DddA Deaminase for In Vivo DNA-Protein Interaction Mapping
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Solution Overview
Problem
Current methods for mapping DNA-protein interactions (DPIs) are limited by their inability to capture interactions in living cells and often require high starting material quantities, crosslinking artifacts, or low resolution, making them unsuitable for single-cell studies.
Innovation Solution
The method involves contacting a double-stranded DNA molecule with a target protein, coupling a double-stranded DNA deaminase (DddA) to the protein, allowing deamination of cytosine residues to uracil, and sequencing to detect deamination events, thereby mapping DPI sites with high resolution and sensitivity in living cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ChIP-seq or Cut&Run methods are used to map DNA-protein interactions, then DPI sites can be identified, but the methods cannot capture interactions in living cells and require high starting material quantities
Solution Approach 1:
The patent replaces mechanical/experimental methods (crosslinking, immunoprecipitation, DNA fragmentation) with an in vivo enzymatic tagging system. A deaminase enzyme is fused to the DNA-binding protein, allowing direct marking of interaction sites within living cells through cytosine deamination, eliminating the need for ex vivo processing and high starting material quantities
Solution Approach 2:
The patent introduces a deaminase enzyme as an intermediary that bridges the DNA-binding protein and the detectable mark (uracil residue). This intermediary enables indirect detection of DPIs through a biochemical cascade that occurs naturally within living cells, avoiding the need for direct isolation and analysis of DNA-protein complexes
2Measurement precision
If DamID method is used to map DPIs, then in vivo interactions can be captured, but resolution is limited to 1 kb due to DAM recognition site frequency
Solution Approach 1:
The patent changes the recognition parameter from DNA sequence specificity (DAM recognizes GATC sites every 8-10 bp) to enzymatic activity specificity (deaminase acts on cytosine residues within the protein-binding domain). This parameter change enables higher resolution mapping because the deaminase only modifies cytosines in the immediate vicinity of the DNA-protein interaction, rather than at fixed recognition sites throughout the genome
Solution Approach 2:
The patent applies local quality by confining the deaminase activity to a specific spatial domain - the region where the DNA-binding protein is bound to DNA. The deaminase modifies cytosine residues locally at the interaction site, creating a focused mark that precisely defines the DPI location rather than distributed marks across a large genomic region
3Measurement precision
If transposon insertion method is used to map DPIs, then in vivo interactions can be detected, but transposon insertions occur at low frequency and are not amenable to single cell studies
Solution Approach 1:
The patent implements continuous useful action by maintaining the deaminase enzyme continuously fused to the DNA-binding protein, allowing constant marking of interaction sites as the protein binds to DNA throughout cell division and growth. This continuous enzymatic activity ensures that every interaction event is captured, providing sufficient signal even in single-cell studies where transposon methods fail due to low insertion frequency
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, referred to as 3D-seq, enables precise mapping of DPIs in vivo at single-cell resolution, offering improved signal-to-noise ratio and reduced toxicity, and can be applied to various cell types, including bacteria and eukaryotes, with potential for genome-wide DPI detection.
Implementation Method 1
permitting deamination of one or more cytosine residues in a domain of the double stranded DNA molecule by the DddA to provide one or more uracil residues within the domain
Data Source
AI summary
The disclosure provides methods and related compositions and kits for mapping DNA-protein interactions (DPIs). In one aspect, the disclosed methods comprise contacting a double stranded DNA molecule with a target protein; coupling a double stranded DNA deaminase (DddA) to the target protein, before or after the contacting step; permitting deamination of one or more cytosine residues in a domain of the double stranded DNA molecule by the DddA to provide one or more uracil residues, wherein the domain comprises a site of interaction between the target protein and the double stranded DNA molecule; determining the sequence of at least a portion of the double stranded DNA molecule; and detecting the domain comprising one or more cytosine deamination events. The method can be controlled by use of DddA inhibitors. The method can also incorporate use of inhibiting a base-excision repair pathway when addressing DPIs in a cellular context.