NanoBRET Assay for Epigenetic PPI in Living Cells

Inquiry

Epigenetic regulation is fundamentally driven by dynamic protein-protein interactions (PPIs) within chromatin-associated complexes. Directly monitoring these interactions in their native, living cellular environment is crucial for understanding their regulation and for developing targeted therapeutics. CD BioSciences offers expert NanoBRET assay services to illuminate these critical epigenetic PPIs in real time.

Overview of Epigenetic Protein-Protein Interactions

Epigenetic protein-protein interactions (PPIs) refer to the specific binding events between writers, erasers, readers, and scaffold proteins that form functional complexes like PRC2, MLL/COMPASS, or the BET transcriptional machinery. These interactions are often transient, regulated by cellular signals and post-translational modifications, and are essential for proper chromatin targeting, enzymatic activity, and gene regulation. Dysregulation of these PPIs is a hallmark of cancer and other diseases. Traditional methods like co-immunoprecipitation (Co-IP) provide only a static, endpoint snapshot and lack the temporal resolution and physiological context of living cells. Therefore, technologies capable of quantifying these dynamic interactions in real time within live cells are indispensable for validating drug targets, characterizing disease mechanisms, and screening for PPI modulators.

PPI inhibition and stabilization mechanisms of ortho- and allosteric structures.

Fig.1 Orthosteric and allosteric mechanisms for PPI inhibition and stabilization. (Lu H, et al., 2020)

Advantages of NanoBRET Technology

Conventional PPI assays often fail to capture the dynamics, specificity, and native context of interactions within living systems. NanoBRET technology represents a significant breakthrough for epigenetic PPI analysis. Its key advantages include:

Live-Cell & Real-Time Monitoring: Enables quantitative tracking of PPIs as they occur in living cells, allowing observation of kinetic changes in response to stimuli or drug treatment.
High Sensitivity & Signal-to-Noise Ratio: Utilizes the bright, stable NanoLuc luciferase as the donor and a cell-permeable HaloTag ligand as the acceptor, facilitating detection of interactions at near-physiological expression levels with minimal background.
Proximity-Based Specificity: Energy transfer occurs only when tagged proteins are within ~10 nm, ensuring detection of direct or very close macromolecular associations specific to true complex formation.
Endogenous Context Compatibility: The system can be adapted for endogenous tagging via gene editing technology, allowing PPI studies at natural expression levels and genomic loci without overexpression artifacts.
High-Throughput Compatibility: The assay is homogenous and readily scalable to 384-well plates, making it ideal for screening compound libraries for PPI inhibitors or stabilizers.

Our Services

Drawing on deep expertise in chromatin biology and biophysical assay development, CD BioSciences specializes in developing and deploying tailored NanoBRET assays to dissect the complex interactome of epigenetic regulation. We bridge the gap between complex biological hypotheses and actionable data by transforming challenging questions about dynamic protein complexes into robust, quantitative, and physiologically relevant insights. This approach accelerates your critical path from target validation and mechanism-of-action studies to the identification of novel PPI modulators in compound screening campaigns.

Customized Solution for NanoBRET Assays

We design and implement fully customized NanoBRET assays tailored to your specific epigenetic protein-protein interaction (PPI) pair and research objectives. Our expertise is uniquely focused on detecting the weak-affinity, post-translational modification (PTM)-dependent, and chromatin-context-sensitive PPIs that define epigenetic regulation. This approach transforms complex biological questions into precise, quantitative readouts, delivering physiologically relevant data directly from living cells.

Typical PPI Pairs	Description
Reader Domain & Modified Histone Interactions	Quantify the binding of epigenetic "readers" to their cognate histone marks, such as between BET bromodomains and acetylated histone tails.
Intra-complex Assembly & Dynamics	Monitor the assembly and stability of multi-subunit epigenetic complexes, for example, the interactions among core subunits of the PRC2 complex (EED, EZH2, and SUZ12).
Enzyme-Substrate/Cofactor Recruitment	Study the functional interactions between epigenetic enzymes and their essential partners, like the association of DNA methyltransferase DNMT1 with its cofactor UHRF1.
Competitive Inhibition Assays	Directly measure how a small-molecule inhibitor competitively disrupts a native PPI, such as a compound interfering with the binding of a BET bromodomain to an acetyl-lysine peptide.

Workflow of NanoBRET Assay Service

Construct Design & Molecular Cloning

We initiate the project by designing and cloning the optimal donor (NanoLuc fusion) and acceptor (HaloTag fusion) expression constructs tailored to your specific target proteins, ensuring proper folding and functionality of the fusion proteins.

Cell Line Establishment & Validation

We establish the assay system by generating stable cell lines expressing both constructs or by optimizing transient transfection conditions. Cell health and consistent expression levels of the fusion proteins are rigorously validated.

Assay Development & Optimization

Our scientists systematically optimize all critical assay parameters, including substrate concentrations, incubation times, and signal detection windows. We validate the assay's robustness by calculating key performance metrics such as the Z'-factor.

Screening & Data Acquisition

The validated assay is deployed for your specific application, whether for high-throughput compound screening or detailed mechanistic profiling. Quantitative BRET ratio data is collected in real-time or at specified endpoints.

Data Analysis & Reporting

We perform in-depth data analysis, which includes calculating normalized BRET ratios, generating dose-response curves (for IC₅₀/EC₅₀ determination), and performing kinetic fitting for time-course studies. A final, detailed report with all raw data, analysis, and biological interpretation is delivered.

Applicable Cellular Models

Our NanoBRET platform is adaptable to a wide range of cellular contexts to ensure your PPI data is biologically meaningful:

Types of Cell Models

Standard Adherent Lines: HEK293, HeLa, MCF-7, etc., for robust assay development and screening.
Suspension & Hematopoietic Cells: Jurkat, K562, THP-1, etc., for immune and blood cancer research.
Stem Cells & Differentiated Progeny: iPSCs, ESCs, neural progenitor cells, etc., for studying development and differentiation.
Patient-Derived or Engineered Disease Models: To capture disease-specific pathophysiology.
Custom Cell Line Development: We can establish stable, clonal cell lines expressing your tagged targets of interest.

Application Scenarios

Epigenetic Compound Screening & Validation: High-throughput screening to identify small-molecule inhibitors or molecular glues that disrupt or stabilize specific PPIs.
Mechanistic Studies (MOA): Quantitative investigation of how mutations, post-translational modifications, or cellular signaling pathways modulate PPI affinity.
Lead Compound Optimization: Guide structure-activity relationship (SAR) studies by quantifying PPI inhibition potency (IC50), enabling the rational optimization of compound efficacy.
Target Identification & Validation: Confirm whether novel epigenetic targets exert their function through specific PPIs.

Our Advantages

High Sensitivity & Low Background: Utilizes the bright NanoLuc luciferase as the donor and HaloTag fusion proteins as the acceptor, enabling high signal-to-noise ratios and detection of interactions within a very close proximity (<10 nm).
Physiological Relevance: Supports endogenous tagging via gene editing technology or optimized transfection, allowing PPI studies at near-native expression levels for biologically accurate insights.
Real-Time Kinetic Monitoring: Enables long-term live-cell imaging to monitor the dynamic changes in PPI strength over time or in response to drug treatment.
High-Throughput Screening Compatibility: The assay is readily adapted to a 384-well plate format, making it ideal for screening small-molecule or fragment libraries for PPI inhibitors or stabilizers.

Leveraging cutting-edge live-cell analysis technology, CD BioSciences provides the NanoBRET assay service, a powerful tool for elucidating dynamic epigenetic protein-protein interactions. To fully characterize epigenetic drug mechanisms, we also provide cell-based histone & tubulin deacetylation assays, histone deacetylase assays, and histone methylation cell assay services. This integrated, customizable suite is designed to meet all your functional validation and screening needs in epigenetic drug discovery. If you are interested in our services, please feel free to contact us for more details and quotation information of related services.

Reference

1. Lu H, Zhou Q, He J, et al. Recent advances in the development of protein–protein interactions modulators: mechanisms and clinical trials[J]. Signal transduction and targeted therapy, 2020, 5(1): 213.