Nucleic Acid Binding Site Mapping via Tagged Nuclease Cleavage
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Solution Overview
Problem
Existing methods for mapping small molecule interactions with nucleic acids, such as chromatin, are limited by high binding affinity and low dissociation rates, leading to low signal and high background, and are not applicable to many probes, especially in rare cell populations, and do not account for differences in accessibility in native chromatin.
Innovation Solution
A method involving a tagged test compound covalently linked to a tag, which binds to nucleic acid or associated protein, followed by specific binding members and an activatable nuclease to cleave and sequence nucleic acid fragments for high-resolution mapping of binding sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional affinity pulldown methods are used to map small molecule binding sites, then binding sites can be identified, but the method requires high binding affinity and low dissociation rates which limits applicability to many probes and results in low signal with high background
Solution Approach 1:
The patent introduces an intermediary system consisting of a tag on the small molecule, a first binding member that recognizes the tag, and a second binding member that recruits the nuclease. This multi-component intermediary system allows probes with varying binding affinities to be effectively captured and mapped, resolving the contradiction between measurement precision and adaptability to different probes.
Solution Approach 2:
The small molecule is pre-modified with a tag before the mapping experiment. This preliminary action ensures that regardless of the probe's natural binding affinity, the tagged molecule can be reliably captured by the specific binding members, thereby improving both mapping precision and broadening probe applicability.
2Reliability
If formaldehyde cross-linking is used to preserve protein-DNA interactions, then binding sites can be captured, but epitope masking occurs and accessibility differences in native chromatin are not accounted for
Solution Approach 1:
The patent replaces the chemical cross-linking mechanism (formaldehyde) with a nuclease-based cleavage mechanism. The nuclease is recruited to the small molecule binding site through the tag and binding members, and cleaves the DNA in proximity to the binding site. This substitution avoids epitope masking while preserving binding site information, thereby improving both reliability and measurement precision.
3Quantity of substance
If large amounts of input material are used to overcome low DNA recovery yields, then sufficient DNA can be recovered for sequencing, but the method cannot be applied to rare cell populations
Solution Approach 1:
The patent employs local enrichment of DNA fragments at the small molecule binding sites through specific nuclease cleavage. Instead of requiring large amounts of total DNA, the method concentrates sequencing reads at the relevant binding sites, thereby improving DNA recovery efficiency and enabling application to rare cell populations without sacrificing binding site mapping quality.
4Stability of the object's composition
If the non-covalent interaction strength is increased to prevent dissociation during processing, then binding sites remain stable, but the method becomes less applicable to probes with weaker binding
Solution Approach 1:
The small molecule is pre-modified with a tag that has high-specificity binding partners (first and second binding members). This preliminary tagging action ensures stable complex formation during processing without requiring the probe itself to have high binding affinity, thereby maintaining complex stability while improving probe compatibility.
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 efficient and high-resolution mapping of small molecule binding sites within nucleic acids, providing insights into pharmacogenetics and enhancing therapeutic targeting.
Implementation Method 1
activating the nuclease, such that the nuclease cleaves the nucleic acid at the one or more binding sites to generate fragments
Data Source
AI summary
This invention relates to mapping the binding sites of a test compound within a nucleic acid. The nucleic acid is contacted with a tagged test compound that binds to the nucleic acid or to protein associated with the nucleic acid at one or more locations. The tagged test compound is contacted with a first binding member that specifically binds to the tag and a second binding member that specifically binds to the first binding member and is attached to an activatable nuclease, such that the second binding member binds to first binding member that is bound to the tagged test compound at the one or more binding sites. The nuclease is then activated to cleave the nucleic acid at the binding sites to generate fragments. The sequence of the generated fragments is indicative of the binding sites of the test compound.