Structure of the human telomere in K+ solution: a stable basket-type G-quadruplex with only two G-tetrad layers

Abstract

Previously, it has been reported that human telomeric DNA sequences could adopt in different experimental conditions four different intramolecular G-quadruplexes each involving three G-tetrad layers, namely, Na(+) solution antiparallel-stranded basket form, K(+) crystal parallel-stranded propeller form, K(+) solution (3 + 1) Form 1, and K(+) solution (3 + 1) Form 2. Here we present a new intramolecular G-quadruplex adopted by a four-repeat human telomeric sequence in K(+) solution (Form 3). This structure is a basket-type G-quadruplex with only two G-tetrad layers: loops are successively edgewise, diagonal, and edgewise; glycosidic conformations of guanines are syn x syn x anti x anti around each tetrad. Each strand of the core has both a parallel and an antiparallel adjacent strands; there are one narrow, one wide, and two medium grooves. Despite the presence of only two G-tetrads in the core, this structure is more stable than the three-G-tetrad intramolecular G-quadruplexes previously observed for human telomeric sequences in K(+) solution. Detailed structural elucidation of Form 3 revealed extensive base pairing and stacking in the loops capping both ends of the G-tetrad core, which might explain the high stability of the structure. This novel structure highlights the conformational heterogeneity of human telomeric DNA. It establishes a new folding principle for G-quadruplexes and suggests new loop sequences and structures for targeting in human telomeric DNA.

Figure 1

Schematic structures of intramolecular G-quadruplexes formed by the human telomeric sequences: (a) basket-type form observed for d[A(GGGTTA)₃GGG] in Na⁺ solution, (b) propeller-type form observed for d[A(GGGTTA)₃GGG] in a K⁺-containing crystal, (c) (3+1) Form 1 observed for d[TA(GGGTTA)₃GGG] in K⁺ solution, and (d) (3+1) Form 2 observed for d[TA(GGGTTA)₃GGGTT] in K⁺ solution. anti guanines are colored cyan; syn guanines are colored magenta.

Figure 2

Imino proton spectra of (a) the natural 22-nt human telomeric d[(GGGTTA)₃GGGT] sequence and (b) the modified d[GGGTTA(^BrG)GGTTAGGGTTAGGGT] sequence in K⁺ solution with assignments for the major forms listed over the spectra.

Figure 3

(a-c) CD spectra of (a) Form 3 (this work), (b) Form 2 and (c) Form 1 intramolecular human telomeric G-quadruplexes (strand concentration, 8 μM; potassium phosphate, 20 mM; KCl, 70 mM; pH 7). Dotted black and continuous red curves are for the natural and ^BrG-modified human telomeric sequences, respectively. (d,e) Melting curves of (d) the natural and (e) ^BrG-modified sequences of Form 1 (continuous line, left axis), Form 2 (dotted line, left axis) and Form 3 (red circles, right axis) monitored by UV absorption at 295 nm (DNA strand concentration, 8 μM; potassium phosphate, 20 mM; pH 7). (f) The fractions of G-quadruplexes as a function of temperature for three different DNA concentrations of ^BrG7-Form 3: 10 μM (continuous line), 50 μM (dotted line) and 250 μM (red circles), in a buffer containing 20 mM potassium phosphate pH7 and 20 mM KCl. In each case, both heating and cooling curves are plotted.

Figure 4

Imino proton spectra and assignments of the natural human telomeric d[(GGGTTA)₃GGGT] sequence in K⁺ solution. Imino protons were assigned in ¹⁵N-filtered spectra of samples, 2% ¹⁵N-labeled at the indicated position. The reference spectrum (ref.) is shown at the top.

Figure 5

H8 proton assignments. (a) Long range J-couplings in a guanine. (b, c) Through-bond correlations between guanine imino and H8 protons via ¹³C5 at natural abundance for (b) the natural d[(GGGTTA)₃GGGT] sequence and (c) the modified sequence, using long range J-couplings shown in (a).

Figure 6

NOESY spectra (mixing time, 300 ms), showing the H8/H6-H1′ connectivity of (a) the natural d[(GGGTTA)₃GGGT] sequence and (b) the modified sequence in K⁺ solution. Intra-residue H8/H6-H1′ NOE cross-peaks are labeled with residue numbers. Weak or missing sequential connectivities are marked with asterisks. For the modified sequence (b), the connectivity is broken at position 7 (shown by dotted line) because the proton H8 of G7 was replaced by bromine.

Figure 7

Determination of G-quadruplex folding topology. (a, b) NOESY spectra (mixing time, 200 ms), showing imino-H8 connectivity of (a) the natural d[(GGGTTA)₃GGGT] and (b) the modified sequence. Cross-peaks that identify the two G-tetrads are framed and labeled with the residue number of imino proton in the first position and that of H8 in the second position. (c) Characteristic guanine imino-H8 NOE connectivity patterns around a G_α•G_β•G_γ•G_δ tetrad as indicated with arrows (connectivity between G_δ and G_α implied). (d) Guanine imino-H8 NOE connectivities observed for G1•G14•G20•G8 and G2•G7•G19•G15 tetrads.

Figure 8

Structure of the human telomeric G-quadruplex Form 3 in K⁺ solution. (a) Stereo view of eight superpositioned refined structures of ^BrG7-Form 3. (b) Ribbon and (c) schematic view of a representative structure of natural Form 3. anti and syn guanines are colored cyan and magenta, respectively; adenines are colored green; thymines, orange; backbone, gray; O4′ atoms, yellow; phosphorus atoms, red. W, M1, M2 and N represent wide, medium 1, medium 2 and narrow grooves, respectively.

Figure 9

Stacking between different layers in the human telomeric G-quadruplex Form 3 in K⁺ solution. (a) View from the side looking into the medium groove 1. (b) Top T•T base pair and top triple. (c) Top triple and top tetrad. (d) The two tetrads. (e) Bottom tetrad and bottom triple. (f) Bottom triple and bottom two T bases.

Figure 10

Folding of the base of T4, T10, and T16 into the hydrophobic groove of the G-quadruplex, viewing from the side looking into (a) the wide groove (W) and (b) the medium groove 2 (M2).

Figure 11

Comparison of (a) the three-tetrad basket-type human telomeric G-quadruplex observed in Na⁺ solution and (b) the Form 3 two-tetrad basket-type human telomeric G-quadruplex observed in K⁺ solution (this work).