Fluorescence and Ultrafast Fluorescence Unveil the Formation, Folding Molecularity, and Excitation Dynamics of Homo‐Oligomeric and Human Telomeric i‐Motifs at Acidic and Neutral pH

: i-Motifs are tetraplex DNAs known to be stable at acidic pH. The structure of i-motifs is important in DNA nanotechnology; i-motif-forming sequences with consecutive cytosine (C) molecules are abundant throughout the human genome. There is, however, little information on the structure of C-rich DNAs under physiologically relevant neutral conditions. The electron dynamics of i-motifs, crucial to both biology and materials applications, also remains largely unexplored. In this work, we report a combined femtosecond and nanosecond broadband time-resolved fluorescence (TRF) and steady-state fluorescence investigation on homo-oligomer dC20 , a human telomeric sequence (HTS) 5'-dC3 (TA2 C3 )3 , and its analogue performed with different excitation at both acidic and neutral pH. Our study provides direct observation of intrinsic fluorescence and the first full probe of the real-time dynamics of the intrinsic fluorescence from i-motifs formed from varied sequences and pH conditions. The results obtained demonstrate concrete evidence for the existence at neutral pH of i-motifs from both dC20 and the HTS. It also identifies that, under neutral conditions, the i-motif from dC20 adopting the bimolecular folding structure is significantly more stable than the HTS i-motif featuring the unimolecular topology. Our femtosecond and nanosecond TRF study unveils excitation dynamics distinctive of the interdigitated architecture of i-motifs with the excited states involved exhibiting deactivation over a remarkably broad timescale through multiple channels involving proton-coupled electron transfer lasting tens of picoseconds, as signified by the solvent kinetic isotope effect, and structure-dependent charge recombination in the hundreds of picoseconds to tens of nanoseconds time regime.