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系統識別號 U0026-1808201512455900
論文名稱(中文) 運用奈米策略發展DNA奈米立體結構達抗菌效果
論文名稱(英文) Development of three-dimensional DNA nanostructure for anti-bacteria strategy
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 103
學期 2
出版年 104
研究生(中文) 謝靜宜
研究生(英文) Ching-Yi Hsieh
學號 T46021025
學位類別 碩士
語文別 英文
論文頁數 62頁
口試委員 指導教授-謝達斌
共同指導教授-吳尚蓉
召集委員-黃一修
中文關鍵字 冷凍電子顯微鏡  DNA摺紙技術  藥物輸送系統  適體  細菌感染 
英文關鍵字 Cryo-electron microscopy  DNA origami  drug delivery system  aptamer  bacterial infection 
學科別分類
中文摘要 奈米生醫為基礎的診療體系之發展已逐漸走向臨床實用為目標。近期研究指出,特定之無機奈米材料對細胞會產生毒性及生物功能障礙等影響。而DNA奈米立體結構為細胞本身俱有之巨分子,因此基本上被認為能發展為具低生物毒性、高生物相容性的奈米載體。
本研究以DNA構成的奈米立體結構為平台,發展一個利用鹼基互補特性自行組合而成的DNA奈米立體結構,外露一段具有標靶能力的DNA適體。我們透過抗菌分子放線菌素D (Actinomycin D, AMD) 會與DNA次凹槽交互作用形成氫鍵而結合的特性,將放線菌素D嵌入DNA奈米立體結構雙股支架裡,達到乘載藥物的目的,並能藉由DNA適體的專一性標靶目標物而達到標靶性釋放藥物的抗菌能力。目前我已經將DNA奈米立體結構組裝完成,並運用DNA洋菜膠體電泳進行確認。我更進一步利用冷凍電鏡及三維結構重組技術分析此DNA奈米立體結構,發現其為空心的球體。接著,我們發現DNA奈米立體結構達到飽和時能攜帶2409.47個放線菌素D。我們也發現DNA奈米立體結構與其攜帶之相同濃度的自由態抗生素具有更強的抗菌效果。在適體結合能力測試中,我們證實適體能專一性的與突變型鏈球菌結合。
英文摘要 The rapid development of nanomedicine based on theranostic has gradually evolved into practical clinical applications. Recently, researchers have reported that certain inorganic nanomaterial may induce cytotoxicity and biological dysfunction. DNA is an intrinsic biological macromolecule thus their nanostructures were regarded as excellent carriers with low toxicity and high biocompatibility.
In my study, I would like to develop an advanced theranostic platform based on a self-assembled DNA nanostructure as scaffold with targeting aptamer and carrying antibiotics. Antimicrobial molecules (Actinomycin D, AMD) will be attached to minor groove of DNA double helix through hydrogen bond. The DNA nanostructure system serves as carriers for targeting delivery of AMD to attack bacteria. Currently, the DNA nanostructure system has been assembled and was confirmed by DNA agarose gel electrophoresis. Cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction technique showed the DNA nanostructure system was a hollow spherical structure. In addition, we found a single DNA nanostructure could carry 2409.47 AMD molecules when saturated. We further found that the anti-bacteria ability of our system had a higher efficacy than free AMD. In the aptamer binding assays, we demonstrated the aptamer target specifically to Streptococcus mutans.
論文目次 中文摘要 ii
Abstract iv
Acknowledgement vi
Table of Contents vii
Figures List xi
Tables List xii
Abbreviations xiii
1. Introduction 1
1.1 Nanotechnology 1
1.1.1 The rapid advancement of DNA based nanomedicine 2
1.1.2 DNA origami 3
1.1.3 DNA nanostructures in drug delivery system 4
1.2 Advanced delivery concept for drugs and functional molecules 5
1.2.1 Aptamers in drug delivery system 5
1.3 An emerging trend of transmission electron microscopy (TEM) in nanotechnology 6
1.3.1 Innovation in technique for Cryo-electron (Cryo-EM) 6
1.3.2 Images analysis using single particle reconstruction approach in EMAN2 7
1.4 Bacterial infection in the gastrointestinal system 8
1.4.1 Antibiotics for the treatment in bacterial infection 8
1.4.2 Dental caries 9
1.5 Rationale 10
2. Materials and Methods 11
2.1 Oligonucleotides 11
2.2 Synthesis of the DNA nanostructure 12
2.3 Characterization of the DNA nanostructure 12
2.3.1 Gel electrophoreses analyze the formation of DNA nanostructure 12
2.3.2 Transmission electron microscopy observation of the native and actinomycin d carrying DNA nanostructure (AMD/DNA nanostructure) 13
2.4 Cryo-electron microscopy (Cryo-EM) 14
2.5 Three-dimensional reconstruction of the DNA nanostructure using EMAN2 software package 14
2.6 Drug loading content analysis 15
2.7 Bacteria strains and media 15
2.8 Minimum inhibitory concentration (MIC) 16
2.9 Minimum bactericidal concentration (MBC) 17
2.10 Aptamer binding assays 17
2.11 DNA nanostructures binding assays 18
3. Results 19
3.1 Scheme of DNA and AMD/DNA nanostructure 19
3.2 Characteristics of DNA and AMD/DNA nanostructure 19
3.3 Three-dimensional reconstruction of DNA nanostructure 20
3.4 Actinomycin D loading capacity 21
3.5 Minimum inhibitory concentration (MIC) of Actinomycin D against oral Streptococcus bacteria 22
3.6 Minimum bactericidal concentration (MBC) of Actinomycin D against oral Streptococcus bacteria 22
3.7 The efficacy of AMD/DNA nanostructure as a drug delivery system 23
3.8 In aptamer-binding assays 23
3.9 In aptamer-binding assay for the DNA nanostructure with aptamers 24
4. Discussion 25
5. Conclusion 30
6. References 32
7. Figures and Legends 39
8. Table 62
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