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Abstract: Background: Telomere is protected by its G-quadruplex, T-loop structure, telomerase, and binding protein complex. Protein POT1 (protection of telomeres 1) is one subunit of telomere binding protein complex Shelterin. POT1 acts as a regulator of telomerase-dependent telomere length, and it can help telomere to form D-loop structure to stabilize telomere. POT1 protects telomere ends from ATR-dependent DNA damage response as well. Methods: Extensive methods were used, including CD, EMSA, ITC, PCR stop assay, luciferase reporter assay, quantitative real-time PCR, Western blot, chromatin immunoprecipitation (Ch-IP), cloning, expression and purification of proteins. Results: We found a new G-rich 30-base-pair long sequence (P-pot1 G18) located from −165 to −136 base pairs upstream of the translation starting site of protein POT1. This sequence in the promoter region of pot1 gene formed G-quadruplex resulting in down-regulation of pot1 gene transcription. This G-rich sequence is close to a binding site “TCCC” for transcription factor hnRNP K (heterogeneous nuclear ribonucleoprotein K), and its conversion to G-quadruplex prevented the access of hnRNP K to this binding site. The binding of hnRNP K could up-regulate pot1 gene transcription. TMPyP4 (meso-tetra(N-methyl-4-pyridyl)porphine) has been widely used as G-quadruplex binding ligand, which stabilized the G-quadruplex in vitro and in cellulo, resulting in down-regulation of pot1 gene transcription. Conclusions: This G-quadruplex might become a potentially new drug target for antitumor agents. General significance: Our results first demonstrated that G-quadruplex formation can affect the binding of transcription factor to its nearby binding site, and thus making additional influence to gene transcription. [Copyright &y& Elsevier]

Abstract: Background: G-quadruplexes are promising therapeutic targets for small molecules. In general, the introduction of steady positive charges through the in situ alkylation of nitrogen atoms within potential G-quadruplex ligands can significantly improve their quadruplex binding and stabilization abilities. However, our previous studies on bisaryldiketene derivatives showed that the derivative M4, whose central piperidone moiety is quaternized, exhibits a poor G-quadruplex stabilization ability. Methods: To clarify this unusual finding, CD, ITC, UV and NMR analyses were performed to determine the binding behaviors of M4 and its non-quaternized analog M2 to G-quadruplex DNA [d(TGGGT)]4. Molecular modeling approaches were also employed to help illustrate ligand–quadruplex DNA interactions. Results: The CD melting and ITC analyses revealed that M2 exhibited much stronger stabilization and binding abilities to [d(TGGGT)]4 compared to M4. Moreover, the CD and ITC analyses in combination with UV, NMR and MD simulations revealed that M2 tended to be end-stacked on the G-quartet, whereas M4 tended to be bound in the groove region. Analysis of the electrostatic potential showed that the charged surface of M4 was more positive than that of M2 and other reported ligands that bind to the G-quadruplex via end-stacking interactions. Conclusions: The results indicated that the different positively charged surfaces of M2 and M4 might be the key reason for their different binding modes. These different binding modes also lead to different binding affinities and stabilization abilities for [d(TGGGT)]4. General significance: These results provide new clues for the rational design of G-quadruplex-binding small molecules with steady positive charges. [Copyright &y& Elsevier]

Abstract: Background: G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied. Methods: Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation. Results: G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle. Conclusions: Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch. General significance: These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates. [Copyright &y& Elsevier]