Split Ribozyme RNA Biosensor for In Vivo Plant Expression Mapping
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Solution Overview
Problem
Current RNA analysis technologies in plants are destructive, labor-intensive, and unable to monitor temporal and spatial patterns of native RNA signals, necessitating a non-destructive approach for in vivo detection of RNA molecules.
Innovation Solution
A split ribozyme biosensor system comprising two expression cassettes with ribozyme fragments and guide RNAs that bind to target RNA, allowing self-splicing and reassembly of reporter protein transcripts for visual output, enabling in vivo RNA expression analysis in plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current RNA analysis technologies (qRT-PCR, in situ hybridization, transcriptome-sequencing) are used, then RNA expression can be detected, but the analysis is destructive, labor-intensive and time-consuming
Solution Approach 1:
The ribozyme is divided into two separate fragments (first ribozyme fragment and second ribozyme fragment) that are independently expressed and must assemble on the target RNA to form a functional ribozyme. This segmentation enables the biosensor to remain inactive until the specific target RNA is present, allowing non-destructive monitoring without requiring complex laboratory protocols
Solution Approach 2:
The split ribozyme system is self-activating through RNA-guided assembly. The guide RNAs bind to the target RNA and facilitate the bringing together of the two ribozyme fragments, which then self-splice to form the active ribozyme that releases the reporter mRNA. This self-service mechanism eliminates the need for destructive sampling and complex external processing
2Measurement precision
If current RNA analysis technologies are used, then RNA expression levels can be measured, but temporal and spatial patterns of native RNA signals cannot be monitored in vivo
Solution Approach 1:
Guide RNAs serve as intermediaries that specifically bind to the target RNA sequence and facilitate the assembly of the split ribozyme fragments at the precise location where the target RNA is present. This intermediary mechanism enables spatially-resolved detection within plant tissues while maintaining the ability to monitor temporal dynamics of RNA expression patterns in vivo
Solution Approach 2:
The invention transitions from bulk RNA analysis to spatially-resolved in vivo monitoring by incorporating tissue-specific promoters and subcellular localization signals. This allows the biosensor to detect RNA expression patterns across different spatial dimensions (tissue types, cellular locations) and temporal dimensions (developmental stages, stress responses) simultaneously
3Ease of operation
If aptamer-based RNA labelling technologies are used, then biosensors for gene expression detection can be built, but modification of input RNA signals is required making them infeasible for monitoring native RNA signals
Solution Approach 1:
The invention extracts and utilizes endogenous plant RNA molecules (guide RNAs and target RNA) instead of requiring exogenous aptamer modifications. The guide RNAs are derived from or designed to match native plant RNA sequences, allowing the biosensor to detect unmodified native RNA signals directly within the plant system without altering the input RNA
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
Enables non-destructive, spatial-temporal monitoring of RNA expression in plants through visual outputs, facilitating early detection of stress and developmental changes without genetic modification of input RNA signals.
Implementation Method 1
the ribozyme removes itself from flanking sequences to allow formation of an mRNA encoding the full length sequence of the first protein
Implementation Method 2
the first guide RNA sequence and the second guide RNA sequence bind to the target RNA, and bring the two ribozyme fragments together
Data Source
AI summary
The present disclosure is directed to a split ribozyme biosensor system. Additionally, a genetically modified plant, plant tissue, or plant cell comprising the split ribozyme biosensor system is described. Methods for examining in vivo RNA expression in plants, plant cells or plant tissues are disclosed. Lastly, disclosed herein is a kit comprising the split ribozyme biosensor system.