Circular RNA Aptamer Expression via Ribozyme Autocatalysis
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Solution Overview
Problem
RNA aptamers struggle to achieve high enough concentrations in cells to effectively modulate proteins due to instability and low intracellular levels, limiting their use in mammalian cells where proteins are expressed at higher concentrations, and existing methods for generating circular RNAs are not suitable for widespread expression across various cell types.
Innovation Solution
A novel system for generating circular RNAs using ribozymes and endogenous RNA ligase RtcB, which autocatalytically processes RNA transcripts to create substrates for ligation, resulting in stable circular RNA molecules that can reach high concentrations and be expressed in virtually any metazoan or bacterial cell without additional enzyme co-expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If linear RNA aptamers are expressed in cells, then they can be produced, but they are degraded by exonucleases and fail to accumulate to high concentrations
Solution Approach 1:
The patent applies circularization of the RNA aptamer to eliminate the 5' and 3' ends that are susceptible to exonuclease degradation. The circular RNA structure allows the molecule to evade degradation pathways while maintaining functional aptamer sequences, thereby achieving both high stability and high intracellular concentration accumulation.
Solution Approach 2:
The patent changes the topological parameter of the RNA from linear to circular configuration. This structural parameter change fundamentally alters the degradation profile of the RNA, protecting it from exonuclease attack while allowing accumulation to micromolar concentrations necessary for effective protein modulation.
2Reliability
If RNA aptamers are expressed at high concentrations to modulate proteins, then effective protein modulation can be achieved, but the aptamers are unstable and degraded
Solution Approach 1:
The circular RNA structure eliminates the ends that are targets for exonuclease degradation, providing continuous protection along the entire molecule. This allows the aptamer to maintain high concentrations without degradation, simultaneously achieving reliable protein modulation and molecular stability.
Solution Approach 2:
The circularization strategy provides preemptive protection against exonuclease degradation before the aptamer can be degraded. By eliminating vulnerable ends in advance, the molecule is cushioned against degradation pathways, allowing it to persist at high concentrations necessary for effective protein modulation.
3Ease of manufacture
If existing circular RNA generation methods are used, then some circular RNA can be produced, but they require additional enzyme co-expression and are not suitable for widespread expression
Solution Approach 1:
The patent employs self-cleaving ribozymes that autonomously process the RNA transcript without requiring external enzyme co-expression. The ribozymes are integrated into the RNA molecule itself, enabling autocatalytic cleavage and circularization, thereby eliminating the need for additional enzymatic systems and simplifying the overall expression platform.
Solution Approach 2:
The patent combines the circularization function directly into the RNA molecule by integrating ribozyme sequences and ligation sequences within the aptamer construct. This merging of circularization functionality into the RNA itself eliminates the need for separate enzymatic systems, reducing device complexity while maintaining ease of manufacture.
4Measurement precision
If aptamers are designed to bind specific protein domains, then precise protein modulation is achieved, but the aptamer concentration remains too low to stoichiometrically bind target proteins
Solution Approach 1:
The circular RNA structure enables accumulation to micromolar concentrations by eliminating degradation pathways. This concentration increase allows stoichiometric binding to target proteins while maintaining the precise domain-specific binding characteristics of the aptamer, thereby resolving the contradiction between precision and quantity.
Solution Approach 2:
The topological change from linear to circular configuration fundamentally alters the concentration parameter of the aptamer. This parameter change enables the aptamer to reach micromolar levels necessary for stoichiometric protein binding, while the sequence-specific binding properties remain intact for precise modulation.
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 enhances the expression of RNA aptamers and metabolite sensors to micromolar levels in mammalian cells, enabling effective protein modulation and metabolite detection, and demonstrates improved stability and functionality of RNA-based devices.
Implementation Method 1
ribozymes and endogenous RNA ligase RtcB, which autocatalytically processes RNA transcripts to create substrates for ligation
Implementation Method 2
endogenous RNA ligase RtcB, which autocatalytically processes RNA transcripts to create substrates for ligation
Data Source
AI summary
The present invention relates to a RNA molecule comprising a first ribozyme, a first ligation sequence, an effector molecule, a second ligation sequence, and a second ribozyme. Methods of producing circular RNA molecules and treatment methods are also disclosed.