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  • br Conclusions br Introduction Bioinformatics analysis shows

    2022-06-22


    Conclusions
    Introduction Bioinformatics analysis shows that there are about 300,000 G-rich sequences in human genome if searching with the consensus sequence (G3+N1-7G3+N1-7G3+N1-7G3+) [1], and their localizations are non-random. These G-rich sequences always localize in functional regions, such as promoters [2], [3], untranslated regions [4], [5] and telomeres [6] to regulate the gene Epalrestat [7], [8]. The unusual topologies of G-quadruplex make it a potential target, for example, the chiral metallo-supramolecule and the carvoxylated single-wall carbon nanotubes could selectively recognize human telomeric G-quadruplex and inhibit telomerase activity [9], [10]. And expression of a given gene could be modulated by selective G-quadruplex binders. More convincingly, studies show that after the addition of G-quadruplex binder (For instance, TMPyP4), the expression of numerous genes showed differences, especially the genes which were enriched in G-rich sequences [11]. About 42.7% of human promoters have one or more G-rich sequences [2], which could affect the transcription progress in different ways. Their existences may impair the transcription by impending the RNA polymerase, or in other situations, may enhance gene expression through the removal of transcriptional suppressor, the recruitment of G-quadruplexes activators or facilitating the re-initiation of transcription by holding the DNA open [12]. c-Myb proto-oncogene is aberrantly expressed in leukemia and some other solid tumors, and it is an important target for cancer therapy [13], [14]. The G-rich sequences in the downstream region of c-Myb transcription start site have been proved to fold into G-quadruplex structures and to regulate c-Myb transcription [15], [16], [17]. Recently, the bioinformatics analysis shows that there are other four G-rich sequences [-80∼ −49 (S1), −112∼ −94 (S2), −352∼ −318 (S3) and −809∼ −775 (S4)] [19] in the upstream region of the transcription start site of c-Myb gene. At the upstream close to the transcription start site, the transcription factor myeloid zinc finger 1 (MZF-1, −52∼ −64 and −162∼ −178) [18] was proven to be a suppressor in the transcription of c-Myb gene through the binding of specific sequence, which coincided with S1 sequence (Fig. 1). We have used ESI–MS and CD spectroscopy to probe the formation of G-quadruplexes from the S1, S2, S3 and S4 sequences and their recognition by small-molecules, revealing that the G-rich sequences (S1-S4) could form stable G-quadruplex structures (Q1-Q4) [19] (Fig. S1), respectively. In this work, Q1 G-quadruplex was proved to function as an enhancer of c-Myb gene. In addition, topotecan was found to bind towards the G-quadruplex with high affinity, and to modulate the function of c-Myb in both mRNA and protein levels, which was based on the stabilization of the G-quadruplex structure. This study provided the possibility to modulate c-Myb transcription by G-quadruplex and topotecan.
    Materials and methods
    Results and discussions The G-rich sequences in the upstream region of the transcription start site of c-Myb gene were proved to form G-quadruplex structures in the NH4OAc or KCl solution by ESI–MS and CD experiments in our previous research [19]. When these G-rich DNA samples (S1-S4) were mixed with NH4OAc solution, the base peak in ESI mass spectra showed up with different adduct ammunium ion numbers [S+nNH4+-mH+](m−n)− (n=1-3, m=5, 8 or 9), and the high-resolution mass spectra identified the charges of the base peaks. The characteristics from ESI mass spectra indicated the formation of G-quadruplex structures [16], [17], [19] (Fig. S1a). When KCl were added to the S1-S4 samples, all sequences could form domanating parallel structures showing positive bands near 260nm and negative bands at 240nm. S1, S3 and S4 were identified to have high thermo stabilities, while the G-quadruplex formation ability of S2 sequence seems to be weaker according to the lower Tm value [19] (Fig. S1d).