Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored, fully C.C+ paired iMs. A higher temperature is necessary to unfold the kinetic forms and enable their substitution by a slowly developing thermodynamic structure. This complicated kinetic partitioning process considerably slows down iM folding, making it much slower than the timeframes of biological reactions and, therefore, unlikely to have any biological relevance. Our data suggest kinetically driven iM species as more likely to be biologically relevant than thermodynamically most stable iM forms.

Central European Institute of Technology Masaryk University Kamenice 5 Brno 625 00 Czech Republic

Department of Biophysics of Nucleic Acids Institute of Biophysics of the Czech Academy of Sciences Královopolská 135 Brno 612 00 Czech Republic

Laboratoire d'Optique and Biosciences Institut Polytechnique de Paris Inserm CNRS Ecole Polytechnique Palaiseau 91128 France

National Centre for Biomolecular Research Faculty of Science Masaryk University Kotlářská 2 Brno 611 37 Czech Republic

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