Functional Molecule Screening Vectors with Cellular Response Reporters
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Solution Overview
Problem
Existing methods for comprehensive phenotypic analysis of functional molecules like RNA and peptides are labor-intensive and time-consuming, requiring long-term culture and complex concentration operations, limiting throughput.
Innovation Solution
A vector system comprising polynucleotides encoding expression systems for RNA or peptides, transcriptional regulatory sequences activated by cellular responses, and reporter systems operably linked to these sequences, allowing simultaneous analysis of multiple molecules in a single cell or population by measuring reporter system expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional phenotypic analysis methods are used to examine gene functions, then comprehensive analysis can be achieved, but enormous labor and time are required
Solution Approach 1:
The patent combines multiple gene expression analysis into a single experimental setup by pooling shRNA or gRNA vectors targeting different genes into one transfection mixture. This allows simultaneous examination of multiple gene functions in parallel, transforming sequential individual gene analysis into concurrent multi-gene analysis, thereby dramatically reducing the time and labor required for comprehensive phenotypic analysis
Solution Approach 2:
The patent creates a universal screening platform using pooled RNAi or CRISPR/Cas9 vectors that can analyze multiple gene functions simultaneously under identical experimental conditions. This multi-functional system replaces the need for separate experiments for each gene, enabling comprehensive phenotypic analysis through a single standardized protocol that handles multiple target genes
2Productivity
If shRNA or gRNA vectors are pooled and introduced into cells for comprehensive phenotypic analysis, then multiple genes can be examined simultaneously, but long-term culture and complex concentration operations are required
Solution Approach 1:
The patent extracts and measures the abundance of specific shRNA or gRNA sequences directly from the cell population using massively parallel sequencing technology. By taking out and quantifying the molecular signatures (RNA sequences) directly rather than requiring physical cell concentration steps, the method simplifies the experimental procedure while maintaining high throughput capability for identifying genes responsible for specific phenotypes
Solution Approach 2:
The patent replaces mechanical cell concentration operations with molecular detection methods. Instead of physically separating and concentrating cells exhibiting specific phenotypes through complex laboratory operations, the invention uses massively parallel sequencing to detect and quantify RNA sequences, substituting mechanical manipulation with automated molecular analysis that reduces procedural complexity
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
Enhances the efficiency and throughput of functional molecule analysis by associating molecules with cellular responses through reporter system expression, enabling rapid functional assessment of multiple molecules concurrently.
Implementation Method 1
a transcriptional regulatory sequence that is activated by a response in a cell that can be caused by perturbation by the RNA or peptide and a reporter system operably linked thereto
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 1E~1F
AI summary
A vector or set of vectors for analyzing a function of a functional molecule includes a polynucleotide encoding an expression system of a candidate molecule of a functional molecule, a polynucleotide encoding a transcriptional regulatory sequence or translational regulatory sequence that is activated by a predetermined response in a cell, and a polynucleotide encoding a reporter system operably linked to the transcriptional regulatory sequence or translational regulatory sequence. A method for searching a functional molecule causing a response in a cell includes introducing the vector or set of vectors into a cell, and measuring expression of a reporter system included in the vector or set of vectors in the cell.