Proximity Proteolysis Detection via DNA Hybridization

The assay is divided into separate functional modules: DNA-protein conjugates serve as capture agents, while DNA-payload conjugates serve as signal generators. These modules operate independently in solution before being brought together through hybridization, eliminating the need for complex solid-phase binding and washing steps while maintaining detection sensitivity.

DNA molecules serve as intermediaries that bridge protein targets and detectable payloads. The DNA-protein conjugates and DNA-payload conjugates hybridize through complementary DNA sequences, enabling signal transduction without direct protein-payload interaction. This intermediary approach simplifies the assay procedure while preserving measurement precision.

The DNA-protein and DNA-payload conjugates are pre-prepared with complementary DNA sequences that enable specific hybridization. This preliminary preparation allows the actual detection to proceed rapidly through simple mixing and hybridization, reducing total assay time from over one day to significantly shorter durations while maintaining detection sensitivity.

The traditional mechanical steps of solid-phase binding, washing, and signal development are replaced with solution-phase hybridization reactions. The DNA complementarity-driven assembly occurs spontaneously in solution, eliminating time-consuming mechanical operations while preserving the precision of target detection.

Multiple assay functions are merged into solution-phase hybridization: target capture, signal generation, and signal transduction all occur simultaneously in a single reaction mixture. The DNA-protein and DNA-payload conjugates self-assemble through hybridization, eliminating the need for separate binding and washing steps, thereby improving ease of operation while maintaining detection sensitivity.

The assay components self-organize through DNA hybridization without requiring external manipulation. The complementary DNA sequences on the protein conjugates and payload conjugates drive spontaneous assembly around the target, eliminating the need for trained personnel to perform complex procedural steps while preserving measurement precision.

The DNA-protein and DNA-payload conjugates are designed with specific local properties: the DNA sequences are engineered for high-affinity, specific hybridization only at the intended target sites. This localized specificity ensures that hybridization occurs preferentially at the protein-DNA-payload complex rather than forming non-specific aggregates in solution, maintaining a high signal-to-background ratio while preserving ease of operation.

The assay uses composite DNA-protein conjugates where the DNA component provides specific recognition and the protein component provides target binding. This composite structure combines the advantages of nucleic acid specificity with protein target affinity, enabling high-precision detection in solution phase while maintaining operational simplicity.