The c-kit gene encodes a receptor tyrosine kinase, whose engagement by its ligand triggers signals leading to cell proliferation. c-kit activity is elevated in gastrointestinal stromal tumors (GISTs), and its therapeutic inhibition by small molecules such as imatinib is clinically validated. We identified a putative quadruplex forming 21-nucleotide sequence upstream of the c-kit transcription initiation site (c-kit21), on the G-rich strand, which occupies a site required for core promoter activity. Here, we show by nuclear magnetic resonance (NMR), circular dichroism (CD), and ultraviolet (UV) spectroscopic methods that c-kit21 forms quadruplexes under physiological conditions. Mutational analysis of c-kit21 has provided insights into its structural polymorphism. In particular, one mutated form appears to form a single quadruplex species that adopts a parallel conformation. The quadruplex-forming sequence shows a high level of sequence conservation across human, mouse, rat, and chimpanzee. The small variation in sequence between the quadruplex in human/chimpanzee as compared to the rat/mouse was examined more closely by biophysical methods. Despite a variation in the sequence and length of loop 2, the quadruplexes showed both comparable CD spectra, indicative of parallel quadruplexes, and also similar thermalstability profiles, suggesting conservation of biophysical characteristics. Collectively, the evidence suggests that this quadruplex is a serious target for a detailed functional investigation at the cellbiology level.It was first proposed over 4 decades ago that guanine bases can associate in a cyclic arrangement to form planar arrangements of four guanines, G-quartets (1), in which each guanine is bonded to two neighboring guanines via hydrogen bonds (2). These G-quartets enable DNA sequences rich in guanines to form complex-folded structures, "quadruplexes", that are further stabilized by cations. The biological relevance of quadruplexes is now being intensively investigated, largely as a consequence of the identification of quadruplexforming sequences in the genome. Tandem-repeat sequences in the telomeres of humans (3), Oxytricha (4), and Tetrahymena (5) have been shown to form quadruplex structures. These have also been proposed in immunoglobin switch regions (6), within human

G-quadruplex nucleic acids have been proposed to play a role in a number of fundamental biological processes that include transcription and translation. We have developed a single-chain antibody that is selective for G-quadruplex DNA over double-stranded DNA, and here show that when it is expressed in human cells, it significantly affects the expression of a wide variety of genes, in a manner that correlates with the presence of predicted G-quadruplexes. We observe cases where gene expression is increased or decreased, and that there are apparent interactions with G-quadruplex motifs at the beginning and end of the genes, and on either strand. The outcomes of this genome-wide study demonstrate that G-quadruplex recognition by the antibody has physiological consequences, and provides insights into some of the complexity associated with G-quadruplex-based regulation.