Patel Brahmmi, Hoang Cailin, Yoo Hyejin, Davis Caitlin M
Department of Chemistry, Yale University, 225 Prospect Street, New Haven, Connecticut 06511-6712, United States.
bioRxiv. 2025 Jul 4:2025.06.30.661982. doi: 10.1101/2025.06.30.661982.
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. Therefore, additional work is necessary to understand how the cellular environment promotes their folding. Here we investigate the folding of the 15-mer thrombin-binding aptamer G-quadruplex (G4) and the designed 35-mer TAA(CTAA) i-motif (iM) inside living osteosarcoma cells. We use fast relaxation imaging, which couples fluorescence microscopy with a laser-induced temperature jump, to quantify the stability and dynamics of FRET-labeled constructs. The melting temperature of G4 and iM inside cells are 44.9 ± 0.9 °C and 46.5 ± 0.6 °C, respectively. Compared to in vitro buffers, inside cells G4 is destabilized by ≈10 °C and iM is stabilized by ≈20 °C. These in-cell stabilities cannot be replicated by simple buffers that account for macromolecular crowding or non-specific interactions in the nuclear environment. Instead, specific interactions with nuclear proteins produce the observed in-cell stability trends. Furthermore, our results reveal that charge-driven condensation regulates the in-cell folding kinetics of both G4 and iM. Our work suggests that the 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.
G-四链体和i-基序是非经典DNA结构，被认为在基因表达调控中起重要作用。尽管这些结构已在体外得到充分研究，但它们通常是在旨在稳定其结构的非生理缓冲液中进行表征的。因此，需要进一步开展工作来了解细胞环境如何促进它们的折叠。在这里，我们研究了15聚体凝血酶结合适体G-四链体（G4）和设计的35聚体TAA(CTAA) i-基序（iM）在活骨肉瘤细胞内的折叠情况。我们使用快速弛豫成像技术，将荧光显微镜与激光诱导温度跃升相结合，以量化FRET标记构建体的稳定性和动力学。细胞内G4和iM的解链温度分别为44.9±0.9°C和46.5±0.6°C。与体外缓冲液相比，细胞内G4的稳定性降低了约10°C，而iM的稳定性提高了约20°C。这些细胞内稳定性无法通过简单的缓冲液来复制，这些缓冲液考虑了核环境中的大分子拥挤或非特异性相互作用。相反，与核蛋白的特异性相互作用产生了观察到的细胞内稳定性趋势。此外，我们的结果表明，电荷驱动的凝聚作用调节了G4和iM在细胞内的折叠动力学。我们的工作表明，i-基序和G-四链体DNA的动力学通过相分离进行了精细调节，以适应细胞内关键调控过程的秒到分钟时间尺度。综上所述，我们的研究结果强调了在近生理条件下研究DNA结构折叠对于其机理理解以及开发靶向DNA药物的重要性。
