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  • br Experimental br Results and discussions


    Results and discussions
    Introduction The noncanonical DNA structures that is, i-motif [[1], [2], [3], [4], [5], [6], [7], [8], [9]] and G-quadruplex [[10], [11], [12], [13], [14]] have recently been carefully studied using either experimental or computational techniques. [[15], [16], [17], [18], [19], [20]] This is because the biological role of these structures appears to be more and more important as the research on them continues. G-quadruplex may form in the guanine rich DNA strand. Moreover, when it forms in the 3′ overhang then it may itself be a DNA damage signal, producing responses analogous to those of other mediators of telomere damage. [21,22] Additionally, the presence of G-quadruplex inhibits the telomerase activity [23] which is responsible for the reconstruction of the telomere length in Probucol mg reveling infinite proliferative capacity. Thus, the G-quadruplex stabilizing ligands are considered as selective inhibitors of cancer cell growth and it strongly suggests that induction of telomere shortening (or inhibition of telomerase activity) is a viable therapeutic strategy against cancer. [24,25] More recently, increasing interest is being paid to the i-motif structure which can form in cytosine rich genome fragments, particularly in telomeres. [1,3] Biological role of i-motif is less recognized and it was believed that it can form only at reduced pH when the semi-protonated cytosine pairs can form. [3,4,26] However, very recently the evidence for i-motif formation in vivo has been provided by in cell NMR experiments [27] and the discovery of an antibody that binds i-motif specifically in the nuclei of human cells. [28] It was thus confirmed that i-motif can exist in regulatory regions of genome in living cells at physiological conditions. Recently, however, Chen et al. [4] described that carboxylated single-walled carbon nanotubes can selectively stabilize human telomeric i-motif DNA. Moreover, they found a subsequent inhibition of telomerase activity in the studied living cells. They proposed that the stabilization of the i-motif structure and the concomitant G-quadruplex formation lead to telomere uncapping and displacement of telomere-binding proteins, generating a DNA-damage response at telomeric level and subsequent cessation of tumor cell growth. [4] G-quadruplex and i-motif are coupled in some way since they may form in the complementary regions of genome. However, in majority of cases these structure were studied independently and conclusions concerning their stabilization were rather limited to such factors like temperature, pH, presence of cations or molecular crowding conditions. There were also a few reports showing that these noncanonical DNA structures cannot exist together due to steric hindrance. [29,30] Though the mutual influence of both structures on their appearance and stabilization was understood [25] to our best knowledge these effects have not been adequately studied in the literature. In our recent paper we found that the presence of the G-quadruplex makes the i-motif containing cytosine-rich strand stable at the neutral pH though it was deteriorating spontaneously without the complementary guanine-rich and G-quadruplex containing strand. [31] We also found that iG structure (i-motif + complementary G-quadruplex structure) is relatively stable at neutral pH and the free energy barrier against i-motif unfolding/refolding is ca. 70 kJ mol−1. This means that spontaneous unfolding of i-motif to the hairpin (and vice-versa) can occur at the neutral pH but the lifetime of each of these structures will be long enough to be observed experimentally [31].
    Methods The analyzed iG structure (i-motif + G-quadruplex) has been composed by utilizing NAB (nucleic acid builder) language from AmberTools16 package [32] and using pdb files from the PDB database. The starting structure of the sequence [5′-GGG(TTAGGG)6]: [5′-CCC(TAACCC)6] has been build using NAB and next the atomic coordinates of the bases from 13 to 33 have been replaced by the corresponding coordinates from pdb files 2JPZ and 1EL2. The 1EL2 structure is a hybrid-type mixed parallel/antiparallel-stranded G-quadruplex (Hybrid-2) and this is intramolecular G-quadruplex structure of a biologically native, unmodified human telomeric sequence [33].