C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of living cells is not currently known. Herein, using state-of-the-art in-cell NMR spectroscopy, we evaluate the stabilities of i-motif structures in the complex cellular environment. We show that i-motifs formed from naturally occurring C-rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i-motif stabilities in vivo are generally distinct from those in vitro. Our results are the first to interlink the stability of DNA i-motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNA biosensors for intracellular applications.

Cancer Research Cambridge Institute University of Cambridge Li Ka Shing Centre Cambridge CB2 0RE UK

CEITEC Central European Institute of Technology Masaryk University Brno 625 00 Czech Republic

Faculty of Science University of South Bohemia 370 05 Ceske Budejovice Czech Republic

Institute of Biophysics Academy of Sciences of the Czech Republic 612 65 Brno Czech Republic

Université de Bordeaux INSERM U1212 CNRS UMR 5320 ARNA Laboratory IECB Pessac France

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