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Quantitative mapping of chromatin associated proteins

A technology for histones and proteins, applied in the field of quantitative mapping of chromatin-related proteins, can solve problems such as inability to map research, lack of capturing representative antibody epitopes, etc.

Pending Publication Date: 2021-11-19
EPICYPHER INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current DNA barcoded recombinant nucleosomes cannot be applied to genome mapping studies of ChAP (e.g., ChIP, ChIC, CUT&RUN, or CUT&Tag) because they lack the epitopes required to capture representative antibodies

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  • Quantitative mapping of chromatin associated proteins
  • Quantitative mapping of chromatin associated proteins

Examples

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Embodiment 1

[0168] Example 1: Generation of ChAP-containing nucleosomes using recombinant methods

[0169] Nucleosomes containing ChAP epitopes can be generated using any method known in the art. Below we introduce two methods. First, ChAP histone fusion proteins can be expressed directly using recombinant methods. Generates a series of nucleosomes (termed "verSaNuc") containing common SPTs, including 3xFLAG, 3xTY1, and 3xHA. For these nucleosomes, SPT was expressed followed by a GGGGS (SEQ ID NO: 8) linker fused to histone H3. This modified histone was then incorporated into the recombinant nucleosome using 250 bp of DNA (approximately 50 bp of adapter DNA on each side of the nucleosome core particle). Similar methods can be used for other ChAP fragments such as CTCF. Second, ChAP-containing histones can be produced by linking synthetic peptides or recombinantly expressed proteins by chemical or enzymatic ligation.

[0170] Nucleosomal incorporations were engineered to contain CTCF,...

Embodiment 2

[0172] Example 2: Generation of ChAP-containing nucleosomes using enzymatic ligation

[0173] To expedite nucleosome manufacture, the Staphylococcus aureus (S. aureus) sortase A (SrtA) transpeptidase can be used to directly attach modified peptides to fully assembled tailless nucleosomes ( Figure 1A ). This method provides two capabilities: a) rapid development of small batches of modified nucleosomes (microgram and milligram scale for standard dNuc assembly); and b) multiplexing of modified nucleosome synthesis. This method is well suited for ChAP-containing nucleosome development, which requires a small number of nucleosomes for each assay, but a large diversity to meet market demands.

[0174] In one example, verSaNuc nucleosomes were assembled comprising: (i) a unique DNA barcode identifier, and (ii) a GGGGS (SEQ ID NO: 8) motif at the N-terminus of H3. Next, use the encoding ChAP epitope (or SPT) and the C-terminal native sortase target motif (LPATG (SEQ ID NO: 9); F...

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Abstract

The present invention relates to DNA-barcoded recombinant nucleosomes and polynucleosomes that have been engineered for use as spike-in controls for the quantitative mapping of chromatin associated proteins using Chromatin ImmunoPrecipitation (ChIP) assays, tethered enzyme-based mapping assays, and other chromatin mapping assays. The invention further relates to methods of using the engineered DNA-barcoded recombinant nucleosomes in ChIP assays, tethered enzyme-based mapping assays, and other chromatin mapping assays.

Description

[0001] priority statement [0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 806,174, filed February 15, 2019, the entire contents of which are incorporated herein by reference. technical field [0003] The present invention relates to DNA barcoded recombinant nucleosomes and polynucleosomes constructed to map chromatin using chromatin immunoprecipitation (ChIP) assays, tethered enzyme-based mapping assays, and other chromatin mapping assays. Incorporation controls for quantitative mapping of related proteins. The invention also relates to methods of using engineered DNA barcoded recombinant nucleosomes in ChIP assays, tethered enzyme-based mapping assays, and other chromatin mapping assays. [0004] Background of the invention [0005] Chromatin immunoprecipitation sequential next-generation sequencing (ChIP-seq) is widely used to profile chromatin elements such as histone post-translational modifications (PTMs) and chromatin-associat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/10C12Q1/6804G01N33/68
CPCC07K19/00C12N15/1034C12Q1/6804G01N33/6872G01N33/6875G01N2800/52C12Q2565/133C12N15/1065
Inventor M·W·考尔斯Z-W·孙M-C·基奥
Owner EPICYPHER INC