Riboregulator Toehold Switch Design for RNA Detection
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Solution Overview
Problem
Previous riboregulator systems are limited by structural constraints, particularly sequence constraints, which restrict their flexibility and functionality, making it difficult to generate programmable riboregulators that can be activated by endogenous RNAs and used simultaneously in a single system without relying on the ribosome binding site (RBS) for structure, thereby limiting their application in controlling gene expression and sensing RNA levels in real-time.
Innovation Solution
Development of programmable riboregulators, also known as toehold switches, which utilize a single-stranded toehold domain, a fully or partially double-stranded stem domain with an initiation codon, and a loop domain comprising a ribosome binding site, allowing activation by RNAs including endogenous ones, and enabling independent regulation of multiple nucleic acids in a cell, with the ability to detect RNA levels and perform complex logic operations without depending on the RBS for structure.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If riboregulators rely on kissing loop structures with specific sequences, then hybridization reaction is driven forward, but the number of possible crRNA designs is severely limited
Solution Approach 1:
The riboregulator is divided into distinct functional domains: a toehold domain for initial RNA binding, a stem domain for structural stability, and a loop domain for ribosome binding site presentation. This segmentation allows independent optimization of each domain's function while maintaining overall system reliability and enabling diverse crRNA designs through variable sequence combinations in each segment.
Solution Approach 2:
Instead of using the conventional kissing loop structure where a loop binds to another loop, this invention uses a linear single-stranded toehold domain that binds to a complementary sequence. This inversion of the binding mechanism eliminates the need for specific kissing loop sequences while maintaining reliable hybridization, thereby expanding design versatility.
2Reliability
If RBS is sequestered within a stem region to impede translation, then gene expression is controlled, but the kinetics of binding with crRNA and dynamic range are decreased
Solution Approach 1:
The ribosome binding site is segregated into a separate loop domain rather than being embedded within the stem region. This segmentation allows the stem to maintain its repressive function through base pairing while the loop domain presents the RBS in an accessible configuration, thereby improving binding kinetics and dynamic range without sacrificing translation control.
Solution Approach 2:
The loop domain acts as an intermediary structure that connects the repressive stem region with the ribosome binding function. It mediates between the need for RBS sequestration (for control) and RBS accessibility (for kinetics), allowing the RBS to be protected when needed while remaining accessible for rapid ribosome binding when the riboregulator is activated.
3Adaptability or versatility
If RBS sequence is varied to tune protein levels, then output characteristics are adjusted, but corresponding modifications in trans-activating RNA sequence are required
Solution Approach 1:
The riboregulator sequence is segmented into functional domains with the RBS located in the loop domain. This segmentation allows the RBS sequence to be independently varied to tune protein output levels without requiring modifications to the trans-activating RNA sequence, as the toehold domain binding interface remains separate and unchanged.
Solution Approach 2:
The toehold domain serves as a universal binding interface that can interact with various trans-activating RNA sequences without requiring sequence changes in the toehold itself. This universality allows the RBS to be optimized for different protein levels while maintaining compatibility with the same trans-activating RNA, reducing the complexity of sequence modifications.
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
These riboregulators provide sensitive control over protein expression, enable real-time detection of endogenous RNA, and facilitate complex logic operations, allowing for simultaneous control of multiple cellular activities with low system cross-talk and high programmability, effectively overcoming the limitations of previous systems.
Implementation Method 1
a first domain that hybridizes to the toehold domain
Implementation Method 2
repression of protein translation has relied on sequestration of the normally single-stranded ribosome binding site (RBS) within a duplex RNA region
Data Source
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.