DNA-Encoded Library Decoding via Segmented Barcode qPCR
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Solution Overview
Problem
Current methods for decoding DNA-encoded libraries in drug discovery, such as Sanger sequencing, DNA arrays, and high-throughput sequencing, face challenges including low throughput, high cost, low sensitivity, and high systemic error, making it difficult to efficiently identify true positives and distinguish them from false positives, especially in large libraries.
Innovation Solution
A method involving a DNA-encoded library with unique barcode sequences divided into multiple coding regions, allowing for quantitative PCR (qPCR) analysis using specific primers that encode information about the enriched DNA-substance conjugates, enabling faster and more cost-effective decoding without the need for DNA sequencing, and providing detailed information on the abundance of specific groups of DNA-substance conjugates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If Sanger sequencing is used to decode DNA barcodes in a DNA-encoded library, then the decoding can be performed with simple equipment, but the throughput is low and the process consumes a lot of time
Solution Approach 1:
The DNA barcode is segmented into multiple coding regions, each independently amplifiable by specific primers. This segmentation allows parallel decoding of multiple barcode regions through qPCR, significantly increasing throughput while maintaining the simplicity of targeted amplification approaches
Solution Approach 2:
The patent introduces coding regions as intermediary elements that can be selectively amplified by specific primers. These coding regions act as mediators between the full DNA barcode and the detection system, enabling high-throughput parallel analysis through qPCR while keeping the equipment requirements relatively simple
2Productivity
If high-throughput sequencing is used to decode DNA barcodes, then the throughput is high, but the cost is high and systemic error is high
Solution Approach 1:
The patent extracts specific coding regions from the full DNA barcode sequence that are sufficient for identification. By amplifying and analyzing only these selected coding regions through qPCR rather than sequencing the entire barcode, the method achieves high throughput while reducing cost and minimizing sequencing-related systemic errors
Solution Approach 2:
The patent uses qPCR primers as disposable, library-specific tools that can be designed for each coding region. These primers enable targeted amplification without the need for expensive high-throughput sequencing infrastructure, providing a cost-effective alternative that maintains high throughput through parallel processing
3Loss of time
If DNA arrays are used to decode DNA barcodes, then the decoding can be performed quickly, but the sensitivity is low and false positives are high
Solution Approach 1:
The patent changes the detection parameter from hybridization-based signal detection (DNA arrays) to amplification-based quantification (qPCR). This parameter change increases sensitivity because the amplification process exponentially enriches the target, enabling detection of rare sequences while maintaining rapid throughput through efficient PCR cycling
4Quantity of substance
If the second part of the DNA barcode differs by only one nucleotide between molecules, then the library diversity is maximized, but the ability to uniquely identify and decode specific conjugates is reduced
Solution Approach 1:
The patent segments the barcode differentiation into multiple coding regions rather than relying solely on single-nucleotide differences in one region. Each coding region can be independently amplified and analyzed, providing redundant identification information that maintains accuracy even when individual nucleotide differences are minimal
Solution Approach 2:
The patent adds another dimension to barcode identification by using multiple coding regions that can be independently amplified. This dimensional expansion transforms the identification problem from relying on single-nucleotide resolution to using combinatorial patterns across multiple regions, significantly improving identification accuracy while preserving library diversity
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 allows for rapid, cost-efficient, and highly specific identification of enriched DNA-substance conjugates, reducing the risk of false positives and improving the detection of true hits, even in large libraries, by constructing two-dimensional matrices with qPCR that deconvolute the mixture of DNA barcodes, thus enhancing the decoding process.
Implementation Method 1
PCR (polymerase chain reaction) is mainly used to amplify the selected compounds
Implementation Method 2
quantitative PCR (qPCR) analysis using specific primers that encode information about the enriched DNA-substance conjugates
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
A method for providing a DNA-encoding library, the DNA-encoding library and a method of decoding a DNA-encoded library are presented. Many different DNA molecules are synthesized which differ from each other by comprising different DNA barcode sequences, wherein each DNA barcode sequence comprises at least a first coding region DNA sequence comprising at least a first part, a second part and a third part, wherein the second part is located between the first and third part and the second part differs between all the DNA molecules by at least two nucleotides. Each of the many different DNA molecules is bonded to at least a specific substance forming different DNA-substance conjugates, wherein the DNA-substance conjugates differ from each other by the specific substance and by their DNA molecules, wherein the first part and the third part encode information regarding the second part of the first coding region and wherein a certain first part and/or a certain third part uniquely codes for a certain group of DNA-substance conjugates which is smaller than the group of all DNA-substance conjugates in the DNA-encoded library. The DNA-encoded library has the advantage that, for example after an enrichment experiment performed with the library, the library may be decoded in a faster and less expensive manner than known DNA-encoded libraries.