Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

Abstract

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K+ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K+ solution is distinct from those reported on the 22 nt Tel22 in Na+ solution and in crystalline state in the presence of K+, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K+, regardless of the presence or absence of Na+. Furthermore, the addition of K+ readily converts the Na+-form conformation to the K+-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K+, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.

Figure 1

Folding topologies of Tel22. (A) Propeller-type parallel-stranded intramolecular G-quadruplex in the presence of K⁺ in crystalline state. (B) Basket-type mixed parallel/antiparallel-stranded intramolecular G-quadruplex in Na⁺ solution determined by NMR.

Figure 2

(A) Different four repeat telomeric DNA sequences. WT-Tel26, Tel22 and Tel24 are the wild-type four repeat telomeric DNA sequences with sizes of 26, 22 and 24 nt, respectively. The numbering system is shown above Tel26. (B) Imino and aromatic regions of 1D ¹H NMR spectra of Tel26 in K⁺ and Na⁺ solutions at 25°C. (C) Imino regions of 1D ¹H NMR spectra of Tel22 and Tel26 in Na⁺ and K⁺ solutions at 25°C.

Figure 3

CD spectra of Tel26, WT-Tel26 and Tel22 in 100 mM Na⁺ or K⁺ solutions at 25°C. The same CD signatures are observed for Tel26 and WT-Tel26 in K⁺ solution, while similarly distinct CD signatures are observed for Tel22 and Tel26 in Na⁺ solution.

Figure 4

(A) Imino proton assignments of Tel26 using 1D ¹⁵N-filtered experiments on site-specific labeled oligonucleotides. Conditions: 25 mM K-PO₄, 70 mM KCl, pH 7.0, 25°C, 0.6–0.7 mM DNA. (B) Long-range through-bond correlations between intraresidue imino and H8 protons via C5 of the guanine base, determined by JR-HMBC experiment at natural abundance (46).

Figure 5

(A) A G-tetrad with H1–H1 and H1–H8 connectivity patterns detectable in NOESY experiments. (B) Schematic diagram of the folding topology of the unimolecular human telomeric G-quadruplex in K⁺ solution. red ball, guanine; red box, (anti) guanine; magenta box, (syn) guanine; green ball, adenine; blue ball, thymine.

Figure 6

The expanded H1–H8/H2/H6 and H1–H1 regions of the exchangeable proton 2D JR-NOESY spectrum of Tel26 in K⁺ solution at 1°C. NOEs are labeled as follows: intra-tetrad NOEs are labeled in red, sequential NOEs are labeled in green, inter-tetrad NOEs are labeled in blue, NOEs between stacking adenine H2 protons and G-tetrad imino protons are labeled in black. Conditions: 1°C, 25 mM K-PO₄, 70 mM KCl, pH 7.0, 2.5 mM DNA.

Figure 7

Titration experiments of K⁺ in the presence of 150 mM Na⁺ for Tel26 (A) and Tel22 (C), and titration experiments of Na⁺ in the presence of 100 mM K⁺ for Tel26 (B) and Tel22 (D), monitored by CD spectroscopy.

Figure 8

(A) Imino proton region of Tel26 in K⁺ solution in a variable temperature study. As seen above 65°C there is no detectable peak intensity in the imino (exchangeable proton) region. The imino protons are labeled on the spectrum recorded at 20°C. (B) D₂O-to-H₂O NMR exchange experiments. The imino region of the 1D ¹H NMR spectrum of Tel26, which was first incubated overnight in 99.96% D₂O and then incubated in H₂O for 30 min, 3.5 h and then overnight at 25°C. Imino protons from the middle layer of the G-tetrad (Figure 5) are the last to appear and are labeled with the residue numbers. (C) Melting profile of Tel26 in K⁺ solution in a CD variable temperature study. The same melting temperature of 55°C is shown by both CD and NMR (A).

Figure 9

Comparison of G-quadruplex-forming sequences. Loops colored in red have been shown to adopt parallel-stranded double-chain-reversal side loops (for more details see text).

Figure 10

Schematic diagram of interconversions between the Na⁺ and K⁺ forms of telomeric G-quadruplexes. (A) For the extended four repeat telomeric sequence Tel26, the hybrid-type G-quadruplex structure is the most stable and thus the predominant form in the presence of K⁺, regardless of the presence or absence of Na⁺. Addition of K⁺ readily converts the preformed Na⁺-form G-quadruplex to the hybrid-type G-quadruplex conformation. Tel26 no longer forms a single stable intramolecular G-quadruplex structure in Na⁺ solution, likely caused by the steric interference of the flanking sequences with the diagonal loop, both of which positioned on the same side of the basket-type G-quadruplex structure. (B) The truncated Tel22 forms a single stable basket-type intramolecular G-quadruplex in Na⁺ solution. However, in the presence of K⁺ Tel22 does not form a single G-quadruplex structure and is likely to have two stable G-quadruplex conformations co-existing. A possible mechanism of the interconversion of the two G-quadruplex conformations is proposed. The exchange rate between the two stable G-quadruplexes is slow on the NMR time scale at 25°C (Figure 2C). Addition of K⁺ to the preformed Na⁺ basket-type G-quadruplex readily converts the conformation to the K⁺-form, thus the two interconvertable K⁺ G-quadruplex conformations are both more stable than the Na⁺-basket-type G-quadruplex.

Figure 11

A schematic model of DNA secondary structure in human telomeres. The hybrid-type telomeric G-quadruplex structure reported in this article can be readily folded and stacked end to end to form a compact-stacking structure for multimers of this conformation in the elongated linear telomeric DNA strand.