Abstract
R-loops, structures that play a crucial role in various biological processes, are integral to gene expression, the maintenance of genome stability, and the formation of epigenomic signatures. When these R-loops are deregulated, they can contribute to the development of serious health conditions, including cancer and neurodegenerative diseases. The detection of R-loops is a complex process that involves several approaches. These include S9.6 antibody- or RNAse H-based immunoprecipitation, non-denaturing bisulfite footprinting, gel electrophoresis, and electron microscopy. Each of these methods offers unique insights into the nature and behavior of R-loops. In our study, we introduce a novel protocol that has been developed based on a single-molecule DNA combing assay. This innovative approach allows for the direct and simultaneous visualization of RNA:DNA hybrids and replication forks, providing a more comprehensive understanding of these structures. Our findings confirm the transcriptional origin of the hybrids, adding to the body of knowledge about their formation. Furthermore, we demonstrate that these hybrids have an inhibitory effect on the progression of replication forks, highlighting their potential impact on DNA replication and cellular function.