Microfluidic Single-Cell Protein-DNA Interaction Sequencing
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Solution Overview
Problem
Current methods for measuring protein-DNA interactions in single cells are limited by their inability to capture dynamic processes in small cell populations and asynchronous single cells, failing to account for biological heterogeneity and spatial localization of interactions within the nucleus.
Innovation Solution
A microfluidic device-based method that isolates and images single cells to determine the cellular location and nucleotide sequence of DNA contacted by a protein of interest, using DNA adenine methyltransferase (Dam) to chemically record protein-DNA interactions and pair this with imaging data for contemporaneous measurement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If bulk methods are used to measure protein-DNA interactions, then large numbers of cells can be processed, but critical epigenomic processes in small numbers of dividing cells and biological heterogeneity are lost
Solution Approach 1:
The invention segments the bulk cell population into individual single cells for separate analysis. Each cell is isolated in a microfluidic device, allowing protein-DNA interactions to be measured in individual cells while preserving biological heterogeneity and critical epigenomic processes that occur in small numbers of dividing cells.
Solution Approach 2:
The invention uses an intermediary chemical marking system where DNA adenine methyltransferase (Dam) enzymatically modifies DNA at protein-DNA interaction sites. This chemical mark serves as a mediator that records interaction information in single cells, enabling subsequent detection and sequencing while maintaining the single-cell context.
2Productivity
If bulk biochemical data are averaged together, then large datasets can be generated, but important epigenomic dynamics in asynchronous single cells during differentiation or cell cycle are missed
Solution Approach 1:
The invention segments the population-level measurement into individual cell measurements. By isolating and analyzing each cell separately in microfluidic chambers, the method captures epigenomic dynamics in asynchronous single cells during differentiation or cell cycle without averaging effects, while still processing multiple cells through parallelization.
Solution Approach 2:
The invention changes the measurement parameter from population-averaged biochemical signals to single-cell-resolved measurements. This parameter change enables detection of epigenomic dynamics in individual cells at different stages of differentiation or cell cycle, preserving temporal and spatial information that would be lost in bulk measurements.
3Measurement precision
If bulk methods are used, then biochemical data can be obtained, but pairing with imaging data that reveals spatial location of protein-DNA interactions is difficult
Solution Approach 1:
The invention merges biochemical measurement and imaging capabilities into a single microfluidic device platform. The device integrates chambers for cell isolation, imaging to capture spatial location of protein-DNA interactions, and sequencing to obtain biochemical data, allowing both measurement types to be performed on the same single cells without complex data pairing procedures.
Solution Approach 2:
The microfluidic device is designed as a universal platform that performs multiple functions: cell isolation, optical imaging, biochemical processing, and DNA sequencing preparation. This multi-functional integration eliminates the need for separate bulk biochemical assays and imaging experiments, directly linking spatial and biochemical information from the same single cells.
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 high-sensitivity, spatially resolved measurement of protein-DNA interactions in single cells, overcoming limitations of bulk methods by providing detailed, dynamic information on chromatin organization and gene expression states.
Implementation Method 1
the protein of interest is a fusion of the protein of interest and (i) DNA adenine methyltransferase (Dam) or a biologically active fragment thereof... Contacting the DNA sequence by the at least one protein of interest results in a modification to the DNA that is detectable by imaging. In one embodiment, the modification is methylation.
Data Source
AI summary
The present disclosure provides materials and methods for co-determining the cellular location and nucleotide sequence of a DNA that is contacted by (or in close proximity to) a protein of interest in a single cell. Thus the present disclosure provides methods and materials wherein the cellular location of the DNA comprising a DNA-binding site or otherwise in close proximity to a protein of interest is coupled to the sequence of said DNA to provide contemporaneous imaging and sequence measurement of a protein-DNA interaction.