Functionalized Recombinant Mononucleosomes for High-Throughput Remodeling Assays
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Solution Overview
Problem
Current nucleosome remodeling assays are slow, low-throughput, and labor-intensive, making them impractical for screening large compound libraries, and they lack the sensitivity and speed required for high-throughput analysis.
Innovation Solution
The development of functionalized recombinant mononucleosomes that can be used in no-wash, high-throughput assays, allowing for the rapid quantification of enzyme activity and identification of compounds that modulate remodeling enzyme activity, by incorporating specific histone post-translational modifications and a signal site for direct readouts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional nucleosome remodeling assays are used, then measurement accuracy is maintained, but productivity is low and the process is labor-intensive
Solution Approach 1:
The patent replaces traditional mechanical/gel-based detection methods with fluorescence-based optical detection. Fluorophore-labeled nucleosomes allow direct measurement of remodeling activity through fluorescence intensity changes, eliminating the need for labor-intensive gel electrophoresis and manual analysis, thereby dramatically increasing throughput while reducing labor requirements
Solution Approach 2:
The patent introduces fluorophore labeling as a new parameter to track nucleosome remodeling. By attaching fluorescent markers to nucleosomes and measuring fluorescence signal changes during remodeling reactions, the assay transforms from a qualitative/semi-quantitative method to a highly sensitive quantitative method that can be automated and performed in high-throughput formats
2Speed
If traditional nucleosome remodeling assays are used, then measurement precision is adequate, but speed is slow
Solution Approach 1:
The patent replaces time-consuming gel electrophoresis and multiple washing steps with real-time fluorescence detection. The fluorophore-labeled nucleosomes allow continuous monitoring of remodeling reactions in solution, reducing assay time from hours to minutes while maintaining or improving measurement precision through sensitive optical detection
Solution Approach 2:
The patent enables continuous monitoring of nucleosome remodeling reactions through real-time fluorescence measurement. Instead of discrete sampling and processing steps, the fluorophore signal provides continuous data on remodeling progression, allowing rapid kinetic analysis and eliminating idle time between measurement steps
3Productivity
If functionalized recombinant mononucleosomes are used, then productivity and sensitivity are improved, but device complexity increases
Solution Approach 1:
The patent uses fluorophore labels as intermediaries to translate nucleosome remodeling events into measurable signals. The fluorophores attach to nucleosomes without interfering with remodeling activity, serving as convenient mediators that convert biological events into optical signals that can be detected by standard plate readers, thereby enabling high-throughput screening without requiring complex specialized instrumentation
Data Source
AI summary
The invention relates to assays for nucleosome remodeling activity using functionalized recombinant mononucleosomes. The functionalized recombinant mononucleosomes comprise a histone octamer comprising recombinant histone H2A, H2B, H3 and H4 proteins and a DNA template comprising a nucleosome positioning sequence that effectively positions the histone octamer and a signal site. The invention further relates to methods of using the assay to quantify enzymatic activity of remodeling enzymes and identifying modulators of remodeling enzyme activity.