Authors
Zou, M., Tian, F., Zheng, W., Jiang, M., Zhang, S., Liu, X., Ma, C., Zhao, M., Dai, L., Li, C., Wang, S.
Abstract
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 15N-labeled ssDNA. This approach combines molecular cloning and enzymatic design processing by propagating plasmids in E. coli cultured with 15NH4Cl, followed by double restriction digestion and isolation target 15N-ssDNA. Using RED, we produced milligram-scale quantities of 96%-enriched 15N-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.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 06 Nov 2025.
Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 37
- Comments 0