Oligonucleotide Primer Pairs for Gene Expression Screening
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Solution Overview
Problem
Current cell-based assays for drug discovery are inefficient, costly, and often require genetic engineering, rely on limited readouts, fail to filter out toxic components early, and lack targets for certain diseases, making the process unapproachable for many conditions.
Innovation Solution
A high-throughput gene signature-based screening method that eliminates the need for RNA isolation, using oligonucleotide primer pairs to hybridize and ligate with mRNA, followed by amplification and sequencing to identify changes in gene expression signatures in response to test compounds, allowing for the identification of drugs that modify cell physiology.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional cell-based assays are used for drug discovery, then specific molecular targets can be screened, but the process becomes inefficient, costly, and requires genetic engineering
Solution Approach 1:
The patent extracts the mRNA of interest directly from cell lysate without requiring RNA isolation purification steps. This is achieved by using biotinylated oligonucleotide probes that specifically bind to target mRNA sequences, allowing direct capture and amplification from crude lysate, thereby eliminating time-consuming RNA extraction while maintaining screening reliability
Solution Approach 2:
The patent introduces biotinylated oligonucleotide probes as intermediaries that mediate between the target mRNA and the detection system. These probes hybridize to specific mRNA sequences and are captured via biotin-streptavidin interaction, enabling specific molecular target screening without complex RNA isolation procedures
2Measurement precision
If RNA isolation is performed in traditional assays, then gene expression can be measured, but the process becomes tedious and unsuitable for high throughput applications
Solution Approach 1:
The patent merges the RNA capture and cDNA synthesis steps into a single reaction. Biotinylated oligonucleotide probes that are complementary to the target mRNA sequence are used as primers for reverse transcription, capturing and converting mRNA to cDNA in one step, eliminating separate RNA isolation operations while maintaining measurement precision
Solution Approach 2:
The patent employs a self-capturing system where biotinylated oligonucleotide probes automatically bind to their complementary mRNA sequences during the reverse transcription reaction. The biotin-streptavidin interaction provides automatic enrichment of target transcripts without requiring manual RNA purification steps
3Device complexity
If limited surrogate readouts are used in screening, then the assay is simpler, but broad and non-specific effects of candidate hits are not captured
Solution Approach 1:
The patent uses a universal gene expression profiling approach that can detect changes in any gene's expression level. By using biotinylated oligonucleotide probes targeting multiple genes simultaneously, the system provides comprehensive information about cellular responses including specific and non-specific effects, while maintaining a relatively simple overall assay structure
4Reliability
If genetic engineering is required to develop reporter cell lines, then specific targets can be monitored, but the process becomes costly and time-consuming
Solution Approach 1:
The patent uses synthetic biotinylated oligonucleotide probes that copy the sequence information of target genes without requiring physical modification of the cell line. These probes hybridize to endogenous mRNA sequences, providing reliable target monitoring through sequence-specific recognition rather than engineered reporter systems
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 approach enables efficient, cost-effective, and high-throughput drug screening without the need for specific molecular targets, allowing for the identification of compounds that alter cell physiology by measuring changes in gene expression signatures, thereby overcoming the limitations of traditional assays.
Implementation Method 1
each primer pair (the first and the second primer) comprises a sequence designed to specifically hybridize (anneal) to a spliced message (mRNA)
Implementation Method 2
when the primer pair is hybridized (annealed) to one strand of the spliced mRNA the first and the second primers are sufficiently adjacent such that they can be ligated by an enzyme
Implementation Method 3
amplification of all or substantially all or a subset of the spliced RNA message (spliced mRNA) in the cells or cells is by a method comprising: (1) providing a plurality of oligonucleotide primer pairs
Data Source
AI summary
The invention provides products of manufacture for screening for compositions that can modify a cell's gene expression profile, and methods for making and using them. In one embodiment, the invention provides products of manufacture and methods comprising a high content, high throughput screening for a composition (e.g., chemicals, small molecules) that can modify a cell's physiology based on the composition's ability to modify the cell's gene expression signature.