Solution Structure of the Biologically Relevant G-Quadruplex Element in the Human c-MYC Promoter. Implications for G-Quadruplex Stabilization

Abstract

The nuclease hypersensitivity element III(1) (NHE III(1)) of the c-MYC promoter strongly controls the transcriptional activity of the c-MYC oncogene. The purine-rich strand of the NHE III(1) element has been shown to be a silencer element for c-MYC transcription upon formation of a G-quadruplex structure. We have determined the predominant G-quadruplex structure of this silencer element in potassium solution by NMR. The G-quadruplex structure adopts an intramolecular parallel-stranded quadruplex conformation with three guanine tetrads and three side loops, including two single-nucleotide side loops and one double-nucleotide side loop, that connect the four guanine strands. The three side loops are very stable and well-defined. The 3'-flanking sequence forms a stable fold-back stacking conformation capping the top end of the G-quadruplex structure. The 5'-flanking A and G bases cap the bottom end of the G-quadruplex, with the adenine stacking very well with the bottom tetrad. This paper reports the first solution structure of a G-quadruplex found to form in the promoter region of an oncogene (c-MYC). This G-quadruplex structure is extremely stable, with a similar melting temperature (>85 degrees C) to that of the wild-type 27-mer purine-rich NHE III(1) sequence of the c-MYC promoter. This predominant quadruplex structure has been shown to be biologically relevant, and the structural information revealed in this research provides an important basis for the design of new drug candidates that specifically target the c-MYC G-quadruplex structure and modulate gene expression.

Diffusion-ordered nuclear magnetic resonance spectroscopy for analysis of DNA secondary structural elements

“…The optimization of this sequence for NMR structure determination has been performed by elongation and mutation, resulting in a modified sequence of (TGAG 3 TG 3 TAG 3 TG 3 TAA) which selected out the predominant G-quadruplex formed in the c-MYC promoter sequence [63]. The structure of this successful NMR candidate has already been solved in our laboratory [3]. The DOSY F1 projection of this sequence can be seen in Figure 6C, representing a sharp dominant peak at the monomeric molecular weight at a millimolar NMR concentration (referencing standards are not shown).…”

“…DNA; NMR; DOSY; quadruplex; bcl-2; human telomere; c-myc Structural analysis of unusual DNA motifs with particular emphasis on G-quadruplexes and imotifs has a central role in the identification and characterization of molecular targets related to these structures [1][2][3][4][5][6][7]. Such structures are implicated in several physiologically important regulatory processes, with special emphasis on carcinogenesis [4][5][6][7][8][9][10][11][12].…”

“…Most of the structural studies on these elements have been performed by NMR spectroscopy. In most cases the related nucleotide sequence bearing the actual physiological function in vivo needs to be truncated/ elongated and/or mutated in order to obtain a successful candidate for NMR structure determination [3][4][5][6][7]13]. This NMR candidate should be a well-defined molecular entity in physiologically relevant solution conditions (ionic strength, pH, temperature) representing the same conformation as the original sequence.…”

“…Sequences comprising multiple guanine runs show a propensity to form G-quadruplex structures [14,15]. The physiologically relevant form of G-quadruplexes is unimolecular as these structures generally fold up at the ends of telomeres from single-stranded DNA [5,7] or loop out in the promoter regions from double-stranded DNA [3,4,6]. Depending on sequence and solution conditions, some sequences form very stable unimolecular quadruplexes in physiological buffer conditions even at millimolar concentrations while other sequences tend to form higher order structures composed of multiple strands.…”

Ruthenation of Non‐stacked Guanines in DNA G‐Quadruplex Structures: Enhancement of c‐MYC Expression

“…Remarkably, irradiation of a mixture of the parallel c‐MYC quadruplex d[TTGAG 3 TG 3 TAG 3 TG 3 TA 3 ]10 (10 μ m ) with 5 equiv of 1 in 10 m m phosphate buffer (pH 7.5) and 100 m m KCl led to the clean formation of a product with a mass corresponding to a monoadduct derivative (Figure 2 A, trace C, peak at 22 min, MYC ‐[Ru], 81 % conversion). We also observed reaction in the absence of light (approx.…”