Innovating Epigenetic Mapping: The Power of Tn5 Technology for Multiple Target Profiling

Abstract

Epigenetic modifications, such as histone modifications, and DNA-binding proteins like CTCF, PRC2, and transcription factors, are crucial in regulating gene expression and are implicated in various diseases, including cancer and neurodegenerative disorders. These specific epigenetic regulators are essential for maintaining cellular function, and any disruption in their regulation can lead to disease. Current methods for epigenomic profiling, while powerful, often fall short in efficiently and simultaneously profiling multiple targets, particularly in capturing the complexity of DNA-binding proteins alongside histone modifications. This limitation poses significant challenges for comprehensive epigenetic analysis. This thesis explores the development and application of a novel epigenetic profiling tool, SNAP-Tn5, which integrates SNAP-tag technology with Tn5 transposase. The SNAP- tag facilitates the targeted labeling of proteins, making it possible to simultaneously profile multiple epigenetic marks by attaching various functional groups, while Tn5 transposase is instrumental in DNA fragmentation and library preparation. The combination of these technologies aims to overcome the current limitations in multi- target profiling. The study first validates the tagmentation activity of SNAP-Tn5, demonstrating its effectiveness in DNA fragmentation and subsequent library preparation. A comparative analysis with the well-established pA-Tn5 enzyme reveals that while SNAP-Tn5 is functional, it initially shows lower efficiency. However, after conjugation with specific oligonucleotides, the efficiency of SNAP-Tn5 significantly improves, indicating that functionalization through SNAP-tag can mitigate the initial performance limitations. These findings suggest that, with further optimization, SNAP-Tn5 holds promise as a versatile tool for high-throughput, multi-target epigenetic profiling. This innovation could significantly advance our understanding of complex epigenetic landscapes and 4 contribute to the development of novel therapeutic strategies for diseases driven by epigenetic dysregulation.