Engineered MCP and PCP Proteins for Live Cell RNA Imaging
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Solution Overview
Problem
Existing methods for live cell RNA imaging, such as the MS2-MCP system, suffer from high background fluorescence due to lingering MCP proteins when not bound to RNA, obscuring image clarity.
Innovation Solution
Engineered MCP and PCP proteins with a hidden degron that is exposed only when not bound to their respective RNA hairpin loops, ensuring protein stability and reduced background fluorescence during live cell RNA imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If MS2-MCP system is used for live cell RNA imaging, then real-time dynamics of mRNA behavior can be observed, but excess MCP proteins linger in the cell and produce high background fluorescence that obscures images
Solution Approach 1:
The patent extracts and removes the harmful component (excess unbound MCP protein) from the system by introducing a degron tag that targets these proteins for degradation. This allows the useful MCP-RNA complexes to remain while eliminating the background fluorescence problem.
Solution Approach 2:
The patent changes the stability parameter of MCP proteins by fusing them to degron sequences. This creates a differential stability system where unbound MCP proteins are rapidly degraded while bound MCP proteins are protected, thereby reducing background fluorescence without compromising imaging function.
2Object-generated harmful factors
If MCP proteins are made unstable to reduce background, then background fluorescence decreases, but the proteins may become too unstable when bound to RNA
Solution Approach 1:
The RNA aptamer sequence acts as an intermediary that mediates between the destabilizing degron tag and the MCP protein. When the aptamer binds to the MCP, it shields the degron from degradation machinery, thereby protecting the MCP-RNA complex while allowing free MCP proteins to be degraded.
Solution Approach 2:
The MCP protein system serves itself by using its own RNA binding activity to protect itself from degradation. The bound MCP proteins automatically gain stability through RNA binding, while unbound proteins are degraded, creating a self-regulating stability system.
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
The engineered proteins maintain stability and functionality when bound to RNA, significantly reducing background noise and enhancing the clarity of live cell RNA imaging.
Implementation Method 1
wherein the degron is hidden upon binding of the engineered RNA-binding domain to an MS2 RNA hairpin loop
Implementation Method 2
when the engineered RNA-binding domain is not bound to the MS2 RNA hairpin loop, the degron is exposed and degradation of the engineered MCP protein increases
Data Source
AI summary
The technology described herein is directed to engineered MCP proteins and engineered PCP proteins, which are degraded when the proteins are not bound to an MS2 or PP7 RNA hairpin loop, respectively. Also described herein are fusion proteins comprising such engineered MCP proteins and engineered PCP proteins linked to various effector proteins. The linkage to the effector proteins can be modulated through of specialized linker domains. In addition, described herein are complexes and systems comprising the fusion proteins in combination with synthetic RNA molecules, in order to modulate the structure and/or function the synthetic RNA molecules.