Hybrid-type and two-tetrad antiparallel telomere DNA G-quadruplex structures in living human cells

Abstract

Although the telomeric sequence has been reported to form various G-quadruplex topologies in vitro and in Xenopus laevis oocytes, in living human cells, the topology of telomeric DNA G-quadruplex remains a challenge. To investigate the human telomeric DNA G-quadruplex in a more realistic human cell environment, in the present study, we demonstrated that the telomeric DNA sequence can form two hybrid-type and two-tetrad antiparallel G-quadruplex structures by in-cell 19F NMR in living human cells (HELA CELLS). This result provides valuable information for understanding the structures of human telomeric DNA in living human cells and for the design of new drugs that target telomeric DNA.

Figure 1.

¹⁹F NMR spectra of ¹⁹F-labeled 22-mer ODN 1 d[AGGG(TTAGGG)₃] in Na⁺ and K⁺ solutions. (A) Chemical structure of ¹⁹F-labeled DNA bearing ¹⁹F group at the 5′ terminal. (B)¹⁹F NMR of ¹⁹F-labeled DNA at different temperatures in Na⁺ solution. (C) ¹⁹F NMR of ¹⁹F-labeled DNA at different temperatures in K⁺ solution. Blue and black spots indicated antiparallel G-quadruplex and single strand, respectively. Green indicated hybrid-1 and hybrid-2 G-quadruplexes conformations. Temperatures indicated on the right. Condition: 0.1 mM DNA in (B) 300 mM NaCl and 20 mM Na-PO₄ buffer (pH 7.0) or (C) 100 mM KCl and 20 mM K-PO₄ buffer (pH 7.0). The sample is kept for 10 min of each temperature for ¹⁹F NMR detection.

Figure 2.

¹⁹F NMR spectra of ¹⁹F-labeled ODN 1 in PEG 200. (A) ¹⁹F NMR of ¹⁹F-labeled 22-mer DNA at different concentration of PEG 200 in K⁺ solution. (B) ¹⁹F NMR of ¹⁹F-labeled ODN 1 at different temperatures in 40% PEG 200. Green indicated hybrid-1 and hybrid-2 G-quadruplexes conformations. Orange and black spots indicated parallel G-quadruplex and single strand, respectively. PEG 200 ratio and temperatures indicated on the right. Condition: 0.1 mM DNA in 100 mM KCl and 20 mM K-PO₄ buffer (pH 7.0). The sample is kept for 10 min of each temperature for ¹⁹F NMR detection.

Figure 3.

In-cell ¹⁹F NMR of ¹⁹F labeled ODN 1. (A) Schematic overview of the SLO treatment cell system for transfection DNA into HeLa cells. (B) Comparison with the position of reference in vitro spectrum provides a reliable determination of intracellular ¹⁹F-labeled DNA. (C) Comparison of ¹⁹F NMR spectra of ODN 1 in K⁺ solution, in HeLa cell, in supernatant, difference spectrum between HeLa cell and supernatant, in K⁺ solution (23 and 60°C) and in 30% PEG 200.