RNA Cleavage Transcript Stabilizer for Rapid Reporter Expression
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Solution Overview
Problem
Existing methods for detecting RNA cleavage activities, such as those mediated by microRNA, involve a 'double-inversion' strategy that results in a time delay between RNA cleavage detection and reporter production due to high levels of RNA cleavage leading to low repressor expression.
Innovation Solution
Genetic circuits and modules that incorporate RNA degradation signals, stabilized by RNA cleavage, to directly respond to RNA cleavage events and produce an output molecule, utilizing RNA cleavers like endoribonucleases, ribozymes, or RNAi molecules to remove degradation signals and enhance RNA stability, thereby facilitating rapid expression of output molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a double-inversion strategy is used to detect RNA cleavage, then RNA cleavage detection is achieved, but a time delay exists between RNA cleavage detection and reporter production
Solution Approach 1:
The patent extracts and removes the problematic time delay by directly coupling RNA cleavage to reporter expression through degradation signals. Instead of using a double-inversion strategy that requires repressor expression, the invention directly links cleavage events to transcript stabilization and immediate reporter production, eliminating the intermediate steps that cause delays.
Solution Approach 2:
The patent incorporates degradation signals and stabilizer sequences into the reporter transcript structure in advance. These pre-configured elements are designed to respond immediately upon RNA cleavage, allowing the transcript to be rapidly stabilized and translated without waiting for repressor expression or other intermediate processes.
2Measurement precision
If high levels of RNA cleavage occur, then RNA cleavage detection is enhanced, but repressor expression decreases leading to delayed reporter production
Solution Approach 1:
The patent inverts the traditional logic by making the cleaved transcript itself the source of reporter expression rather than using a repressor system. Instead of high cleavage leading to low repressor expression, the invention designs the system so that cleavage directly removes degradation signals, stabilizing the transcript and immediately increasing reporter production in direct proportion to cleavage activity.
3Adaptability or versatility
If RNA degradation signals are present, then RNA transcript is unstable, but RNA cleavage removes these signals stabilizing the RNA
Solution Approach 1:
The patent applies local quality by placing degradation signals and stabilizer sequences at specific locations within the transcript structure. The degradation signals are positioned in regions that will be removed or rendered non-functional upon cleavage, while stabilizer sequences are placed to protect the reporter-coding region. This localized arrangement allows the transcript to be unstable when needed for regulation but stable when the cleavage event occurs.
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 solution enables immediate and sensitive detection of RNA cleavage activities and allows for therapeutic or diagnostic applications by stabilizing RNA transcripts and enhancing the expression of output molecules, such as therapeutic agents, in response to RNA cleavage events.
Implementation Method 1
RNA cleavage is an important process during cellular RNA processing
Implementation Method 2
The RNA degradation signals lead to the degradation of the RNA molecule
Data Source
AI summary
Provided herein are genetic circuits and encoded RNA transcripts that produce an output molecule in response to an RNA cleavage event that removes a degradation signal. In some embodiments, the genetic circuits described herein may be used for detecting RNA cleaver activities (e.g., in a cell). Methods of using the genetic circuits described herein in diagnostic or therapeutic applications are also provided.