Toehold Riboregulator Design for Programmable RNA Detection
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Solution Overview
Problem
Previous riboregulator systems are constrained by sequence specificity, limiting their ability to be activated by endogenous RNAs and requiring modifications to the ribosome binding site (RBS) sequence, which complicates tuning and activation kinetics.
Innovation Solution
The development of toehold riboregulators with a single-stranded toehold domain, a fully or partially double-stranded stem domain, and a loop domain comprising a ribosome binding site, allowing for activation by RNAs with arbitrary sequences and independent modification of the RBS, enabling sensitive control over protein translation and detection of endogenous RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If loop-linear interactions are used to drive crRNA/trans-RNA hybridization, then hybridization is facilitated through kissing loop structure, but sequence specificity severely limits the number of possible crRNA designs
Solution Approach 1:
The crRNA is segmented into distinct functional domains: a toehold domain for initial binding, a stem domain for structural stability, and a loop domain for target recognition. This segmentation allows independent optimization of each domain's function while maintaining overall hybridization efficiency and expanding design versatility.
Solution Approach 2:
The toehold domain acts as an intermediary element that facilitates the initial binding between crRNA and trans-RNA before the main hybridization occurs. This toehold-mediated mechanism enables controlled activation while preserving the ability to design diverse crRNA sequences with different specificities.
2Reliability
If RBS is sequestered within a hairpin structure to impede translation, then gene expression is controlled, but RBS sequence modifications require corresponding changes in trans-RNA sequence
Solution Approach 1:
The riboregulator is divided into separable functional modules: the RBS is placed in the loop domain while the stem domain contains the sequence that binds trans-RNA. This modular design allows independent modification of the RBS sequence without requiring changes to the trans-RNA binding region, simplifying device construction and tuning.
Solution Approach 2:
The RBS sequence is extracted from the trans-RNA binding region and placed in the loop domain. This separation allows the RBS to be independently optimized for translation control while the stem domain maintains its binding function, eliminating the need for coordinated sequence modifications.
3Reliability
If RBS is sequestered within stem regions of taRNA, then translation is controlled, but binding kinetics with crRNA and dynamic range are decreased
Solution Approach 1:
The taRNA is segmented into a toehold domain that binds to the crRNA toehold and a second domain that binds downstream. This segmentation allows the RBS to be positioned in the crRNA loop domain rather than sequestered in the taRNA stem, maintaining fast binding kinetics while preserving translation control through the crRNA hairpin structure.
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 allows for programmable and sensitive control of protein translation, real-time detection of endogenous RNA levels, and simultaneous control of multiple cellular activities with low system cross-talk, enhancing the diversity and orthogonality of riboregulators.
Implementation Method 1
a single-stranded toehold domain, a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain comprising a ribosome binding site
Implementation Method 2
a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain comprising a ribosome binding site
Data Source
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AI summary
The invention provides novel and versatile classes of riboregulators, including inter alia activating and repressing riboregulators, switches, and trigger and sink RNA, and methods of their use for detecting RNAs in a sample such as a well and in modulating protein synthesis and expression.