Human genomic DNA is widely interspersed with i-motif structures

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles. Synopsis i-motifs (iMs) are knot-like DNA structures structures formed in the nuclei of human cells and believed to provide critical genomic regulation. This study uses immunoprecipitation and next-generation sequencing to identify i-motif structures in human DNA on a genome-wide scale. DNA i-motif structures are common in human genomic DNA. ~53,000 iMs are observed among three human cells lines (MCF7, U2OS, HEK293T). iMs are widely distributed throughout the human genome and frequent in genes upregulated in G0/G1 phase. Genome-wide i-motif immunoprecipitation and next-generation sequencing shows that sequences capable of forming these knot-like DNA structures are widely distributed and common in G0/G1-expressed genes.