Detection of SARS-CoV-2 using RNA multi-arm junction logic gates
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Solution Overview
Problem
Existing ribocomputing devices face limitations in monitoring a wide range of input RNAs and producing output proteins due to sequence constraints and inefficient translation, particularly in detecting natural transcripts lacking complementarity and requiring adapter strands.
Innovation Solution
The development of loop-mediated riboregulators with multi-arm junctions that expose ribosome binding sites and start codons upon target RNA binding, enabling sequence-independent activation of gene expression for AND and OR logic operations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If ribocomputing devices use hybridization between multiple input RNAs for implementing AND logic, then logic operations can be performed, but sequence constraints limit the range of input RNAs that can be monitored
Solution Approach 1:
The device is segmented into multiple independent sensor arms, each capable of binding to different input RNAs. This segmentation allows the system to monitor a wide range of input RNAs without requiring complex interdependencies between them, thereby resolving the sequence constraint limitation while maintaining logical operation reliability
Solution Approach 2:
Each sensor arm is designed with universal binding capability to recognize various input RNA sequences through complementary base pairing. This multi-functionality enables the same ribocomputing device to monitor diverse input RNAs (including viral transcripts) without requiring device redesign, thus expanding the range of monitorable inputs while maintaining reliable logic operations
2Adaptability or versatility
If adapter strands are used to enable input RNA binding, then sequence constraints are reduced, but system output is reduced
Solution Approach 1:
The adapter strand function is extracted and integrated directly into the sensor arm structure itself. Each sensor arm contains its own binding site for input RNAs, eliminating the need for separate adapter strands. This integration maintains sequence independence while preserving full system output capability through direct binding to the ribosome binding site
Solution Approach 2:
The functions of input RNA binding and ribosome binding site access are merged into a single integrated sensor arm structure. This combination allows direct interaction between input RNAs and the translation machinery without intermediate adapters, thereby maintaining both sequence independence and high system output
3Adaptability or versatility
If long open reading frames with high secondary structure are placed upstream of the output gene sequence to encode OR logic, then logic operations can be performed, but ribosome processivity is impeded and translation efficiency decreases
Solution Approach 1:
The ribocomputing device employs dynamic sensor arms that can transition between bound and unbound states. When input RNAs bind to sensor arms, the structure dynamically reconfigures to expose the ribosome binding site. This dynamic behavior enables complex OR logic operations while maintaining high translation efficiency through rapid conformational changes that do not impede ribosome processivity
Solution Approach 2:
The device utilizes changes in RNA secondary structure parameters (folding/unfolding of sensor arms) to encode logic operations. By controlling the structural state of sensor arms through input RNA binding, the system achieves complex logic functionality without requiring long static open reading frames, thus maintaining ribosome processivity and translation efficiency
4Adaptability or versatility
If multi-arm junctions are used to conceal RBS and start codon, then sequence constraints are eliminated, but the structure becomes more complex
Solution Approach 1:
The ribocomputing device employs nested sensor arms within a multi-arm junction structure. Each sensor arm is nested within the overall junction framework, allowing compact packaging of multiple binding sites. This nesting achieves sequence independence while managing structural complexity through efficient spatial organization of functional elements
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 riboregulators achieve high dynamic range and orthogonality in gene expression without sequence constraints, allowing for accurate detection of SARS-CoV-2 RNA sequences and differentiation between HIV subtypes using paper-based cell-free assays.
Implementation Method 1
The multi-arm junctions comprise from 5' to 3': a first base stem region, at least two sensor arms, and a second base stem region. Importantly, the first base stem region is at least partially complementary to the second base stem region, such that the first and second base stem regions pair to form a base stem.
Implementation Method 2
a portion of the loop region is at least partially complementary to a target RNA sequence from SARS-CoV-2. Binding of one or more target RNA sequences to one or more loop regions unwinds at least a portion of the secondary structure to expose the RBS and start codon
Data Source
AI summary
The present invention provides loop-mediated riboregulators for the detection of SARS-CoV-2. Also provided are DNA constructs encoding the loop-mediated riboregulators and methods of using the loop-mediated riboregulators to detect the presence of SARS-CoV-2 in a sample.