%0 Journal Article
%T Variable-Temperature Size Exclusion Chromatography for the Study of the Structural Changes in G-Quadruplex
%A Sanae Benabou
%A Ramon Eritja
%A Raimundo Gargallo
%J ISRN Biochemistry
%D 2013
%R 10.1155/2013/631875
%X The conformational equilibria of a guanine-rich sequence found at the promoter region of the human c-kit oncogene are studied by means of circular dichroism spectroscopy (CD) and variable-temperature size exclusion chromatography (SEC). It is shown that the wild sequence ckit21 exists as a mixture of monomeric and multimeric G-quadruplexes. Appropriate mutation of several bases in the wild sequence produces the shift from parallel to antiparallel G-quadruplex, as well as the disappearance of multimeric species. The shift from the antiparallel to the parallel conformation induced by temperature is reflected in both CD and SEC profiles. 1. Introduction Guanine-rich regions can fold into complex structures known as G-quadruplexes, whose building block is the tetrad, an almost planar arrangement of four guanine bases bonded by hydrogen bonds [1]. Cations, such as Na+ or K+, are known to strongly stabilize such structures by intercalation in the middle or among the tetrads. Depending on the relative orientation of the guanine tracts, antiparallel, parallel, or mixed topologies can be found. The formation of G-quadruplexes in sequences corresponding to the telomeres and to the promoter regions of several oncogenes has been deeply studied in vitro by means of disparate instrumental techniques [2, 3]. Recently, the presence in vivo of these structures has been observed [4]. In their pioneer work, Fernando et al. identified a G-quadruplex forming sequence upstream of the c-kit transcription initiation site [5]. The wild sequence (herein identified as ckit21, Figure 1) contains three tracts of three guanines and a fourth tract comprising four guanines. Based on CD, NMR, and UV-monitored melting experiments, the mutated sequence ckit21T18 was shown to be less heterogeneous than ckit21, whereas a homogeneous parallel G-quadruplex was proposed for the mutated sequence ckit21T21. In a later work, Hsu et al. studied the conformational space of ckit21 and ckit21T21 by means of NMR [6]. Their results confirmed that, in presence of a concentration of K+ around 100£żmM, ckit21 existed as an ensemble of monomeric structures that share a parallel-stranded propeller-type conformation. Schematically, these structures produced two similar folding patterns named dangling-end and blunt-end (Figure 1). Hence, the sequence ckit21T18 should form the blunt-end structure, including a two-base third loop, whereas the sequence ckit21T21 should form the dangling-end structure, including a single-base reversal-loop. Almost simultaneously to the previous paper, the existence of a
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