京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第19415号 / エネ博第323号 / 新制||エネ||65(附属図書館) / 32440 / 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 木下 正弘 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19527号 / 理博第4187号 / 新制||理||1601(附属図書館) / 32563 / 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

The treatment and/or prevention of cancer by selective down regulation of cancer causing gene (oncogene) transcription would represent a significant advance in the area of anticancer drug design. Non-canonical higher order DNA structures formed in oncogene promoter regions are novel targets for the modulation of oncogene expression. An obvious advantage of selectively targeting oncogene expression would be that general cytotoxicity would be minimized and the negative side effects of current chemotherapy approaches could be minimized or eliminated. To provide a foundation for the design of drugs that target oncogene promoter G-quadruplexes and i-Motifs, the basic understanding is required of the folding of guanine and cytosine rich sequences and how small molecules bind to these structures. The research reported here focuses on higher order DNA structures of two oncogenes, K-ras that is overexpressed in pancreatic cancer, and Bcl-2 that is overexpressed in a number of cancers, and one non-oncogene, HAR1. We have probed the overall structure, stability, and binding of a model drug compounds to G-quadruplex and i-Motif DNA structures in these genes. The overall objectives of this work were: 1) to understand the relationship between oligonucleotide sequence and intramolecular folding topology and stability, and 2) to understand the mechanisms for the selective binding of small molecules to these structures. Biophysical techniques including: microcalorimetry, spectroscopy, analytical ultracentrifugation, gel electrophoresis, and computational methods were used to characterize both the folding and the binding interactions. We have shown that the native K-ras purine and pyrimidine rich sequences form stable G-quadruplexes and i-Motifs. We have also characterized four G-rich sequences found within the reading frame of the human HAR1 gene. This is the first report on the formation of stable G-quadruplex motifs within the RF of any gene. The model drug, TMPyP4, binds to the Bcl-2, K-ras, and HAR1 G-quadruplexes by two different binding modes, end binding and intercalation. The significance of this research is that the results of the K-ras and Bcl-2 studies could lead to the design of drugs that selectively target oncogenes while the HAR1 results could provide new approaches to the treatment of Schizophrenia and Alzheimer’s disease.

<p>c-Myc is one of the most important oncogenes. G-quadruplex DNA secondary structure formed in the proximal promoter region of c-Myc functions as a transcription silencer and is targetable by small molecules. Therefore, the c-Myc promoter G-quadruplex (MycG4) is an attractive anticancer drug target. Protein recognition of MycG4 is essential for its transcriptional regulating. Nucleolin was discovered as a major MycG4 binding protein in 2009. It shows a remarkably higher binding affinity for MycG4 over its known substrate NRE_RNA and overexpression of nucleolin represses the activity of the c-Myc promoter. However, little is known about its molecular recognition of MycG4. Here, we use X-ray crystallography combined with other biochemical and biophysical methods to understand how nucleolin recognizes MycG4. Nucleolin is a 77 kD protein with a modular organization. The four RNA-binding domains (RBD) of nucleolin are the minimal domains for high affinity binding with MycG4. We show that nucleolin prefers the c-Myc parallel G-quadruplex with a 6-nt central loop (Myc161) that is the thermodynamically favored conformation. Using a custom G4 DNA microarray, we optimized the MycG4 sequence with over 10-fold increased binding affinity to nucleolin. Fabs are widely used tools to facilitate crystallization and we have discovered Fabs that specifically bind the nucleolin-MycG4 complex using a phage display screening. This approach enabled us to obtain crystals of the nucleolin-MycG4-Fab ternary complex diffracted at 2.6 Å and we determined the crystal structure. In the structure, the parallel MycG4 is very well-defined with two K⁺ between the three G-treads. The central 6-nt loop residue protrude from the G4-core and extensively recognized by the nucleolin. Only RBD1 and RBD2 of nucleolin are seen in the crystal structures and interact extensively with the 6-nt central loop and 5′-flanking of MycG4. The binding surface and area of the globular MycG4 by nucleolin is much more extensive than NRE_RNA and involves an extra binding site. Fab binds to both RBD1 and 3′-end of MycG4 to stabilize the complex. The well-defined partial RBD2-3 linker and a cavity close to the 1-nt T19 loop suggest that the missing RBD3 likely binds the 3^rd loop of MycG4. This structure is the first MycG4-protein complex structure. It will help understand MycG4 and nucleolin interactions and the development of MycG4 targeted cancer therapeutics. This structure also provides novel insights into how proteins recognize the globular G-quadruplexes, highlighting the potential of G-quadruplexes as a platform for multivalent interactions such as with multiple tandem RBDs.</p>

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24805号 / 医科博第150号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 上野 英樹, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The objective of this project was to develop a DNA hybridization sensor that can detect the presence of E. coli and reveal its resistance to the drug gentamicin. This probe will enable rapid and user-friendly diagnostics of E. coli infections and analysis of bacterial gentamicin-susceptibility profile by interrogation of a fragment of E. coli 16S rRNA bearing a substitution in the gentamicin-resistant cells. The sensor is promising for the point-of-care use to provide a timely UTI diagnostic solution. A quick diagnosis of E. coli infection and antibiotic resistance is crucial for treatment. To design a hybridization probe, we proposed a split approach for target interrogation and catalytic activity of a peroxidase-like deoxyribozyme (PDz) as a signal reporter. PDz contains a series of guanine residues in a strand and has been shown to form a parallel guanine-quadruplex (G4). This G4, with the addition of a hemin cofactor, catalyzes the reaction similar to that of horseradish peroxidase. If a colorless organic indicator is added to the G4-PDz-hemin containing solution and mixed H2O2, a colored oxidation product is formed (e.g., a dark blue/green). The color change reports the presence of the catalytically active G4, which occurs only when the nucleotide sequence of the target is a perfect match. When the target is not a perfect match, for example, in the case of the drug-causing nucleotide substitution, the G4 does not form, and there is no color change. The probes tested in this paper show promising results of such a sensor by being able to catalyze the described colorimetric reaction to generate a strong signal in the presence of a "gentamicin-susceptible" target and show selectivity against the "gentamicin-resistant" target.

No description available.

No description available.

It is now widely known that some nucleic acid molecules, either DNA or RNA, are capable of forming intricate three-dimensional structures and carrying out functions of molecular recognition and catalysis. Most of known functional nucleic acids are isolated from DNA or RNA pools with random sequences using the technique of in vitro selection. With intensive research for the past three decades, a variety of functional nucleic acids have been discovered and examined for potential applications. The general objective of this thesis is to expand the repertoire of functional nucleic acids via new in vitro selection experiments and pursue their biosensing applications. I started by asking the question of whether it is possible to develop a new kind of functional nucleic acids: chimeric RNA/DNA substrates that have high activity for ribonuclease H2 from the important bacterial pathogen Clostridium difficile but much reduced activity towards the same enzymes from other bacterial species. The key rationale behind pursuing these special functional nucleic acids is my hypothesis that these molecules can eventually be developed into useful biosensors for diagnosing Clostridium difficile infection. For this reason, in my first project, I applied the in vitro selection technique to a random-sequence DNA pool, obtained several highly selective chimeric RNA/DNA substrates, and carried out in-depth analysis of their reactivities and their structural properties. During this study, I accidentally discovered a family of highly guanine-rich DNA molecules that are able to form an unusual guanine-quadruplex structure in 7 molar urea, a strong denaturing condition for nucleic acid structures. This discovery constitutes a novel observation and therefore, in my second project, I fully characterized the sequence and structural properties of these special DNA molecules and established the conditions that allow these molecules to create stable structures in 7 molar urea. I then got interested in devising a unique application to take advantage of the urea-resistant property exhibited by these molecules. Towards this end, in my third project, I used one such DNA molecule to set up a DNA detection method capable of detecting single nucleotide polymorphism in very long DNA sequences, a desired application that has never been demonstrated before. The findings made in these projects contribute to the ever-growing appreciation of functional capability and practical utility of nucleic acids. / Thesis / Doctor of Philosophy (PhD)