Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.
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The authors thank S. Thompson (Leica Microsystems Australia) and P. Young (University of Sydney) for access to the Leica 3XSTED instrument. This work was supported by Program Grants 1113904, Project Grant 1148051, Development Grants 1113790 and 1076356 and Fellowship 105146 from the National Health and Medical Research Council (NHMRC) and Discovery Grants 160104915 and 140103465 from the Australian Research Council (ARC).
Material and Method section, supplementary tables outlining oligonucleotide sequences and phage display specifics, eight supplementary figures, and a supplementary method describing protocols for the selection of i-motif specific antibody fragments using phage display are described. In addition, we outline methods for counting nuclear foci using the FIJI software package
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