Ligandable Tag Labeling Enzyme for In-Cell Binding Partner Detection
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Solution Overview
Problem
Current methods for profiling protein-small molecule interactions on a network scale are lacking, missing interactions due to the use of lysates that do not accurately represent cellular contexts, and there is a need for comprehensive methods to elucidate these interactions.
Innovation Solution
The development of compositions and methods utilizing a ligandable tag, linker, and labeling enzyme system, where the linker attaches the ligandable tag to the labeling enzyme, enabling the detection and identification of binding partners of molecules of interest through labeling and detection of binding events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If lysates are used for detecting protein-small molecule interactions, then the detection process is simplified, but the accuracy of representing cellular contexts deteriorates
Solution Approach 1:
The patent introduces a proximity labeling enzyme (e.g., biotin ligase) as an intermediary that bridges the small molecule of interest and the detection system. The enzyme is recruited to the binding site through the small molecule, then labels nearby proteins in situ within intact cells, thereby maintaining cellular context accuracy while enabling simplified detection through subsequent biotin-streptavidin purification and mass spectrometry analysis.
2Measurement precision
If conventional affinity-based methods are used, then the identification of binding partners is achieved, but comprehensive network-scale profiling is missed
Solution Approach 1:
The patent creates a universal platform that combines the specificity of affinity-based methods with the scalability of proteomics. The proximity labeling enzyme can be recruited by any small molecule of interest, making the method universally applicable. When combined with mass spectrometry, it simultaneously identifies multiple binding partners and their interaction networks, achieving both accurate binding partner identification and comprehensive network-scale profiling.
Solution Approach 2:
The patent merges affinity-based detection with proximity labeling technology and proteomics. The small molecule serves as an affinity reagent to recruit the labeling enzyme, which then labels interacting proteins. The labeled proteins are purified using affinity tags and analyzed by mass spectrometry, combining multiple techniques into a unified workflow that achieves both specificity and scalability.
3Measurement precision
If DARTS or SPROX methods are used, then protein stability differences are detected, but some relevant interactions are missed due to lysate limitations
Solution Approach 1:
The patent performs preliminary recruitment of the proximity labeling enzyme to the binding site before any detection or analysis steps. The small molecule of interest is used to pre-recruit the labeling enzyme to the target protein's binding pocket within intact cells, ensuring the enzyme is in position to label interacting proteins before they are subjected to lysis or other processing that might alter their stability or interactions.
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 rapid and accurate identification of binding partners and protein-small molecule interactions on a network scale, overcoming limitations of previous methods by providing a more accurate representation of cellular interactions.
Implementation Method 1
when the molecule of interest binds to a binding partner in the sample, the labeling enzyme labels the binding partner
Data Source
AI summary
Disclosed are compositions comprising a ligandable tag, a linker, and a labeling enzyme, wherein the linker attaches the ligandable tag to the labeling enzyme. Disclosed are nucleic acid sequences capable of encoding one or more of the disclosed protein based compositions. Disclosed are compositions comprising a ligand conjugated to a molecule of interest. Disclosed are systems comprising a labeling composition and a targeting composition, wherein the labeling composition comprises a ligandable tag, a linker, and a labeling enzyme, wherein the linker attaches the ligandable tag to the labeling enzyme, wherein the targeting composition comprises a ligand conjugated to a molecule of interest, wherein the ligand of the targeting composition is a ligand for the ligandable tag of the labeling composition. Disclosed are methods of using the disclosed compositions and systems.