A pH-dependent bolt involving cytosine bases located in the lateral loops of antiparallel G-quadruplex structures within the SMARCA4 gene promotor

Abstract

Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its promoter region, a 44-nucleotides long guanine sequence prone to form G-quadruplex structures has been studied by means of spectroscopic techniques (circular dichroism, molecular absorption and nuclear magnetic resonance), size exclusion chromatography and multivariate analysis. The results have shown that the central 21-nucleotides long sequence comprising four guanine tracts of disparate length is able to fold into a pH-dependent ensemble of G-quadruplex structures. Based on acid-base titrations and melting experiments of wild and mutated sequences, the formation of a c•c + base pair between cytosine bases present at the two lateral loops is shown to promote a reduction in conformational heterogeneity, as well as an increase in thermal stability. The formation of this base pair is characterized by a pK a value of 7.1 ± 0.2 at 20 °C and 150 mM KCl. This value, higher than those usually found in i-motif structures, is related to the additional stability provided by guanine tetrads in the G-quadruplex. To our knowledge, this is the first thermodynamic description of this base pair in loops of antiparallel G-quadruplex structures. Nucleic acids can adopt structures other than the Watson-Crick double helix, such as the i-motif or G-quadruplex structures with biologically relevant roles. The i-motif is formed within cytosine-rich (C-rich) sequences, being its building block the C•C + base pair. As the protonation of some cytosine bases is needed for its formation, the overall stability of the i-motif structure is strongly dependent on pH 1,2. Because of this fact, the potential role in vivo of i-motif structures is still under investigation 3,4. On the other hand, G-quadruplexes are formed by DNA or RNA sequences that are particularly rich in guanine bases. The building block of these structures is the G-quartet (or G-tetrad), which consist on four guanine bases held together by Hoogsteen-type hydrogen bonds in a planar arrangement (Fig. 1a). G-quadruplex structures are mainly stabilized by intramolecular or intermolecular stacking of these G-quartets, as well as by electrostatic interactions with cations (such as K + or Na +) placed within the structure. G-quadruplex structure variability depends strongly on the length of the guanine tracks, the loops connecting the stacked G-quartets, the syn/anti preference of the purine bases, and the presence of ligands or modifiers. Hence, G-quadruplexes may be classified into 'antiparallel' , 'parallel' or even 'hybrid' arrangements (Fig. 1b) 5. The in vitro formation of such structures in DNA sequences corresponding to the end of telomeres and to the promoter regions of several oncogenes has previously been shown, as well as their in vivo presence 6,7. Hence, the role of G-quadruplex structures in biological processes like cancer or aging seems to be clear 8-12. Accordingly, research is being made to identify ligands that could selectively bind to G-quadruplex structures to modulate g...

Influence of pH and a porphyrin ligand on the stability of a G-quadruplex structure within a duplex segment near the promoter region of the SMARCA4 gene

“…The evolution of CD spectra along the titration reflects the presence of several equilibria in the studied pH range. As described previously [9], the interpretation of data of this kind by just plotting the variation of the signal (ellipticity or absorbance, see insets in Figure 3) at one single wavelength may result in a poor approach because minor contributions could be lost. Hence, the analysis of the whole data set (i.e., multivariate analysis) by means of appropriate mathematical methods may result a much better approach to solve it into the contributions of all the species present.…”

“…This may be because SMG01 is a longer sequence than SMG03, which shows more complex polymorphism, as denoted by SE-HPLC analysis (Figure S4 and reference [9]). The calculated stoichiometries and binding constants for the interaction of TMPyP4 with the antiparallel G-quadruplex formed by SMG03 agreed with previously reported values for antiparallel telomeric G-quadruplex [25].…”

“…Whereas the stability of G-quadruplex structures has been shown to depend strongly on the nature and concentration of appropriate cations, like potassium, the stability of i-motif structures is largely pH-dependent. However, recent works have described the existence of G-quadruplex-forming sequences that show also pH-dependent polymorphism [9] [10]. In vivo, the stability of G-quadruplex or i-motif structures is also strongly dependent on the presence of the complementary strand.…”

“…This gene is involved in some diseases, like small cell carcinoma of hypercalcemic type (SCOOHT), a type of ovarian cancer [11]. The study of SMG01 revealed the formation of G-quadruplex structures by the central 21-nucleotide long sequence (coded as SMG03) [9]. Surprisingly, it was found that the topology of these G-quadruplex structures was pH-dependent, with an apparent pH-transition midpoint around 7.4 (150 mM KCl, 25 o C).…”

The parallel-stranded d(CGA) duplex is a highly predictable structural motif with two conformationally distinct strands

“…The hydrogen bonding and base stacking interactions that stabilize anti-parallel duplexes also allow DNA to access other non-canonical conformations, some of which have known biological implications, including G-quadruplexes (3), i-motifs (4), and triplexes (5). Further, the formation of many non-canonical structures can be controlled by nucleotide sequence composition and several environmental factors including pH, the presence and concentration of cations, and temperature (6)(7)(8)(9)(10)(11). The alternative structures formed by genomic DNA triplet repeat sequences (12)(13)(14) have been implicated in their ability to expand and cause genetic instabilities (15,16).…”