converting subtle protein loss into an amplified, programmable signal could provide a scalable way to identify new protein-degrading drugs while also revealing previously difficult-to-detect changes in cellular protein regulation.
