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 dC 20 , a human telomeric sequence (HTS) 5′‐dC 3 (TA 2 C 3 ) 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 dC 20 and the HTS. It also identifies that, under neutral conditions, the i‐motif from dC 20 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.