Phase Separation Tunes the Stability and Dynamics of G-quadruplex and i-Motif DNA in the Nuclei of Living Cells

G-quadruplexes and i-motifs are non-canonical DNA structures that are important not only as targets of gene regulation in cancer therapy, but also as nanotools with biomedical applications. However, their apparent in vitro stabilities and dynamics cast doubt upon their physiological relevance and utility in vivo. Here we investigate folding of the thrombin-binding aptamer G-quadruplex and the TAA(C ₅ TAA) ₄ i-motif. We use fast relaxation imaging, which couples fluorescence microscopy with laser-induced temperature jumps, to quantify the stability and dynamics of FRET-labelled constructs in vitro and in the nuclei of living cells. Our work suggests that the stability and kinetics of i-motif and G-quadruplex DNA are carefully tuned by phase separation to fit the seconds-to-minutes timescales of key regulatory processes inside cells. Taken together, our findings underscore the importance of studying the folding of DNA structures under near-physiological conditions to their mechanistic understanding as well as for the development of DNA-targeted drugs.