Methylated Polynucleotide Gene Function Determination
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Solution Overview
Problem
Current methods for determining the biological function of a target gene, such as siRNA and traditional genetic manipulations, are costly, unstable, and time-consuming, making them inefficient for high-throughput functional analysis of genes.
Innovation Solution
A method involving the separate culturing of cells with and without the introduction of a methylated polynucleotide complementary to the target gene's promoter and first exon region, allowing for the comparison of biological differences to determine the gene's function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional genetic manipulations (knock-out, knock-in, siRNA) are used to determine gene function, then the biological function can be interrogated, but the cost is high, stability is poor, and the process is time-consuming
Solution Approach 1:
The patent changes the chemical parameter of the polynucleotide by introducing methylation at the 5-position of cytosine residues. This methylation modification enables the polynucleotide to bind to the promoter region and inhibit gene transcription, providing a stable and efficient method for gene function determination that avoids the time-consuming construction processes of traditional methods
Solution Approach 2:
The patent uses a polynucleotide sequence that is complementary to the target gene's promoter region, creating a molecular copy that can specifically bind to and silence the target gene. This copying approach allows for high-specificity gene function interrogation without requiring complex genetic manipulation constructions
2Ease of manufacture
If siRNA is used to interfere with gene expression, then specific gene function can be targeted, but the cost is high and stability during delivery is poor
Solution Approach 1:
The patent modifies the polynucleotide by introducing methylation at cytosine residues, which enhances the stability of the molecule during delivery and transfection. The methylated polynucleotide maintains structural integrity and functional activity better than conventional siRNA, while remaining easy to synthesize and apply
Solution Approach 2:
The patent creates a composite molecular structure by combining the polynucleotide sequence with methyl groups at specific positions. This composite structure provides both the sequence-specific binding capability needed for targeted gene silencing and the enhanced stability required for reliable delivery and function
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 method effectively identifies the biological functions of target genes by inducing methylation and altering cellular states, demonstrating its efficiency in analyzing gene functions, as shown by its application to the Trip10 gene in mesenchymal stem cells, promoting differentiation and preventing cell death.
Implementation Method 1
the first population of the cell has accepted the introduction of a methylated polynucleotide
Implementation Method 2
a methylated polynucleotide selected from the group consisting of: (i) a single-stranded DNA molecule, which has a nucleotide sequence identical to or fully complementary to that of a portion of the target gene's nucleotide sequence at the promoter and/or the first exon region
Data Source
AI summary
Disclosed herein is a method to determine the biological function(s) of a target gene in a cell, the steps of which involve separately culturing a first population and a second population of the cell under same culturing conditions, the first population of the cell differing from the second population of the cell in that the first population of the cell has accepted the introduction of a methylated polynucleotide; comparing the first population and the second population of the cell to determine which biological difference(s) is/are present therebetween; and determining which biological function(s) the target gene is associated with based on the determined biological difference(s) between the first population and the second population of the cell.