Photocleavable Dimerizing Agents for Rapid Protein Control
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Solution Overview
Problem
Current methods for inactivating proteins within cells are either slow, cause collateral damage, or have limited applicability due to the need for specific protein susceptibility and susceptibility to photoregulation, and existing Chemical Inducers of Dimerization (CIDs) may not effectively control protein activity with minimal side effects on other cellular components.
Innovation Solution
Development of photocleavable dimerizing agents that can be cleaved by light, allowing precise control over protein function by linking two ligands with a photocleavable linker, enabling the activation or inactivation of proteins within cells using light exposure.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If RNAi constructs are used to inactivate proteins, then protein levels decline, but the method is slow to take effect and may cause cell death or activate compensatory mechanisms
Solution Approach 1:
The invention divides the protein into two separate fragments that are expressed in the cell but remain inactive. These fragments are brought together by a dimerizing agent to form the active protein only when needed, enabling rapid and controlled activation without affecting the entire protein continuously
Solution Approach 2:
A dimerizing agent serves as an intermediary that brings two protein fragments together to form the active protein. This intermediary can be removed or inactivated to rapidly stop protein activity, providing precise temporal control without the slow turnover of RNAi
2Ease of operation
If Chemical Inducers of Dimerization (CIDs) are used to control protein activity, then protein dimerization can be induced, but the time course depends on slow cellular entry and washout rates
Solution Approach 1:
The invention extracts the dimerization function from a small molecule CID and transfers it to a genetically encoded system using a peptide tag and its binding partner. This allows the system to behave like a small molecule (rapid onset) while being genetically controllable
Solution Approach 2:
The invention changes the kinetic parameters of protein activation by using a genetically encoded peptide system instead of small molecule CIDs. The peptide-tag system achieves rapid cellular penetration and fast reversibility by controlling peptide expression levels and degradation tags
3Speed
If light-based methods like CALI are used to inactivate proteins, then rapid inactivation can be achieved, but collateral damage occurs to proteins in close proximity
Solution Approach 1:
The invention segments the target protein into two fragments that are spatially separated in the cell. Only when both fragments are present and brought together by the dimerizing agent does the active protein form, reducing off-target effects and collateral damage
Solution Approach 2:
The invention applies local quality control by using a genetically encoded peptide tag system that provides specific, localized control of protein activity. The peptide tag is incorporated only into the target protein, ensuring that activation or inactivation occurs only at the specific location where the tagged protein resides
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 precise modulation of protein activity with minimal side effects on other cellular components, allowing for the study of protein function and regulation by controlling when and where the protein link is broken, and can be used for both inactivation and activation purposes.
Implementation Method 1
Y is a linker providing a covalent linkage between R1 and R2, wherein Y is photocleavable
Data Source
AI summary
The instant invention provides photolysable compounds, and their use in reversible chemical induced dimerization and light-induced regulation of proteins.