Scaffold RNA Dynamic Control of Protein Colocalization
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Solution Overview
Problem
Current synthetic metabolons lack dynamic control over metabolic flux distribution after assembly, lacking feedback loops to prevent substrate depletion or product accumulation, which limits their efficiency in metabolic engineering applications.
Innovation Solution
The use of scaffold RNA molecules with specific binding motifs, hybridization sequences, and toehold sequences allows for the dynamic colocalization and separation of heterologous proteins through toehold-mediated strand displacement (TMSD), enabling controlled metabolic flux management.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If scaffold RNA molecules are used to assemble synthetic metabolons, then protein colocalization and metabolic flux control are improved, but dynamic control capability and feedback mechanisms are lost
Solution Approach 1:
The patent applies dynamics by designing scaffold RNA molecules with toehold sequences that enable conditional assembly and disassembly of synthetic metabolons. The scaffold RNA can dynamically respond to cellular conditions through toehold-mediated strand displacement, allowing the metabolon structure to transition between assembled and disassembled states based on metabolic needs, thus providing both structural organization and dynamic adaptability
Solution Approach 2:
The patent implements feedback mechanisms by incorporating sensor domains into the scaffold RNA design that can detect metabolic intermediates or end-products. When specific metabolic conditions are detected, the sensor domains trigger conformational changes or recruit nucleases that cleave the scaffold RNA, thereby disassembling the metabolon and providing feedback control over metabolic flux to prevent substrate depletion or product accumulation
2Ease of manufacture
If enzymes are introduced through genetic vectors, then metabolic pathway introduction is achieved, but optimization of turnover and expression rates is required
Solution Approach 1:
The patent uses scaffold RNA molecules as intermediaries to organize enzymes into metabolons, which facilitates substrate channeling and improves metabolic flux without requiring extensive optimization of individual enzyme expression levels. The scaffold RNA acts as a mediator that brings enzymes into close proximity, allowing the system to achieve high productivity even with moderate expression levels of individual pathway enzymes
Solution Approach 2:
The patent segments the metabolic pathway into modular enzyme complexes organized on scaffold RNA molecules. Each enzyme can be independently introduced via genetic vectors and then organized into functional metabolons through the scaffold RNA, allowing for simplified introduction of the complete pathway while maintaining the ability to optimize individual enzyme components separately
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
This approach enables dynamic assembly and disassembly of protein scaffolds, allowing for precise control of metabolic flux and improving product yields by preventing substrate depletion and product accumulation, thus enhancing metabolic engineering efficiency.
Implementation Method 1
expressing the first heterologous protein and a first scaffold RNA molecule in the cell, wherein the first scaffold RNA molecule comprises a first binding motif, a hybridization sequence and a toehold sequence, the first heterologous protein is bound to the first binding motif
Implementation Method 2
the first hybridization sequence is bound to a first sequence complementary with the hybridization sequence
Implementation Method 3
the first scaffold RNA molecule comprises a first binding motif, a hybridization sequence and a toehold sequence... the trigger RNA molecule comprises a first trigger sequence complementary with the toehold sequence... whereby the first trigger sequence complementary with the toehold sequence is bound to the toehold sequence
Data Source
AI summary
The present invention provides a method for controlling colocalization of two or more proteins in a cell. The method comprises expressing the proteins, scaffold RNA molecules having binding motifs for the proteins, and a trigger RNA molecule in the cell. In the presence of the trigger RNA molecule, a scaffold may be assembled (ON) by the scaffold RNA molecules via hybridization such that the proteins may be colocalized; or disassembled (OFF) such that the proteins may be separated and not colocalized. The proteins may provide a biological activity when colocalized or not colocalized.