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.
This is a preview of subscription content
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Parkinson, G. N., Lee, M. P. & Neidle, S. Crystal structure of parallel quadruplexes from human telomeric DNA. Nature 417, 876–880 (2002).
Phan, A. T., Gueron, M. & Leroy, J. L. The solution structure and internal motions of a fragment of the cytidine-rich strand of the human telomere. J. Mol. Biol. 299, 123–144 (2000).
Huppert, J. L. & Balasubramanian, S. Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33, 2908–2916 (2005).
Maizels, N. & Gray, L. T. The G4 genome. PLoS Genet. 9, e1003468 (2013).
Chambers, V. S. et al. High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat. Biotechnol. 33, 877–881 (2015).
Bochman, M. L., Paeschke, K. & Zakian, V. A. DNA secondary structures: stability and function of G-quadruplex structures. Nat. Rev. Genet. 13, 770–780 (2012).
Rhodes, D. & Lipps, H. J. G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res. 43, 8627–8637 (2015).
Biffi, G., Tannahill, D., McCafferty, J. & Balasubramanian, S. Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 5, 182–186 (2013).
Day, H. A., Pavlou, P. & Waller, Z. A. i-Motif DNA: structure, stability and targeting with ligands. Bioorg. Med. Chem. 22, 4407–4418 (2014).
Leroy, J. L., Gueron, M., Mergny, J. L. & Helene, C. Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif. Nucleic Acids Res. 22, 1600–1606 (1994).
Nonin-Lecomte, S. & Leroy, J. L. Structure of a C-rich strand fragment of the human centromeric satellite III: a pH-dependent intercalation topology. J. Mol. Biol. 309, 491–506 (2001).
Garavis, M., Escaja, N., Gabelica, V., Villasante, A. & Gonzalez, C. Centromeric alpha-satellite DNA adopts dimeric i-motif structures capped by AT Hoogsteen base pairs. Chemistry 21, 9816–9824 (2015).
Brooks, T. A., Kendrick, S. & Hurley, L. Making sense of G-quadruplex and i-motif functions in oncogene promoters. FEBS J. 277, 3459–3469 (2010).
Gurung, S. P., Schwarz, C., Hall, J. P., Cardin, C. J. & Brazier, J. A. The importance of loop length on the stability of i-motif structures. Chem. Commun. 51, 5630–5632 (2015).
Benabou, S. et al. Understanding the effect of the nature of the nucleobase in the loops on the stability of the i-motif structure. Phys. Chem. Chem. Phys. 18, 7997–8004 (2016).
Fujii, T. & Sugimoto, N. Loop nucleotides impact the stability of intrastrand i-motif structures at neutral pH. Phys. Chem. Chem. Phys. 17, 16719–16722 (2015).
Jin, K. S. et al. pH-dependent structures of an i-motif DNA in solution. J. Phys. Chem. B 113, 1852–1856 (2009).
Benabou, S., Avino, A., Eritja, R., Gonzalez, C. & Gargallo, R. Fundamental aspects of the nucleic acid i-motif structures. RSC Adv. 4, 26956–26980 (2014).
Cui, J., Waltman, P., Le, V. H. & Lewis, E. A. The effect of molecular crowding on the stability of human c-MYC promoter sequence I-motif at neutral pH. Molecules 18, 12751–12767 (2013).
Rajendran, A., Nakano, S. & Sugimoto, N. Molecular crowding of the cosolutes induces an intramolecular i-motif structure of triplet repeat DNA oligomers at neutral pH. Chem. Commun. 46, 1299–1301 (2010).
Li, H., Hai, J., Zhou, J. & Yuan, G. The formation and characteristics of the i-motif structure within the promoter of the c-myb proto-oncogene. J. Photochem. Photobiol. B 162, 625–632 (2016).
Sun, D. & Hurley, L. H. The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression. J. Med Chem. 52, 2863–2874 (2009).
Wright, E. P., Huppert, J. L. & Waller, Z. A. E. Identification of multiple genomic DNA sequences which form i-motif structures at neutral pH. Nucleic Acids Res. 45, 2951–2959 (2017).
Fleming, A. M. et al. 4n–1 is a ‘sweet spot’ in DNA i-motif folding of 2′-deoxycytidine homopolymers. J. Am. Chem. Soc. 139, 4682–4689 (2017).
Mir, B. et al. Prevalent sequences in the human genome can form mini i-motif structures at physiological pH. J. Am. Chem. Soc. 139, 13985–13988 (2017).
Takahashi, S., Brazier, J. A. & Sugimoto, N. Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase. Proc. Natl Acad. Sci. USA 114, 9605–9610 (2017).
Kang, H. J., Kendrick, S., Hecht, S. M. & Hurley, L. H. The transcriptional complex between the BCL2 i-motif and hnRNP LL is a molecular switch for control of gene expression that can be modulated by small molecules. J. Am. Chem. Soc. 136, 4172–4185 (2014).
Kendrick, S. et al. The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure. J. Am. Chem. Soc. 136, 4161–4171 (2014).
Sutherland , C., Cui, Y., Mao, H. & Hurley , L. H. Mechanosensor mechanism controls the G-quadruplex/i-motif molecular switch in the MYCpromoter NHE III1. J. Am. Chem. Soc. 138, 14138–14151 (2016).
Kaiser, C. E. et al. Insight into the complexity of the i-motif and G-quadruplex DNA structures formed in the KRAS promoter and subsequent drug-induced gene repression. J. Am. Chem. Soc. 139, 8522–8536 (2017).
Brown, R. V. et al. The consequences of overlapping G-quadruplexes and i-motifs in the platelet-derived growth factor receptor beta core promoter nuclease hypersensitive element can explain the unexpected effects of mutations and provide opportunities for selective targeting of both structures by small molecules to downregulate gene expression. J. Am. Chem. Soc. 139, 7456–7475 (2017).
Dudgeon, K. et al. General strategy for the generation of human antibody variable domains with increased aggregation resistance. Proc. Natl Acad. Sci. USA 109, 10879–10884 (2012).
Rouet, R., Dudgeon, K. & Christ, D. Generation of human single domain antibody repertoires by Kunkel mutagenesis. Methods Mol. Biol. 907, 195–209 (2012).
Rouet, R., Dudgeon, K., Christie, M., Langley, D. & Christ, D. Fully human VH single domains that rival the stability and cleft recognition of camelid antibodies. J. Biol. Chem. 290, 11905–11917 (2015).
Manzini, G., Yathindra, N. & Xodo, L. E. Evidence for intramolecularly folded i-DNA structures in biologically relevant CCC-repeat sequences. Nucleic Acids Res. 22, 4634–4640 (1994).
Dhakal, S., Lafontaine, J. L., Yu, Z., Koirala, D. & Mao, H. Intramolecular folding in human ILPR fragment with three C-rich repeats. PLoS One 7, e39271 (2012).
Kim, B. G. & Chalikian, T. V. Thermodynamic linkage analysis of pH-induced folding and unfolding transitions of i-motifs. Biophys. Chem. 216, 19–22 (2016).
Han, X., Leroy, J. L. & Gueron, M. An intramolecular i-motif: the solution structure and base-pair opening kinetics of d(5mCCT3CCT3ACCT3CC). J. Mol. Biol. 278, 949–965 (1998).
Guo, K. et al. Formation of pseudosymmetrical G-quadruplex and i-motif structures in the proximal promoter region of the RET oncogene. J. Am. Chem. Soc. 129, 10220–10228 (2007).
Guo, K., Gokhale, V., Hurley, L. H. & Sun, D. Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene. Nucleic Acids Res. 36, 4598–4608 (2008).
Li, T. & Famulok, M. I-motif-programmed functionalization of DNA nanocircles. J. Am. Chem. Soc. 135, 1593–1599 (2013).
Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).
Karsisiotis, A. I., O’Kane, C. & Webba da Silva, M. DNA quadruplex folding formalism—a tutorial on quadruplex topologies. Methods 64, 28–35 (2013).
Ambrus, A., Chen, D., Dai, J., Jones, R. A. & Yang, D. Solution structure of the biologically relevant G-quadruplex element in the human c-MYC promoter. Implications for G-quadruplex stabilization. Biochemistry 44, 2048–2058 (2005).
Dai, J., Chen, D., Jones, R. A., Hurley, L. H. & Yang, D. NMR solution structure of the major G-quadruplex structure formed in the human BCL2 promoter region. Nucleic Acids Res. 34, 5133–5144 (2006).
Agrawal, P., Hatzakis, E., Guo, K., Carver, M. & Yang, D. Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes. Nucleic Acids Res. 41, 10584–10592 (2013).
Phan, A. T., Kuryavyi, V., Luu, K. N. & Patel, D. J. Structure of two intramolecular G-quadruplexes formed by natural human telomere sequences in K+ solution. Nucleic Acids Res. 35, 6517–6525 (2007).
Luu, K. N., Phan, A. T., Kuryavyi, V., Lacroix, L. & Patel, D. J. Structure of the human telomere in K+ solution: an intramolecular (3+1) G-quadruplex scaffold. J. Am. Chem. Soc. 128, 9963–9970 (2006).
Schultze, P., Macaya, R. F. & Feigon, J. Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG). J. Mol. Biol. 235, 1532–1547 (1994).
Kuryavyi, V., Phan, A. T. & Patel, D. J. Solution structures of all parallel-stranded monomeric and dimeric G-quadruplex scaffolds of the human c-kit2 promoter. Nucleic Acids Res. 38, 6757–6773 (2010).
Jackman, J. & O’Connor, P. in Current Protocols in Cell Biology Ch. 8 (John Wiley and Sons, 1998).
Simon, S., Roy, D. & Schindler, M. Intracellular pH and the control of multidrug resistance. Proc. Natl Acad. Sci. USA 91, 1128–1132 (1994).
Huang, Z. & Huang, Y. The change of intracellular pH is involved in the cisplatin-resistance of human lung adenocarcinoma A549/DDP cells. Cancer Invest. 23, 26–32 (2005).
Smogorzewska, A. et al. Control of human telomere length by TRF1 and TRF2. Mol. Cell Biol. 20, 1659–1668 (2000).
Kendrick, S., Akiyama, Y., Hecht, S. M. & Hurley, L. H. The i-motif in the bcl-2 P1 promoter forms an unexpectedly stable structure with a unique 8:5:7 loop folding pattern. J. Am. Chem. Soc. 131, 17667–17676 (2009).
Roy, B. et al. Interaction of individual structural domains of hnRNP LL with the BCL2 promoter i-motif DNA. J. Am. Chem. Soc. 138, 10950–10962 (2016).
Aronheim, A., Shiran, R., Rosen, A. & Walker, M. D. The E2A gene product contains two separable and functionally distinct transcription activation domains. Proc. Natl Acad. Sci. USA 90, 8063–8067 (1993).
Schaffitzel, C. et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl Acad. Sci. USA 98, 8572–8577 (2001).
Xu, B., Devi, G. & Shao, F. Regulation of telomeric i-motif stability by 5-methylcytosine and 5-hydroxymethylcytosine modification. Org. Biomol. Chem. 13, 5646–5651 (2015).
Cui, Y., Kong, D., Ghimire, C., Xu, C. & Mao, H. Mutually exclusive formation of G-quadruplex and i-motif is a general phenomenon governed by steric hindrance in duplex DNA. Biochemistry 55, 2291–2299 (2016).
Wells, R. D. Non-B DNA conformations, mutagenesis and disease. Trends Biochem. Sci. 32, 271–278 (2007).
Zeraati, M. et al. Cancer-associated noncoding mutations affect RNA G-quadruplex-mediated regulation of gene expression. Sci. Rep. 7, 708 (2017).
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).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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
About this article
Cite this article
Zeraati, M., Langley, D.B., Schofield, P. et al. I-motif DNA structures are formed in the nuclei of human cells. Nature Chem 10, 631–637 (2018). https://doi.org/10.1038/s41557-018-0046-3
Nature Protocols (2022)
Nature Protocols (2022)
Nature Communications (2022)
Nano Research (2022)
Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS
Scientific Reports (2021)