4315179

Folding of a G-quadruplex and i-motif DNA in the nuclei of living cells

Date
August 20, 2025

G-quadruplexes and i-motifs are non-canonical DNA structures that are thought to play an important role in the regulation of gene expression. Although these structures have been well studied in vitro, they are often characterized in non-physiological buffers intended to stabilize their structure. In fact, until recently the existence of G-quadruplexes and i-motifs in vivo remained speculative. Therefore, in vivo work is necessary to understand how the cellular environment promotes folding and stability of these structures. Here, we investigate the folding of the 15-mer thrombin-binding aptamer (TBA) G-quadruplex and the 35-mer C5TAA i-motif inside living osteosarcoma cells. We use Fast Relaxation Imaging (FReI), which couples fluorescence microscopy with a laser-induced temperature jump to quantify the stability and dynamics of FRET-labeled constructs. The melting temperature of fTBA and fC5TAA inside cells are 46 ± 4 °C and 50 ± 4 °C, respectively. In vitro, under physiological crowding and salt conditions, the stability of fTBA, 50.4 (±1.2) °C, is near native but C5TAA is highly destabilized, 28.1 (±0.28) °C. While the in-cell stability of fC5TAA can be mimicked in vitro at pH values <7, these are far from physiological conditions. Recent in silico studies suggest that the presence of a complementary G-quadruplex can stabilize the i-motif. Indeed, that the i-motif folds in vivo supports a model where nuclear DNA stabilizes i-motif folding. Taken together, our work suggests that the folding of i-motifs and G-quadruplexes should be considered in native-like environments.

Presenter

Speaker Image for Caitlin Davis
Yale University

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