Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays
June 14th, 2024
We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.
Artios is developing novel anti-cancer medicines targeting the DNA damage response. To enable this, we developed a range of novel assays to measure cellular DNA repair activity. These assays had a major impact on our programs leading to clinical stage assets being tested in patients with high unmet medical needs.
Accurately measuring on-target cellular activity is essential for progressing novel targeted therapeutics. Generating assays for DNA repair factors with the required properties for fueling an efficient drug discovery campaign has been challenging, whether it's a need for high throughput, robust signal-to-noise, rapid turnaround, and ideally, portability to different biological systems. We have developed a suite of robust quantitative and titratable luminescence-based reporter assays to measure the proficiency of four major double-strand break repair pathways, homologous recombination, non-homologous end joining, microhomology-mediated end joining, and single-strand annealing.
These reporter assays are validated as reading out pathway-dependent repair, are sensitive to pharmacological modulation, and have a fast turnaround time. The repair substrates we have developed are extrachromosomal so they can be transiently transfected into models of interest and easily scaled into a high-throughput format. We believe our assay systems have an important place in enabling DDR drug discovery, but also in furthering our understanding of DDR biology more broadly.
In this respect, we hope the methods help accelerate the identification and prosecution of new drug targets, leading to medicines for diseases, such as cancer, where there is high unmet medical need. To begin, generate each reporter substrate and check their concentration using a spectrophotometer. Obtain 200 nanograms of reporter substrate.
Electrophorese each reporter substrate alongside a high molecular weight DNA ladder in a 0.7%agarose TAE gel containing a fluorescent double-stranded DNA stain. Verify that defined bands of the correct size are observed for each reporter substrate. If assessing the impact of compounds on the reporter assay, dispense compounds dissolved in the vehicle into the wells of a 96-well plate.
To prepare the transfection mix for a 96-well plate, dilute the Firefly control plasmid and NanoLuc double-strand break repair reporter substrate in 500 microliters of transfection buffer in a 1.5-milliliter tube. After adding the lipid-based transfection reagent, vortex the DNA mixture briefly and incubate for 10 minutes at room temperature. To harvest the cells, trypsinize and resuspend them in fresh medium containing 10%fetal bovine serum or FBS.
Then, determine the cell count and resuspend three times 10 to the power of six cells in 8.5 milliliters of medium with FBS in a fresh 15-milliliter tube. Add the DNA transfection mix into the cell suspension and invert the tube several times to mix. Plate 80 microliters of cell suspension per well and incubate the 96-well plate at 37 degrees Celsius with 5%carbon dioxide for 24 hours.
Prepare the control luciferase and the reporter luciferase reagent according to the manufacturer's instructions. Add substrate into an appropriate volume of assay buffer at a 1:100 ratio and mix. After the plate and reagents reach room temperature, add 80 microliters of control Firefly luciferase reagent per well.
Shake the plate for three minutes on an orbital shaker at 450 revolutions per minute. Place the plate in the luminescence plate reader and measure the control luciferase luminescence signal. To measure the reporter luciferase signal, add 80 microliters of NanoLuc reporter luciferase reagent per well.
After a three-minute incubation in an orbital shaker at 450 revolutions per minute, leave the plate to rest at room temperature for seven minutes. Finally, measure the reporter luciferase luminescence signal in a luminescence plate reader. Evaluation of the component NanoLuc and Firefly luminescence signals showed that the observed repair defect was driven by a reduction in the NanoLuc signal while the Firefly control signal was unperturbed.
Overview
This article presents protocols for assessing double strand break repair pathway proficiency in cells using luminescence-based extrachromosomal reporter substrates. The developed assays are crucial for understanding DNA damage response and facilitating drug discovery in oncology.
Key Study Components
Area of Science
- DNA damage response
- Cellular repair mechanisms
- Oncology drug discovery
Background
- Double strand breaks in DNA are critical lesions that need efficient repair.
- Understanding repair pathways is essential for developing targeted cancer therapies.
- Existing assays for measuring DNA repair activity have limitations in throughput and sensitivity.
- Novel assays can enhance drug discovery efforts by accurately measuring cellular activity.
Purpose of Study
- To develop robust assays for measuring proficiency in major double-strand break repair pathways.
- To facilitate the identification of new drug targets in cancer treatment.
- To improve the understanding of DNA damage response biology.
Methods Used
- Development of luminescence-based reporter assays for four repair pathways.
- Transient transfection of extrachromosomal substrates into cell models.
- Measurement of luminescence signals to assess repair proficiency.
- Validation of assays for sensitivity to pharmacological modulation.
Main Results
- Assays demonstrated pathway-dependent repair activity.
- Quantitative measurements showed sensitivity to drug modulation.
- Fast turnaround times were achieved for assay results.
- Assays are scalable for high-throughput screening.
Conclusions
- The developed assays are valuable tools for drug discovery in cancer.
- They enhance the understanding of DNA repair mechanisms.
- These methods can accelerate the identification of new therapeutic targets.
Frequently Asked Questions
Executive Industry Relevance
Quantitative luminescence-based reporter assays for DNA double strand break repair enable high-throughput, pathway-specific measurement of cellular DNA repair activity, directly supporting target validation and mechanistic de-risking in oncology drug discovery. These assays provide rapid, reproducible, and scalable outputs essential for advancing DNA damage response (DDR) therapeutics. Their integration into discovery pipelines enhances predictive confidence and portfolio triage for DDR-targeted assets.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of DDR pathway activity for functional target validation.
- Supports mechanistic de-risking by distinguishing pathway-specific repair proficiency.
- Facilitates predictive confidence in target engagement and biological relevance.
- Accelerates identification and prioritization of novel DDR drug targets.
Screening & Assay Development
- Provides validated, quantitative readouts for robust assay standardization.
- Delivers high-throughput, scalable formats suitable for compound screening.
- Ensures reproducibility and titratability across diverse biological systems.
- Enables reliable evaluation of pharmacological modulation of DNA repair pathways.
Translational & Preclinical Research
- Aligns pathway-specific repair measurements with disease-relevant DDR biology.
- Supports continuity from discovery through preclinical validation of DDR targets.
- Informs risk-adjusted advancement decisions for DDR-focused portfolios.
- Provides mechanistic insights to guide translational biomarker strategies.
Pipeline & Workflow Integration
These assays position within the discovery continuum from early target validation through lead identification and preclinical evaluation of DDR-targeted therapeutics.
- Discovery Biology: Supports hypothesis testing and pathway clarification for DDR mechanisms.
- Screening: Delivers assay readiness, reproducibility, and quantitative outputs for compound evaluation.
- Analytics: Provides sensitive luminescence measurements to compare repair activity across conditions.
- Translational Research: Enables alignment of cellular repair proficiency with disease models when relevant.
- Enterprise Reuse: Offers a portable, scalable platform adaptable to multiple cell systems and discovery programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in DDR target validation.
- Operational Value: Standardizes and accelerates assay workflows with high-throughput scalability.
- Strategic Value: Improves go/no-go decisions and capital efficiency by enabling robust biological readouts.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of DDR-targeted assets.
Implementation Considerations
- Requires expertise in molecular biology and luminescence assay design.
- Needs access to luminescence plate readers and standard cell culture infrastructure.
- Demands cross-team standardization for assay reproducibility and data comparability.
- Adaptable to various cell models via transient transfection of extrachromosomal substrates.
- Dependent on validated controls to distinguish pathway-specific repair activity.
