RNA-Associated Chromatin DNA-DNA Interaction Method

RNA-Associated Chromatin DNA-DNA Interaction Method (RDD) is a targeted strategy for mapping chromatin interactions anchored by a specific RNA. It simultaneously identifies genomic loci associated with the RNA of interest and resolves long-range DNA-DNA contacts among these loci, enabling inference of spatial organization and regulatory relationships. In RDD, biotinylated antisense probes complementary to the target RNA are used to enrich RNA-chromatin complexes. Cells are crosslinked, chromatin is fragmented and end-repaired, and proximity ligation is performed using a designed linker to join DNA fragments that are spatially adjacent within the captured complexes. The ligation products are purified, converted into sequencing libraries, and analyzed by high-throughput sequencing to generate RNA-anchored interaction maps. RDD is applicable to endogenous RNAs as well as exogenous RNAs derived from host-infecting microorganisms and viruses, and it is compatible with diverse cell types and experimental conditions. The method yields locus-specific, high-resolution contact profiles suitable for dissecting enhancer-promoter pairing, long-range regulation, and three-dimensional genome architecture associated with regulatory coding and non-coding RNAs. By integrating biochemical enrichment with proximity ligation and next-generation sequencing, RDD provides a robust and reproducible workflow to reveal how RNAs organize chromatin and influence gene expression.