Nucleic Acid Interaction Detection via Recombinase Fragmentation
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Solution Overview
Problem
Current methods for identifying nucleic acid interactions, such as Chromosome Conformation Capture (3C) and Hi-C, face challenges when working with low cell numbers, requiring large cell quantities and resulting in complex libraries that are difficult to analyze for specific interactions like those between promoters and enhancers.
Innovation Solution
A method involving crosslinking, fragmentation, biotin marking, single-step fragmentation and oligonucleotide insertion using a recombinase enzyme, followed by enrichment and sequencing to identify interacting nucleic acid segments, which allows for focused data on specific interactions with fewer steps and reduced material requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Hi-C method is used to capture genome-wide interactions, then all interactions within the nucleus can be detected, but the resulting libraries are extremely complex and difficult to analyze at required resolution
Solution Approach 1:
The patent divides the genome into specific regions of interest (ROIs) and uses multiple targeted 3C assays, each focusing on a specific ROI. This segmentation approach breaks down the complex genome-wide analysis into manageable targeted assays, reducing library complexity while maintaining comprehensive coverage of regulatory elements and their target genes.
Solution Approach 2:
The patent extracts and enriches for specific interaction types by using biotinylated RNA baits that hybridize to target sequences. This extraction process isolates relevant interactions from the complex background, enabling focused analysis of promoter-enhancer interactions without the noise of all genomic interactions.
2Measurement precision
If conventional 3C or Hi-C methods are used, then nucleic acid interactions can be identified, but large numbers of cells (30-40 million) are required
Solution Approach 1:
The patent performs preliminary enrichment of target sequences using biotinylated RNA baits before the actual 3C library preparation and sequencing. This preliminary action concentrates the relevant genomic regions of interest, allowing detection of interactions from much smaller cell numbers by pre-amplifying the signal of interest.
Solution Approach 2:
The patent introduces biotinylated RNA baits as intermediaries that bridge the target genomic sequences and the detection system. These baits hybridize to specific genomic regions, enabling selective enrichment and amplification of target sequences, thereby reducing the cell number requirement while maintaining detection sensitivity.
3Measurement precision
If targeted approaches are used to focus on specific interactions, then analysis resolution improves, but the method requires previous knowledge of the interaction
Solution Approach 1:
The patent creates a universal platform using biotinylated RNA baits that can be applied to any genomic region of interest. The same core methodology and reagents can target different promoters, enhancers, or genomic elements, providing both high resolution for specific interactions and versatility to study any regulatory element without requiring prior knowledge of specific interactions.
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 enables the identification of nucleic acid interactions with reduced sample requirements, faster processing, and increased specificity, capturing over 22,000 promoters and their interacting genomic loci in a single experiment with a more quantitative readout, suitable for rare cell types or early developmental samples.
Implementation Method 1
performing single step fragmentation and oligonucleotide insertion on the ligated fragments using a recombinase enzyme
Implementation Method 2
enriching for fragments comprising the biotin moiety of step (d)
Implementation Method 3
crosslinking the nucleic acid composition
Data Source
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AI summary
The present invention relates to a method for identifying nucleic acid segments which interact with a target nucleic acid segment or segments as well as kits for performing the method. The invention also relates to a method of identifying one or more interacting nucleic acid segments that are indicative of a particular disease.