I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

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

Centre for Advanced Materials Application Slovak Academy of Sciences 845 11 Bratislava Slovakia

Department of Chemistry McGill University Montreal QC H3A0B8 Canada

Institute of Biophysics Czech Academy of Sciences Brno 612 00 Czech Republic

Instituto de Química Física 'Blas Cabrera' CSIC C Serrano 119 28006 Madrid Spain

Laboratoire d'Optique and Biosciences Institut Polytechnique de Paris Inserm CNRS Ecole Polytechnique Palaiseau 91120 France

National Centre for Biomolecular Research Masaryk University 625 00 Brno Czech Republic

Pharmaceutical and Pharmacological Sciences Department University of Padova 35131 Padova Italy

Slovenian NMR Centre National Institute of Chemistry SI 1000 Ljubljana Slovenia

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Protein structure and interactions elucidated with in-cell NMR for different cell cycle phases and in 3D human tissue models

In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells