Splicing Variant Screening with Antisense Oligonucleotide Switching
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current technologies lack a high-throughput platform for functional genomics studies to effectively screen and characterize splicing variants or events, particularly for therapeutic targeting, given the vast number of isoform variants and splice events uncovered by next-generation sequencing.
Innovation Solution
A method using splice-switching steric hindrance antisense oligonucleotides (stAONs) is employed to induce and reverse splicing events in target genes, allowing for the characterization of splicing variants and their biological functions, including the use of a plate template for hybridization and characterization of cell phenotypes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If microarray technology is used to analyze full-length cDNA clones, then comprehensive splicing variant detection is possible, but the procedure becomes prohibitively complex and time-consuming
Solution Approach 1:
The patent divides the full-length cDNA into multiple overlapping short fragments (e.g., 50-200 nucleotides). Each fragment is independently analyzed on the microarray, and the results are computationally assembled to reconstruct the complete splicing variant structure. This segmentation reduces the complexity of individual hybridization reactions while maintaining comprehensive detection capability.
Solution Approach 2:
The patent performs preliminary fragmentation of cDNA into standardized short oligonucleotide sequences before microarray analysis. These pre-processed fragments are designed to cover all possible exon-combining events, allowing the system to detect splicing variants without analyzing the entire full-length cDNA sequence in one complex reaction.
2Measurement precision
If conventional methods are used to detect splicing variants, then accuracy can be maintained, but the number of false positives from background hybridization signals increases
Solution Approach 1:
By segmenting the cDNA into many short overlapping fragments, the patent increases the statistical confidence of detection. Each fragment independently hybridizes to its complementary sequence on the microarray, and true splicing variants will consistently produce hybridization signals across multiple overlapping fragments, while background noise remains random and does not consistently replicate.
Solution Approach 2:
The patent changes the key parameter of fragment length to very short sequences (50-200 nucleotides). This parameter change reduces the probability of non-specific hybridization and background noise while maintaining sufficient specificity for accurate variant identification through the cumulative evidence from multiple overlapping fragments.
3Adaptability or versatility
If comprehensive microarray analysis is performed on all cDNA fragments, then all splicing events can be detected, but the time and resources required become excessive
Solution Approach 1:
The patent uses partial action by analyzing only the necessary overlapping short fragments required to detect splicing variants, rather than sequencing or analyzing every possible cDNA fragment. The fragment set is designed to be sufficient for detecting all exon-combining events without being excessively comprehensive, optimizing the balance between detection coverage and analysis efficiency.
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 high-throughput screening and characterization of splicing variants, identifying disease-relevant isoforms and their effects on cell phenotypes, facilitating therapeutic targeting.
Implementation Method 1
hybridization of a set of short oligonucleotide sequences to a microarray
Data Source
Figure 1a
Figure 1b
Figure 2
AI summary
The present invention relates to a high-throughput method of screening splicing variants of target genes as drug targets or for characterisation of their biological functions. The disclosure provides a method for the screening of splicing variants, comprising: (a) providing a first antisense oligonucleotide capable of inducing a first splice event on the target gene to express a first splicing variant, and a second antisense oligonucleotide capable of inducing a second splice event on the target gene to express a second splicing variant; (b) hybridising the first and second antisense oligonucleotides to a pre-mRNA of the target gene; and (c) characterising the effect of the splice event. In one embodiment, the first antisense oligonucleotide switches the splice event that expresses the second splicing variant towards one that expresses the first splicing variant, while the second antisense oligonucleotide switches the splice event that expresses the first splicing variant towards one that expresses the second splicing variant.