<div>G-quadruplexes (G4s) are non-canonical secondary structures formed in single-stranded guanine-rich nucleic acid sequences, such as those found in oncogene promoters and telomeres. <i>MYC</i>, one of the most critical oncogenes, has a DNA G4 (MycG4) in its proximal promoter region that functions as a transcriptional silencer. MycG4 is very stable and the pathological activation of <i>MYC</i> requires its active unfolding. However, it remains unclear what drives MycG4 unfolding in cancer cells. We have studied the interactions of DDX5 with the MycG4 at both molecular and cellular levels and discovered that DDX5 actively unfolds the MycG4 and is involved in the <i>MYC</i> gene transcriptional regulation, which is described in the first part of this dissertation. DDX5 is extremely proficient at unfolding the MycG4 and ATP hydrolysis is not directly coupled to the G4-unfolding of DDX5. In cancer cells, DDX5 is enriched at the <i>MYC</i> promoter and activates <i>MYC</i> transcription. G4-interactive small molecules inhibit the DDX5 interaction with the <i>MYC</i> promoter and DDX5-mediated <i>MYC</i> activation. The second part of this dissertation describes the study of interactions of indenoisoquinoline anticancer drugs with MycG4. The MycG4 transcriptional silencer is a very attractive therapeutic target. Compounds that bind and stabilize the MycG4 have been shown to repress <i>MYC</i> gene transcription and are antitumorigenic. Indenoisoquinolines are human topoisomerase I inhibitors in clinical testing. However, some indenoisoquinolines with potent anticancer activity do not exhibit strong topoisomerase I inhibition, suggesting a separate mechanism of action. Our studies show that indenoisoquinolines strongly bind and stabilize MycG4 and lower <i>MYC</i> levels in cancer cells. Moreover, the analysis of indenoisoquinoline analogues for their <i>MYC</i> inhibitory activity, topoisomerase I inhibitory activity, and anticancer activity reveals a synergistic effect of <i>MYC</i> inhibition and topoisomerase I inhibition on anticancer activity. Besides the MycG4, human telomeric G4s are also attractive targets for anticancer drugs due to their ability to inhibit telomere extension in cancer cells. The last part of this dissertation reviews two recent solution structural studies on small molecule complexes with the hybrid-2 telomeric G4 and the hybrid-1 telomeric G4. Structural information of those complexes can advance the design of telomeric G4-interactive small molecules in the cancer therapeutic areas.</div>

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18101号 / 理博第3979号 / 新制||理||1574(附属図書館) / 30959 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

Molecular recognition, particularly as it applies to strong binding interactions between complementary ligand/receptor molecules in solution, is important in such varied areas as molecular biology, pharmacology, synthetic chemistry, and chemical detection. Strong binding is the additive result of a number of specific, weak, non-covalent interactions occurring between complementary molecules. This dissertation reports on the energetics of forming complexes between small molecules and model DNA constructs. Ligands included cationic and metallated cationic porphyrins and polyheterocyclic ruthenium compounds. DNA receptors included double stranded B-DNAs (hairpin and short linear sequences) as well G-quadruplex DNAs. Thermodynamic data were collected using isothermal titration calorimetry, circular dichroism spectropolarimetry, ultraviolet-visible spectroscopy, and mass spectrometry. The measured thermodynamic parameters included the changes in free energy, enthalpy and entropy for ligand/receptor complex formation as well as the stoichiometry of the stable complexes. The first section of this dissertation reports that the binding of cationic porphyrins to model G-quadruplex DNA may proceed through two pathways, end stacking and intercalation. Modulating the number of pyridinium groups on a pyridinium substituted porphyrin yielded differing binding thermodynamics leading to the understanding that a balance of surface area, charge, and geometry affect the ability of a porphyrin to bind to G-quadruplex DNA. Further investigations into the binding of metallated porphyrins developed the understanding that the geometry of the central metal ion affected not only the thermodynamics but could also inhibit the intercalative mode. It was previously shown that the high affinity binding for binuclear polyheterocyclic ruthenium compounds proceeds through an intercalative mode. To further understand the binding process and the structureunction relationship of the ligand components, the binding of smaller mononuclear complexes that were representative of portions of the binuclear complex was examined in this dissertation. While limiting the intercalative ability lowered the binding affinity, the mononuclear complex with the full intercalating bridge was able bind to DNA with a higher affinity than the binuclear complex. These studies have been successful in part in determining the contributions of numerous weak interactions including: charge (Coulombic interactions), H-bonding, hydrophobic interactions, and solvent structure (solvation changes), to the overall energetics of this molecular recognition process. The first section of this dissertation reports that the binding of cationic porphyrins to model G-quadruplex DNA may proceed through two pathways, end stacking and intercalation. Modulating the number of pyridinium groups on a pyridinium substituted porphyrin yielded differing binding thermodynamics leading to the understanding that a balance of surface area, charge, and geometry affect the ability of a porphyrin to bind to G-quadruplex DNA. Further investigations into the binding of metallated porphyrins developed the understanding that the geometry of the central metal ion affected not only the thermodynamics but could also inhibit the intercalative mode. It was previously shown that the high affinity binding for binuclear polyheterocyclic ruthenium compounds proceeds through intercalation. To further understand the binding process and the structureunction relationship of the ligand components, the binding of smaller mononuclear complexes that were representative of portions of the binuclear complex was examined in this dissertation. While limiting the intercalative ability lowered the binding affinity, the mononuclear complex with the full intercalating bridge was able bind to DNA with a higher affinity than the binuclear complex. These studies have been successful in part in determining the contributions of numerous weak interactions including: charge (Coulombic interactions), H-bonding, hydrophobic interactions, and solvent structure (solvation changes), to the overall energetics of this molecular recognition process.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24125号 / 理博第4853号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)准教授 板東 俊和, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24326号 / エネ博第454号 / 新制||エネ||85(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Peptide hormones are important biomolecules that transduce downstream effects such as cell proliferation, regulation, and gene expression. Their levels have been upregulated in various disorders such as cancer, yet detection methods are lacking. We designed two split aptamer-based assays for the detection of a peptide hormone – Somatostatin (SST) – with different signal readouts: fluorescent readout based on light-up aptamers and the colorimetric readout of ABTS peroxidation from a G-quadruplex. We used an already selected split-aptamer –SSTA5–for SST for our designs and we had expected the developed detection systems to exhibit detection and quantification capabilities that would hopefully allow their use for SST monitoring in clinical samples. However, our experiments did not support the hypothesis of this project and SST was not able to be detected using either of our fluorescent or colorimetric methods. To determine if the SSTA5 aptamer could bind SST appropriately, Förster resonance energy transfer (FRET) was used. We verified that there was no energy transfer between two covalently-attached light-sensitive molecules (one attached to each part of the split SSTA5 aptamer); thus, we theorize that the aptamer does not hybridize in the presence of the tetra decapeptide SST. Therefore selection of another, more appropriate, aptamer for SST will be needed for further aptameric-based detection methods. Once this is accomplished, our methodologies could be re-applied for detection of SST which could lead to real-time detection of essential hormonal levels in patients.

No description available.

Gene expression regulation encompasses a wide range of mechanisms that govern cellular processes. Among these, post-transcriptional regulation, including translation control, plays a pivotal role in ensuring precise protein synthesis, timing, and quantity. Perturbations of mechanisms such as RNA modifications, and interactions between RNA-binding proteins (RBPs) and specific RNA motifs, can lead to dysregulation of essential cellular processes. These alterations contribute to the development of various disorders, including cancer, neurodegenerative diseases, and metabolic disorders. Many publicly available datasets and studies offer opportunities to investigate the link between alterations in these mechanisms and disease manifestations. However, the limited availability of datasets for certain conditions or notable inconsistencies among reported associations prevent complete understanding of the underlying processes. Therefore, extending the investigations to encompass a diverse range of genes and/or diseases will enhance our comprehension of these intricate regulatory and disease mechanisms, aiding in the identification of potential therapeutic targets and innovative interventions to mitigate pathological conditions. In particular, we focused on three separate aspects involved in gene expression regulation: RNA modifications, RBPs interactions with RNA secondary structures, and the Kozak consensus sequence as a translational modulator. Each part uncovers essential mechanisms that govern post-transcriptional control of gene expression, shedding light on their roles in cellular processes and disease contexts. At first, we performed a comprehensive exploration of 15 RBPs involved in the regulation of the N6-methyladenosine (m6A) methylation. Leveraging data from The Cancer Genome Atlas (TCGA), we conducted a pan-cancer analysis across 31 tumor types to uncover the distribution of alterations of these factors, and we developed a user-friendly web application to enable users to conduct similar analyses. Additionally, we performed a parallel analysis focused on neuroblastoma, using data from publicly available and in-house datasets. These investigations unveil the potential impact of a subset of m6A factors on cancer development and progression. While in the first case, VIRMA and YTHDF reader proteins, emerged as the most frequently altered genes with significant pan-cancer prognostic implications, in the context of neuroblastoma, the writer METTL14 and the reader ALKBH5, showed the most prominent roles. Subsequently, our focus shifted to a distinct subset of RBPs capable of interacting with RNA secondary structures, particularly with RNA G-quadruplexes (RG4s). We established a comprehensive database cataloging RBPs with potential RG4-binding capabilities. This resource represents a valuable tool for researchers aiming to explore the intricate interplays between RBPs and RG4s, and their putative implications in diverse biological processes and diseases. Finally, attention was directed to the Kozak sequence, a pivotal determinant of the regulation of translation initiation. Exploiting the power of base editors, we developed a method to optimize translation initiation by modifying the Kozak sequence. This strategy offers promise in addressing haploinsufficiency-related disorders, where enhancing the functional protein is essential. Overall, these findings present opportunities for the identification of potential therapeutic targets and precision medicine strategies to alleviate a spectrum of pathological conditions, thus fostering advancements in the field of molecular biology and disease management.