Non-Hydrolysable ADP-Ribose Probe for High-Throughput Binder Detection
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Solution Overview
Problem
Current assays for detecting ADP-ribosylated substrates are not suitable for non-hydrolysing binders of ADP-ribose and are complex, expensive, or not suited for high-throughput processes, limiting the understanding of ADP-ribosyl signalling machinery and the development of inhibitors for cancer and viral infections.
Innovation Solution
A method and kit for detecting ADP-ribosyl-binders using a non-hydrolysable ADP-ribose probe, where an ADP-ribose group is coupled to a peptide or protein via an S-glycosidic bond, allowing for the measurement of binding interactions using labeled entities and various assay technologies, including FRET, BRET, and biolayer interferometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If previous assays for detecting ADP-ribosylated substrates are used, then detection of hydrolysing binders is possible, but they cannot be applied to non-hydrolysing binders and are complex and expensive
Solution Approach 1:
The patent creates a universal assay system using a non-hydrolysable ADP-ribose probe that can detect both hydrolysing and non-hydrolysing binders through a common mechanism. The probe design with S-glycosidic bond and C-terminal Gαi peptide tag provides a platform that works across different binder types without requiring separate assay protocols, thereby achieving multi-functionality and reducing overall assay complexity.
Solution Approach 2:
The invention introduces a non-hydrolysable ADP-ribose probe as an intermediary molecule that mediates detection between the binder proteins and the detection system. This probe acts as a stable surrogate for endogenous ADP-ribose modifications, enabling indirect detection of both hydrolysing and non-hydrolysing binders through a unified approach that simplifies the overall detection methodology.
2Productivity
If previous assays are used, then some binding detection is possible, but they are not suited for high-throughput processes
Solution Approach 1:
The patent replaces complex mechanical and procedural assay steps with a simplified biochemical detection system based on FRET (fluorescence resonance energy transfer). By using fluorescently labeled probes and measuring energy transfer signals, the assay eliminates cumbersome manual operations and enables automated high-throughput screening while maintaining ease of operation through standardized protocols.
3Adaptability or versatility
If a non-hydrolysable ADP-ribose probe is used, then both hydrolysing and non-hydrolysing binders can be detected, but requires development of new assay system
Solution Approach 1:
The patent performs preliminary actions by pre-designing and validating a non-hydrolysable ADP-ribose probe with specific structural features (S-glycosidic bond to cysteine, C-terminal Gαi peptide tag) before applying it to detect various binders. This upfront development of a universal probe platform reduces subsequent assay development effort, as the pre-validated probe can be directly applied to screen different binder types without requiring extensive re-engineering.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the detection of both hydrolysing and non-hydrolysing ADP-ribose binders, facilitating the development of inhibitors for cancer and viral infections, and providing a robust assay technology for high-throughput screening.
Implementation Method 1
an amino acid sequence comprising a cysteine residue whereto at least one ADP-ribosyl group or an analog thereof is coupled via an S-glycosidic bond
Implementation Method 2
measurement of binding interactions using labeled entities and various assay technologies, including FRET
Implementation Method 3
measurement of binding interactions using labeled entities and various assay technologies, including FRET, BRET
Data Source
AI summary
The present invention is directed to a method, kit, system and fusion protein for detecting binding to an ADP-ribosyl group or a polymer thereof, wherein said group or polymer is coupled to a peptide or protein, the method comprising the steps of: i) providing a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine residue whereto at least one ADP-ribosyl group or an analog thereof is coupled via an S-glycosidic bond; ii) contacting in an assay said first entity with a second entity, said second entity being or suspected of being capable of binding to an ADP-ribosyl group or polymer thereof coupled to a peptide or protein; and iii) measuring a signal derived from said first label or localized by said tag, wherein the signal detected is different or is localized differently when said second entity binds to said at least one ADP-ribosyl group of the first entity from the signal detected when the binding interaction between said second entity and said ADP-ribosyl group has not occurred. The kit of the present invention provides means to perform the method of the invention.