單股DNA奈米模版發展與應用

Abstract

在生物科技日新月異的今天, DNA不再只是攜帶遺傳指令，引導生物發育與運作生命機能。由於其具有四種去氧核醣核酸排列的序列，在現今DNA 奈米生物科技（DNA nanobiotechnology）的世界，DNA成為一種具有奈米尺度的解析度，並可合成微米以上的巨分子，儼然成為新世代組成新穎材 料的奈米積木。本論文主要在於利用滾動核酸放大生物技術（Rolling Circle Amplification）發展長鏈具有重複序列的單股DNA，寬度為2奈米，而長度可達數微米。而後將該奈米模版應用於奈米尺度分子操控、新穎材料以及生物感測器等三大領域。在奈米尺度分子操控領域，此奈米模版將藉由短鏈互補序列使得此單股DNA於特定位置結合上兩種以上的生物分子如酵素。同時於奈米尺度下，利用熱力學原理，可將原有的酵素功能移除，形成未結合的ssDNA奈米模版，亦或是直接替換另一種酵素。可應用於生物晶片表面改質，取代傳統高溫強酸鹼處理，以將雙股DNA裂解形成單股DNA。第二部分，單股DNA奈米模版亦可結合上不同尺寸奈米金屬粒子，製備成奈米金粒子鏈，形成一種軟性電子材料，同時該金屬材料具有可微調功能的光學材料，滿足奈米金粒子的需求。第三部分於生物感測器應用領域，由於此論文中滾動核酸放大技術乃是利用癌症指標蛋白，即血小版衍生因子（PDGF）誘導DNA適體構形改變。因此在形成長鏈DNA過程時，亦可結合場效電晶體式生物感測器，藉以放大感測器靈敏度達千倍以上。綜觀國內外發展之DNA奈米生物科技，主要在於設計DNA序列形成奈米尺度下特殊形狀，實際應用解決生物醫學困難，為現今科學家努力的目標與趨勢。此論文特別由材料表面改質、感測器開發應用，延伸至具可調控光學性質的材料，以有效掌握並控制奈米複合材料製作，期待DNA奈米生物科技能提供生物材料、生物感測器與光學元件開發等關鍵前瞻技術。

Nowadays, DNA is used more than just as a carrier of genetic codes of life; it is also served as a generic building block for nanostructures. Based on the Watson–Crick base-pairing strategy, Long and single-stranded DNA (ssDNA) with repetitive sequence was demonstrated as a versatile nanotemplate for introducing biological activity through self-assembled manner. In this study, we characterized the ssDNA nanotemplates, and applied the nanotemplates into three fields, including molecular manipulation, biosensors and material science. In the first chapter, time-dependent synthesis of single ssDNA nanotemplate prepared from rolling circle amplification (RCA) was observed and visualized by atomic force microscopy. Since RCA reaction is triggered by conformation switch of PDGF aptamer which recognized by PDGF. A biosensor for protein detection can be developed by complementary metal–oxide–semiconductor field-effect transistor (CMOS FET), the sensitivity of CMOS FET for PDGF detection was increased by 1000 folds. In the application of material science, functionalization of DNA nanotemplates were prepared from hybridizing wuth enzyme-tagged complementary DNA probes. Re-functionalization and rejuvenation of the ssDNA nanotemplate was achieved in mild biological conditions without using high temperature and strong alkaline to denature DNA. In this innovated strategy, similar procedures can be used to incorporate various other enzymes and biomolecules such as antibodies or receptor proteins to produce specific biological functions on the ssDNA nanotemplate. In the last chapter, we developed flexible metallic nanoparticle chains. Because metallic nanoparticles (NPs) are attractive units for constructing versatile and advanced materials for optical applications, physicochemical responses of NPs can be tuned depending on the structure, shape and morphology of the NP composites. Self-assembled gold nanoparticles (AuNP) chains were generated on the ssDNA nanotemplate backbone. Therefore, we expect the ssDNA nanotemplates can present an original and significant step towards a type of toolbox for DNA-based nanobiotechnology and resolve the unmet needs from ultrasensitive analytics, customized enzymatic units, and optical material development.