A new signal-amplifying tool at Stanford detects small changes in protein abundance by converting tiny differences into large, detectable signals, enabling more sensitive, high-throughput measurements.

Early demonstrations show the tool can amplify signals from degradation of target proteins, providing a clearer view of biology that was previously obscured by low abundance.

Led by Steven Banik, Stanford researchers report a tunable system that links target protein degradation to amplified outputs, addressing a long-standing sensitivity limitation in protein measurement.

In high-throughput drug discovery, the platform was used to identify molecular glue degraders targeting oncogenic proteins, illustrating its potential to streamline development of protein-degrading therapeutics.

Development involved integrating cellular engineering with high-throughput screening at Stanford’s Nucleus facility, in collaboration with researchers like David Solow-Cordero and Melissa Gray.

The system supports multiple readouts and is tunable, with options such as different colored fluorescent outputs or RNA barcodes readable by sequencing, increasing versatility.

This tunable design could enable independent multi-color readouts or alternative outputs, facilitating complex studies of protein interactions and disease mechanisms.

In a proof-of-concept, a target protein fused to an inhibitor of a transcription factor yields a measurable output as the target and inhibitor degrade and the transcription factor activates a reporter gene.

Multiple independent RTA circuits were built in single cells and paired with different reporters to monitor several protein-degradation events simultaneously, enabling multiplexed readouts.

RTA can detect degradation across multiple mechanisms at endogenous protein levels and link degradation to outputs like fluorescent signals, cell survival, or RNA barcodes.

Sensitivity depends on the ratio of the inhibitory fusion to the transcription factor, not just target abundance, allowing tunable and multiplexable signaling.

The platform enables high-throughput detection and parallel testing of hundreds of proteins and drugs, boosting efficiency in discovery and mechanistic studies.