Sortase-Linked Tn5 Antibody Complexes for Single-Cell Chromatin Profiling
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Solution Overview
Problem
Traditional methods for analyzing co-occurring histone post-translational modifications (PTMs) in chromatin, such as ChIP-seq, ChIP-re-ChIP, and co-ChIP, require large input materials and are laborious, while CUT&Tag suffers from low efficiency and variability due to weak antibody attachment to Tn5 transposase.
Innovation Solution
Development of engineered Tn5 transposase complexes with a poly-glycine sequence covalently linked to specific antibodies via sortase, enabling direct and strong conjugation to recombinant antibodies, allowing high-efficiency profiling of co-occurring chromatin features at single-cell resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional ChIP-seq or ChIP-re-ChIP methods are used to profile co-occurring chromatin features, then comprehensive detection of histone PTMs is achieved, but large amounts of starting input materials are required and the protocols are laborious
Solution Approach 1:
The invention extracts and isolates specific chromatin features of interest using antibodies that bind to histone PTMs, separating them from the bulk chromatin material. This allows focused analysis of co-occurring features without requiring large amounts of total chromatin input, as the method enriches for specific epitopes before sequencing.
Solution Approach 2:
The invention introduces an antibody intermediary that specifically binds to histone PTMs of interest. This antibody mediator enables selective capture and enrichment of chromatin segments containing co-occurring modifications, allowing efficient profiling with minimal input material by focusing sequencing capacity on relevant genomic regions.
2Quantity of substance
If CUT&Tag method is used to reduce input requirements, then low cell numbers can be processed, but weak attachment of antibodies to the fusion proteins results in low efficiency and variability
Solution Approach 1:
The invention segments the antibody attachment process into distinct, optimized components: a protein A or protein G fusion partner with engineered properties for enhanced antibody binding, combined with Tn5 transposase activity. This segmentation allows independent optimization of each component's function, resulting in more reliable and consistent antibody attachment compared to the unified fusion protein approach.
Solution Approach 2:
The invention changes key parameters of the fusion protein, specifically modifying the protein A/G component to enhance antibody binding affinity and stability. By adjusting these molecular parameters, the method achieves stronger and more consistent antibody attachment to the Tn5 complex, reducing variability while maintaining the low input cell number advantage.
3Measurement precision
If sequential ChIP or co-ChIP methods are used to study co-occurrence of histone PTMs, then co-occurring modifications can be detected, but repeated washing of pulled-down chromatin requires large amounts of input materials
Solution Approach 1:
The invention merges multiple ChIP steps into a single CUT&Tag reaction. By combining antibody binding, chromatin crosslinking, and tagmentation into one integrated process, the method detects co-occurring PTMs without requiring repeated washing and re-pulling steps, thereby reducing input material requirements while maintaining co-occurrence detection capability.
Solution Approach 2:
The invention performs preliminary enrichment of specific chromatin regions containing co-occurring PTMs before sequencing. By using antibodies to pre-target and capture regions with multiple modifications in a single step, the method eliminates the need for subsequent repeated washing and re-enrichment steps, reducing material loss and input requirements.
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
The engineered Tn5 transposase complexes provide consistent and efficient profiling of multiple chromatin features with reduced variability, enabling low-input, single-cell resolution analysis of co-occurring histone PTMs.
Implementation Method 1
at least one antibody that is covalently linked to the at least one Tn5 transposase by a sortase
Implementation Method 2
covalently linking the at least one Tn5 transposase including poly-glycine sequence with at least one antibody including a LPXTG-motif tag by a sortase
Implementation Method 3
Fusion proteins comprising a modified Tn5 transposase and a binding domain, such as protein A or protein G
Implementation Method 4
which then introduces sequencing adapters to the locus
Data Source
AI summary
In some embodiments, provided is an engineered Tn5 transposase complex, comprising: at least one Tn5 transposase, comprising a poly-glycine sequence; and at least one antibody that is covalently linked to the at least one Tn5 transposase by a sortase, wherein the at least one antibody that is specific to a target site of a chromatin sequence. Other example embodiments are described herein. In certain embodiments, provided engineered Tn5 transposase complexes, systems, methods and kits provide outstanding performance in identifying chromatin features especially co-occurrence of chromatin features in samples and enable identifying multiple chromatin features at single cell resolution.