Micro- and nanofluidic devices for complexed DNA analysis at the single molecule level

應用微奈米流體元件於單分子DNA複合體之分析

史利南(Sriram, K. K.)

指導教授 ： 周家復 ； 蘇育全

國立清華大學/原子科學院/工程與系統科學系/博士(2014年)

摘要

Single molecule DNA studies have developed into a useful tool over the past two decades. Unlike conventional experiments that provide collective information of an ensemble, it gives us the opportunity to manipulate individual DNA molecules, observe and analyze their behavior upon interaction with other biomolecules like proteins. DNA is a semi-flexible polymer, which exists in its physiological random-coil state in a bulk environment, making direct visualization of DNA and its interactions to proteins or drugs challenging. Stretching and immobilization of DNA molecules thus becomes a critical requirement for single molecule studies. Techniques like optical trapping, magnetic trapping, flow stretching, molecular combing, and confinement induced stretching using micro- and nanofluidic devices have been widely used to stretch and immobilize DNA molecules.
In this thesis, we developed two independent micro/nanofluidic devices that share a similar fabrication scheme, but with different approaches for effective DNA immobilization and stretching, other than the previously demonstrated techniques. The first one is a proof-of-concept method adopting nanoslit, bioconjugation, and single molecule imaging for direct mapping of transcriptional factor (TF) binding sites on field-stretched single DNA molecules. TFs are proteins that bind to specific loci of DNA using DNA binding domains to carry out the process of transcription. Hence, the identification of TF binding sites is essential for understanding the regulatory circuits that control cellular processes such as cell division and differentiation as well as metabolic and physiological balance. The nanoslit devices used in this work are fabricated in fused silica substrate, with tens of nanometers in depth, and are conformably sealed by a polymer-coated coverslip. This device is used for the mapping of TF (E. coli RNA polymerase holoenzyme, or RNAP) binding sites along lambda phage DNA, where two promoters and three pseudo-promoters sites of E. coli RNAP have been identified with ~ 100 nm resolution. The advantages of our nanoslit device include multiplexing, quick mapping of TF binding sites, and the ability to distinguish real complexes from non-specific ones.
The second work is the demonstration of a new DNA combing scheme using oxygen (O2) plasma-modified polysilsesquioxane (PSQ) polymer coated surface to effectively immobilize and stretch DNA molecules. The increase in surface roughness and surface potential due to low-pressure O2 plasma treatment are proposed to be the major factors in effective DNA combing. The advantage of this method is extremely simple, and the property of the surface can be fine-tuned with O2 plasma treatment, to anchor DNA molecule at the end or multiple points. Plasma treated PSQ surface can be directly used for room-temperature bonding of micro/nanofluidic devices, which in turn can be used directly for DNA combing experiments without further surface modifications. In this work, we used our DNA combing platform for fluorescence single molecule imaging of anti-cancer drug cisplatin complexed DNA molecules. Our observations show intra-strand crosslinking of DNA molecules in the presence of cisplatin, leading to DNA condensation.
Both platforms enable single molecule DNA studies and open up the possibility of multitude of DNA interaction studies involving other biomolecules, like proteins, or drugs. In addition, we have also demonstrated the possibility of carrying out transverse current measurements using a device with a tandem array of electrode nanogaps embedded co-planar into a nanofluidic channel, to map DNA binding sites of proteins, which, with further development, could well serve as a tool for single molecule DNA analysis and a wide variety of other applications.

關鍵字

DNA 單分子研究 ； 奈米解析

並列摘要

過去二十年來，DNA 單分子研究已經成為實用的研究工具。相較於傳統實驗技術觀察眾多分子的平均整體表現，單分子研究可以操控、觀察與分析單一 DNA 分子與其他生物分子之互動。例如：DNA 作為半彈性聚合物在生理溶液的環境下形成不規則捲曲狀態，可以直接觀察 DNA 及其與蛋白質或藥物互動。因此，伸展與固定化 DNA 分子為單分子研究之必要步驟。相關技術例如：光鑷夾、磁鑷夾、利用流體伸展、分子篩、奈米或微米流道已經被廣泛地用來伸展與固定 DNA 分子。
本論文發展兩型獨立的微奈米流道裝置。相較於其他技術，本研究使用相似製程並提供兩種有效的 DNA 固定與伸展。第一部份為使用奈米流道、生物橋接與單分子影像技術直接觀察轉錄因子於展開之單一DNA 分子上的結合位置。轉錄因子於轉錄時會結合到 DNA 特定基因座。因此，鑑別轉錄因子的結合位對於了解基因迴路與調節機制非常重要，例如細胞分裂與分化以及代謝與生理平衡機制。本研究之奈米流道使用熔融石英基材，流道深度為數十奈米，使用有聚合物塗層之蓋玻片封裝，觀察轉錄因子（大腸桿菌 RNA 聚合酶）在噬菌體 DNA 上的結合位。已知結合位包括兩個啟動子與三個 pseudo-promoters在本研究中可以 100 奈米解析度觀察。此奈米流道裝置可以多工、快速地標定結合位，並可區別真正結合位與非特異性結合位。
第二部份展示新的 DNA 分子梳技術。使用氧電漿修飾之polysilsesquioxane 表面塗層，有效固定與伸展 DNA 分子。表面粗糙度與電位因為低壓氧電漿處理而上升，推測是 DNA 分子梳的主要原因。此技術的優點是製程簡易，並且表面的性質可以藉由微調氧電漿處理的參數，來調整 DNA 固定在末梢或者多點固定。氧電漿處理的 PSQ流道封裝，不需要額外的表面修飾，可直接用來做 DNA 分子梳實驗。本研究中，我們使用 DNA 分子梳平台與單分子螢光影像技術觀察抗癌藥物 cisplatin 結合 DNA 分子。研究結果觀察到 cisplatin 結合在 DNA 雙股中，造成 DNA 凝聚現象。兩個技術平台均使用單分子 DNA 技術觀察 DNA 與生物分子如蛋白質、藥物的互動。此外，我們也在奈米流道中裝置多重平面化奈米電極 做為電流偵測系統來偵測蛋白質在 DNA 上的結合位，未來可望作為 DNA 單分子分析工具或其他未來應用潛能。

並列關鍵字

Micro and nanofluidics ； Single molecule imaging and analysis ； DNA-protein complexes ； Fluorescence imaging

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