Methodologies for R-loop research: from detection to functional analysis.

R-loops are three-stranded nucleic acid structures composed of an RNA : DNA hybrid and a displaced single-stranded DNA strand, and they exert dual roles in transcriptional regulation, DNA damage repair, and the maintenance of genome stability. Over the past decade, a diverse array of techniques has been developed to detect and characterize R-loops at three complementary levels: genome-wide mapping, subcellular localization imaging, and interactome analysis. This review summarizes the principal methodological routes in these three areas, compares the basic principles and applicable scenarios of different strategies, and discusses their differences in specificity, resolution, sample requirements, and possible sources of assay-dependent variation. On this basis, we further consider the major challenges and future directions in the field, with the aim of providing a methodological reference for understanding the biological functions of R-loops and their relevance to disease diagnosis and treatment.