Ligand-Responsive Ribozyme Selection via Self-Cleavage
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Solution Overview
Problem
Existing methods for selecting ligand-responsive ribozymes are labor-intensive, require chemical modification and immobilization of target molecules, and have limited success in finding aptamers that function correctly in the context of RNA devices, making them difficult to automate and scale down.
Innovation Solution
A method involving in vitro transcription of a DNA library, reverse transcription to cDNA, selective amplification of cleaved versus uncleaved cDNA, and repeated cycles to enrich for ligand-responsive ribozymes, allowing for automation and use of unmodified target molecules, enabling selection of ribozymes sensitive to specific ligands without predetermining their components.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing selection methods are used to find aptamers that function as sensors, then binding aptamers can be obtained, but they do not function correctly in the context of the device and require chemical modifications
Solution Approach 1:
The ribozyme sensor performs self-cleavage when bound to the target molecule, eliminating the need for external chemical modifications or immobilization. The aptamer-ribozyme conjugate autonomously converts binding events into catalytic cleavage of the reporter molecule, making the system self-sufficient and easier to manufacture.
Solution Approach 2:
The invention changes the functional parameter from simple binding to catalytic cleavage. By incorporating a ribozyme domain that undergoes conformational change upon target binding, the system transforms the binding event into a catalytic reaction that cleaves the reporter, providing a more reliable and modification-free sensing mechanism.
2Ease of manufacture
If chemical modification and immobilization of target molecules are required, then selection can be performed, but the methods become difficult to implement and limit the target molecules that can be used
Solution Approach 1:
The ribozyme sensor performs self-cleavage when bound to the target molecule, eliminating the need for external chemical modifications or immobilization. The aptamer-ribozyme conjugate autonomously converts binding events into catalytic cleavage of the reporter molecule, making the system self-sufficient and easier to manufacture.
Solution Approach 2:
The invention creates a universal sensing platform where the ribozyme component can detect various target molecules without requiring specific chemical modifications of those targets. The same ribozyme architecture can be adapted to different targets by changing only the aptamer recognition element, greatly expanding target molecule applicability.
3Extent of automation
If SELEX-based processes with fixed ribozyme devices are used, then aptamers can be selected, but the processes require labor-intensive steps that are not amenable to automation
Solution Approach 1:
The invention merges the aptamer selection and ribozyme functional validation into a single SELEX process. The aptamer-ribozyme conjugate is selected and tested for catalytic function simultaneously, eliminating the need for separate labor-intensive validation steps and enabling full automation of the selection process.
Solution Approach 2:
The selection process maintains continuous useful action by performing in vitro transcription and self-cleavage assays throughout the SELEX rounds. This continuous functional testing ensures that only aptamers that actually function as sensors in the device context are selected, while the entire process remains amenable to automation.
4Reliability
If many rounds of selection are performed to isolate desirable sensor sequences, then functional ribozymes can be obtained, but the processes have limited practical utility due to limited automation
Solution Approach 1:
The ribozyme sensor performs self-cleavage when bound to the target molecule, eliminating the need for external chemical modifications or immobilization. The aptamer-ribozyme conjugate autonomously converts binding events into catalytic cleavage of the reporter molecule, making the system self-sufficient and easier to manufacture.
Solution Approach 2:
The invention incorporates feedback by using the cleavage activity itself as the selection criterion. The in vitro transcription and self-cleavage steps provide immediate feedback on whether an aptamer-ribozyme conjugate is functional, allowing rapid enrichment of desirable sequences and improving selection efficiency.
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 method enables efficient selection of ribozymes that are functional under selection conditions and likely functional in other contexts, allowing for rapid automation and scalability, reducing the need for chemical modifications and immobilization, and enabling differential selection against complex mixtures.
Implementation Method 1
The ribozyme-based device framework supports the design of robust genetic controllers in different organisms, responsive to diverse ligands
Implementation Method 2
Other techniques that use a SELEX-based process to find aptamers that function as sensors in the context of an otherwise fixed ribozyme-based device, such as 'allosteric selection'
Implementation Method 3
Existing work in finding aptamers that function as sensors typically use aptamers found using methods based on binding
Implementation Method 4
in vitro transcribing a DNA library to produce RNA
Implementation Method 5
reverse transcribing the RNA to cDNA
Implementation Method 6
selectively amplifying the cDNA such that cDNA molecules corresponding to RNA molecules that have been cleaved are amplified by a different amount relative to cDNA molecules corresponding to uncleaved RNA
Data Source
AI summary
Provided herein, among other things, is an automatable procedure that employs in vitro directed evolution to create DNA sequences that encode a ligand-responsive ribozyme and which, when transcribed, can control expression of genes they are coupled to. The method also allows creation of functional RNA sequences that bind target molecules, without requiring any modification or immobilization of the target.