Nanopore-Enabled STructure detection (NEST): High Resolution Genome-wide Mapping of DNA Secondary Structures

DNA is known for its iconic double helix structure, but it can also form alternative, non-canonical structures like G-quadruplexes (G4s), intercalated motifs, and hairpins. These secondary structures are important because they play key roles in regulating processes like gene expression. However, they can also cause problems by interfering with fundamental processes such as DNA replication, leading to genomic instability. Cells produce specialized proteins called helicases to manage and unwind these secondary structures, but many questions remain unanswered about how these helicases function and which structures they target. This project addresses the challenge of accurately detecting DNA secondary structures across the entire genome in different biological contexts. Current methods for mapping these structures are either biased or have limited resolution. Our goal is to develop a new technique using nanopore sequencing, which will allow us to detect and map these structures at single-nucleotide resolution without the need for amplification or complex reagents. The technology we are developing, called Nanopore-Enabled Structure detection (NEST), exploits the fact that secondary structures cause DNA molecules to pause as they are threaded through nanopores, and employs machine learning to pinpoint the exact location of these secondary structures in our genome. Our research has three main aims: Develop NEST using synthetic DNA and biological samples to reliably detect a wide range of secondary structures, starting with G4s. Map DNA structures in the absence of different helicases to understand how these helicases unwind G4s and how these structures are regulated during DNA replication. Investigate the role of the chromatin remodeller SMARCA4, which appears to protect cells from G4-induced DNA damage, especially in the context of cancer. The results of this work have far-reaching potential applications. By better understanding the biology of DNA secondary structures, this research could lead to new therapeutic strategies for treating cancers and other diseases linked to genome instability. It could also provide insights into viral and bacterial genomes, offering new ways to combat infections. The development of NEST could become a valuable tool for the scientific community, enabling more accurate and widespread detection of these structures. This project aligns well with BBSRC’s long-term research priorities by advancing our understanding of fundamental molecular biology and contributing to new innovations in biotechnology and health.