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Enhancing the stability of DNA origami nanostructures by enzymatic and chemical ligation methods / 酵素および化学ライゲーション反応によるDNAオリガミナノ構造体の安定化に関する研究KRISHNA MURTHY, KIRAN KUMAR 24 July 2023 (has links)
京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24854号 / エネ博第463号 / 新制||エネ||87(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 森井, 孝, 教授 片平, 正人, 教授 佐川, 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM
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ENGINEERED 3D DNA CRYSTALS: CHARACTERIZATION, STABILIZATION AND APPLICATIONSZhe Li (6581093) 10 June 2019 (has links)
In recent years, DNA nanotechnology has emerged as one of the most
powerful strategies for bottom-up construction of nanomaterials. Due to
the high programmability of DNA molecules, their self-assembly can be
rationally designed. Engineered 3D DNA crystals, as critical products
from the design of DNA self-assembly, have been proposed as the
structural scaffolds for organizing nano-objects into three-dimensional,
macroscopic devices. However, for such applications, many obstacles
need to be overcome, including the crystal stability, the
characterization methodology, the revision of crystal designs as well as
the modulation of crystallization kinetics. My PhD research focuses on
solving these problems for engineered 3D DNA crystals to pave the way
for their downstream applications.<br>In this thesis, I started by
enhancing the stability of engineered 3D DNA crystals. I developed a
highly efficient post-assembly modification approach to stabilize DNA
crystals. Enzymatic ligation was performed inside the crystal lattice,
which was designed to covalently link the sticky ends at the crystal
contacts. After ligation, the crystal became a covalently bonded 3D
network of DNA motifs. I investigated the stability of ligated DNA
crystals under a wide range of solution conditions. Experimental data
revealed that ligated DNA crystals had significantly increased
stability. With these highly stabilized DNA crystals, we then
demonstrated their applications in biocatalysis and protein
encapsulation as examples.<br>I also established electron microscope
imaging characterization methods for engineered 3D DNA crystals. For
crystals from large-size DNA motifs, they are difficult to study by
X-ray crystallography because of their limited diffraction resolutions
to no better than 10 Å. Therefore, a direct imaging method by TEM was
set up. DNA crystals were either crushed or controlled to grow into
microcrystals for TEM imaging. To validate the imaging results, we
compared the TEM images with predicted models of the crystal lattice.
With the advance in crystal characterization, DNA crystals of varying
pore size between 5~20 nm were designed, assembled, and validated by TEM
imaging.<br>The post-assembly ligation was further developed to prepare
a series of new materials derived from engineered 3D DNA crystals,
which were inaccessible otherwise. With the directional and spatial
control of ligation in DNA crystal, I prepared new DNA-based materials
including DNA microtubes, complex-architecture crystals, and an
unprecedented reversibly expandable, self-healing DNA crystal. The
integration of weak and strong interactions in crystals enabled a lot of
new opportunities for DNA crystal engineering.<br>In the final chapter,
I investigated the effect of 5’-phosphorylation on DNA crystallization
kinetics. I found that phosphorylation significantly enhanced the
crystallization kinetics, possibly by strengthening the sticky-ended
cohesion. Therefore, DNA crystals can be obtained at much lower ionic
strength after phosphorylation. I also applied the result to controling
the morphology of DNA crystals by tuning the crystallization kinetics
along different crystallographic axes. Together with previously methods
to slow down DNA crystallization, the ability to tune DNA
crystallization kinetics in both ways is essential for DNA crystal
engineering.
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Enzymatische Ligation von Peptiden, Peptidnucleinsäuren und ProteinenPritz, Stephan 13 January 2009 (has links)
Peptide und Proteine sind wichtige Untersuchungsobjekte der biochemischen Forschung. Es wurden in den letzten Jahren eine Reihe von Ligationsmethoden entwickelt, um weitgehend entschützte, gereinigte Peptidsequenzen im wässrigen Milieu zu koppeln. Vor diesem Hintergrund von besonderem Interesse für einen möglichen Einsatz bei Ligationen ist die bakterielle Transpeptidase Sortase A. Dieses Enzym ist in vivo an der Anknüpfung von Proteinen an das bakterielle Peptidoglycan beteiligt, wobei es Substrate an einem LPXTG-Motiv zwischen Threonin und Glycin spaltet und auf ein Oligoglycin-Nucleophil überträgt. Zur Untersuchung der Enzymaktivität wurde in dieser Arbeit ein einfacher HPLC-basierter Assay etabliert. Die an Peptidmodellen gewonnenen Resultate wurden schließlich für den Aufbau eines löslichen Rezeptors genutzt. Ein Schlüsselschritt war die Sortase-vermittelte Ligation des in E. coli exprimierten, gefalteten Rezeptor-N-Terminus an ein 3-Loop-Konstrukt. Das erhaltene 23 kDa große Rezeptormimetikum war nach chromatographischer Reinigung homogen gemäß HPLC und MS. Es zeigte eine spezifische, hoch affine Bindung zu natürlichen Peptidliganden des CRF1-Rezeptors. Weiterhin konnte demonstriert werden, dass sich Sortase für die selektive Markierung von Proteinen eignet. So wurde ein Fluoreszenzlabel C-terminal an das 50 kDa Protein NEMO geknüpft. Als weiteres Anwendungsbeispiel der Sortase-vermittelten Ligation diente die Darstellung von PNA–CPP-Konjugaten. Die Verwendung von Überschüssen des Peptides und die Entfernung der niedermolekularen Abgangsgruppe durch Dialyse erwies sich als sehr effektiv und gestattete gute bis hervorragende Kupplungsausbeuten von bis zu 94%. Die biologische Wirkung der erhaltenen CPP–PNA-Konjugate konnte in Aufnahmeuntersuchungen an Zellen gezeigt werden. / Peptides and proteins are important research objects in biochemical research. Therefore, several ligation methods to couple unprotected, purified peptide sequences in aqueous media have been developed during the last years. At a special interest in this case is the bacterial transpeptidase sortase A. This enzyme couples proteins in vivo to the bacterial peptidoglycan by cleavage at a LPXTG-recognition motif between threonine and glycine and subsequent transfer to an oligoglycin nucleophile. In order to investigate the enzymatic activity, a simple HPLC-based assay was established in this work. Results obtained with model peptides were used for the assembly of a soluble receptor. A key step was the sortase-mediated ligation of the folded receptor N-terminus (expressed in E. coli) to the 3-loop-construct. The resulting receptor mimic of 23 kDa was homogeneous according to HPLC and MS. It showed specific binding to natural peptide ligands of the CRF1-receptor with high affinity. Furthermore, it could be shown that sortase is usable for selective protein labeling. For this purpose, a fluorescence label was attached C-terminally to the 50 kDa protein NEMO. As a further example of sortase-mediated ligation served the synthesis of PNA-CPP-conjugates. The use of an excess of the peptide and dialyzing away the small leaving group proved to be very effective and coupling yields up to 94% could be achieved. The biological activity of the CPP-PNA-conjugates could be shown by uptake studies in cells.
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