R-loops are three-stranded RNA:DNA hybrid structures essential for many normal and pathobiological processes. Previously, we generated a quantitative R-loop forming sequence (RLFS) model, quantitative model of R-loop-forming sequences (QmRLFS) and predicted ∼660 000 RLFSs; most of them located in genes and gene-flanking regions, G-rich regions and disease-associated genomic loci in the human genome. Here, we conducted a comprehensive comparative analysis of these RLFSs using experimental data and demonstrated the high performance of QmRLFS predictions on the nucleotide and genome scales. The preferential co-localization of RLFS with promoters, U1 splice sites, gene ends, enhancers and non-B DNA structures, such as G-quadruplexes, provides evidence for the mechanical linkage between DNA tertiary structures, transcription initiation and R-loops in critical regulatory genome regions. We introduced and characterized an abundant class of reverse-forward RLFS clusters highly enriched in non-B DNA structures, which localized to promoters, gene ends and enhancers. The RLFS co-localization with promoters and transcriptionally active enhancers suggested new models forin cisandin transregulation by RNA:DNA hybrids of transcription initiation and formation of 3D-chromatin loops. Overall, this study provides a rationale for the discovery and characterization of the non-B DNA regulatory structures involved in the formation of the RNA:DNA interactome as the basis for an emerging quantitative R-loop biology and pathobiology.