Sequencing of epigenetic modifications across the genome by single-molecule detection of oligonucleotide binding kinetics

PROJECT SUMMARY/ABSTRACT DNA methylation is a crucial epigenetic modification that plays a pivotal role in regulating gene expression and maintaining genomic stability. Dysregulated DNA methylation is linked to various pathological conditions, including cancer, autoimmune diseases, and neurological disorders, underscoring its significance in both health and disease. Accordingly, analysis of DNA methylation is fundamental in biomedical research and has growing clinical uses, particularly as a cancer biomarker. Next-Generation Sequencing (NGS), the cornerstone of current genetic and epigenetic analysis methods, is incapable of directly reading methylation. As a result, indirect techniques like bisulfite sequencing (BS-Seq) are used. BS-Seq is limited by low accuracy, high losses of input DNA, and an inability to concurrently read genetic sequence. Alternative conversion approaches and 'third generation’ sequencing resolve some issues but fail to significantly improve upon BS-Seq's accuracy and have drawbacks in other areas. The limitations of current DNA methylation analysis methods become increasingly apparent with the development of various methods that allow for the determination of DNA sequence at increasingly high accuracy. For example, using unique molecular identifiers (UMI) analysis of DNA sequence at accuracy exceeding 99.999% is possible (albeit at the cost of complex workflows and high sequencing costs). By contrast, all current methylation analysis methods deliver accuracy below 99% and are incompatible with error suppression methods like UMI. Thus, for key applications like Liquid Biopsy, which require high accuracy to detect cancer DNA present as an extreme minority of total DNA, no highly accurate method is available for the detection of methylation. XGenomes is developing a radically novel epigenetic analysis method which overcomes key limitations of existing technology. XGenomes’ single-molecule platform measures the transient repetitive binding of short (5- letter), fluorescently labeled probes to single-stranded target DNA. Probes exhibit a repetitive kinetic binding pattern, which alters in the presence of methylation. This effect is replicated across all probes binding a methylated site. Combining data from multiple probes allows for determination of the methylation status of each position in the target DNA molecule with unprecedented accuracy. XGenomes’ prior work has validated the fundamental approach. This Phase I project will build out the technology through two specific aims. In Aim 1, the chemistry of the probe library and buffer composition will be optimized for methylation discrimination. In Aim 2, we will refine, validate, and demonstrate our epi-sequencing platform in comparison with existing technologies.