Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter

Abstract

The c-kit oncogene is an important target in the treatment of gastrointestinal tumors. A potential approach to inhibition of the expression of this gene involves selective stabilization of G-quadruplex structures that may be induced to form in the c-kit promoter region. Here we report on the structure of an unprecedented intramolecular G-quadruplex formed by a G-rich sequence in the c-kit promoter in K+ solution. The structure represents a new folding topology with several unique features. Most strikingly, an isolated guanine is involved in G-tetrad core formation, despite the presence of four three-guanine tracts. There are four loops: two single-residue double-chain-reversal loops, a two-residue loop, and a five-residue stem-loop, which contain base-pairing alignments. This unique structural scaffold provides a highly specific platform for the future design of ligands specifically targeted to the promoter DNA of c-kit.

Figure 1

Spectra and assignments of c-kit87up. (a) The 600 MHz imino proton spectrum of the c-kit87up sequence in K⁺ solution at 25 °C. Unambiguous assignments are indicated. (b) Imino protons were assigned in ¹⁵N-filtered spectra of samples, 2% ¹⁵N-labeled at the indicated positions. (c) Long-range J couplings in a guanine used for imino-H8 correlation. (d) H8 proton assignments of c-kit87up by through-bond correlations between imino and H8 protons via ¹³C5 at natural abundance, using long-range J couplings shown in (c).

Figure 2

G-tetrad alignments and folding topology of c-kit87up. (a) Schematic of a G-tetrad with characteristic guanine imino-H8 connectivity patterns indicated by arrows. (b) NOESY spectrum (mixing time, 200 ms). The characteristic guanine imino-H8 cross-peaks for G-tetrads are framed and labeled with the imino proton assignment in the first position and that of the H8 proton in the second position. Peaks k, l, m, and n show close distances between G20(NH₂) and the imino protons of guanines from the bottom tetrad; peaks p, q, r, and s show close distances between A1(H2) and the imino protons of guanines from the top tetrad. (c) Summary of characteristic guanine imino-H8 patterns observed for c-kit87up. (d) Topology of c-kit87up based on the G-tetrad alignments in (c).

Figure 3

Solution structure of the c-kit87up quadruplex in K⁺ solution. (a) Stereoview of 11 superpositioned refined structures. Guanine bases in the G-tetrad core are colored cyan; other bases, green; the backbone, gray; phosphorus atoms, red; O4′atoms, yellow. (b) Ribbon and (c) surface views of a representative refined structure.

Figure 4

Base pairs in loops. Stacking (a) between the Watson-Crick A1•T12 pair and the G2•G6•G10•G13 tetrad, (b) between the Watson-Crick A16•G20 pair and the G4•G8•G22•G15 tetrad, (c) between the Watson-Crick A16•G20 pair and the sheared G17•A19 pair, and (d) between the sheared G17•A19 pair and the single-base G18.

Figure 5

Highlight of the C9-G10-C11-T12 fragment within the c-kit87up quaduplex.

Figure 6

The 600 MHz imino proton spectra of the modified c-kit87up sequences in K⁺ solution at 25 °C.

Figure 7

Comparison between G-quadruplex structures observed in (a, b) c-kit (this work) and (c, d) c-myc promoters. In (a) and (c): color coded as in Figure 3, except that snapback residues are colored orange; the blue arrows indicate break points within interrupted G-tetrad cores. In (b) and (d): loops are colored red; anti and syn guanines are colored cyan and magenta, respectively.