Bio-synthesis of ¹⁵ N-Labeled G-Quadruplexes to Investigate the Structure and Interactions in the Cell Lysate using Nuclear Magnetic Resonance

G-quadruplexes (G4) play key roles in biology, making it critical to understand their structure and ligand-binding behavior in cellular environments for advancing G4-targeted therapeutics. While in-cell nuclear magnetic resonance (NMR) is a powerful technique for studying G4 in situ, its application is limited by the challenge of producing isotope-labeled single-stranded DNA (ssDNA). Here, we introduce Restriction Endonuclease Digestion (RED), a simple and cost-effective method to generate ¹⁵ N-labeled ssDNA. This approach combines molecular cloning and enzymatic design processing by propagating plasmids in E. coli cultured with ¹⁵ NH ₄ Cl, followed by double restriction digestion and isolation target ¹⁵ N-ssDNA. Using RED, we produced milligram-scale quantities of 96%-enriched ¹⁵ N-labeled human telomeric G4 ssDNA (wtTel23c, CTAGGG(TTAGGG)3), ideal for NMR analysis. The NMR spectra revealed that wtTel23c adopts G4 topology and undergoes multiple conformations of wtTel23c in potassium-containing solutions and in Xenopus laevis cell lysate. Interaction studies with the ligand TMPyP4 showed distinct binding profiles in cellular and dilute environments. In dilute solution, TMPyP4 binds to the top tetrad of wtTel23c, while it binds to the loop in cellular environments. The RED method offers an efficient strategy for producing stable isotope-labeled ssDNA, opening new avenues for studying G4 structures and their ligand interactions in complex biological contexts.