Different loop arrangements of intramolecular human telomeric (3+1) G-quadruplexes in K+ solution

Abstract

Intramolecular G-quadruplexes formed by the human telomeric G-rich strand are promising anticancer targets. Here we show that four-repeat human telomeric DNA sequences can adopt two different intramolecular G-quadruplex folds in K+ solution. The two structures contain the (3+1) G-tetrad core, in which three G-tracts are oriented in one direction and the fourth in the opposite direction, with one double-chain-reversal and two edgewise loops, but involve different loop arrangements. This result indicates the robustness of the (3+1) core G-quadruplex topology, thereby suggesting it as an important platform for structure-based drug design. Our data also support the view that multiple human telomeric G-quadruplex conformations coexist in K+ solution. Furthermore, even small changes to flanking sequences can perturb the equilibrium between different coexisting G-quadruplex forms.

Figure 1

Examples of imino proton spectra of human telomeric sequences in K⁺ solution showing recognizable spectral patterns of two different G-quadruplex forms (labeled with asterisk (*) and hash (#), respectively): (a) d[TTAGGG(TTAGGG)₃TT], (b) d[TAGGG(TTAGGG)₃TT], (c) d[TTAGGG(TTAGGG)₃] and (d) d[TAGGG(TTAGGG)₃].

Figure 2

Imino proton spectra of human telomeric sequences in K⁺ solution, (a) d[TAGGG(TTAGGG)₃TT] and (b) d[TAGGG(TTAGGG)₃] (34). In each case, peaks for the major form are labeled with residue numbers obtained from unambiguous assignments.

Figure 3

Imino proton spectra and assignments of the 25-nt human telomeric d[TAGGG(TTAGGG)₃TT] sequence in K⁺ solution. (a) Guanine imino proton spectra with assignments over the reference spectrum (ref). Imino protons were assigned in ¹⁵N-filtered spectra of samples, 2% ¹⁵N-labeled at the indicated positions. (b) Imino proton spectra after 1 h in D₂O at 25°C.

Figure 4

H8 proton assignments of the 25-nt human telomeric d[TAGGG(TTAGGG)₃TT] sequence. (a) Long range J-couplings in a guanine. (b) Through-bond correlations between guanine imino and H8 protons via ¹³C5 at natural abundance, using long range J-couplings shown in (a).

Figure 5

Determination of G-quadruplex topology for the 25-nt human telomeric d[TAGGG(TTAGGG)₃TT] sequence in K⁺ solution. (a) NOESY spectrum (mixing time, 200 ms). Imino-H8 cross peaks that identify three G-tetrads (colored green, red and blue) are framed and labeled with the number of imino protons in the first position and that of H8 in the second position. (b) NOESY spectrum (mixing time, 100 ms). Rectangular H8-H1′ patterns for 5′-syn-anti-3′ steps are highlighted by black lines. Downfield-shifted peaks for A(H8-H1′) are framed in a red box. Some peaks of G3 and G15 in (a) and (b) are broadened at 25°C, probably reflecting a motion at the top of the structure. (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) Characteristic guanine imino-H8 NOE connectivities observed for G3•G21•G15•G11 (green), G4•G10•G16•G22 (red) and G5•G9•G17•G23 (blue) tetrads. (e) Schematic structure of Form 2 human telomeric G-quadruplex. anti and syn guanines are colored cyan and magenta, respectively.

Figure 6

Schematic structures of possible intramolecular human telomeric (3+1) G-quadruplexes. (a) Form 2 observed for the d[TAGGG(TTAGGG)₃TT] sequence in K⁺ solution (this work). (b) Form 1 observed for the d[TAGGG(TTAGGG)₃] sequence in K⁺ solution (34). (c and d) Models of intramolecular (3+1) G-quadruplexes with two double-chain-reversal loops. Loops are colored red; anti and syn guanines are colored cyan and magenta, respectively.