建立蛋白質-去氧核醣核酸交互作用的檢測平台：以p53與Bax基因啟動子之交互作用檢測為例

Abstract

如何建立一個快速且靈敏之蛋白質-去氧核醣核酸交互作用的檢測平台，為當今生物感測研究所亟欲達成之目標。欲達成此一目的的開發，選用適當的載體與測試指示信標的方便性與靈敏度為重要的環節。 奈米鑽石為高度生物相容性的奈米材料。奈米鑽石表面具有很大比例的易活化扭曲石墨鍵結(distortion graphite bond)，有利於化學修飾成特定的活化官能基，此外它具有獨特的光譜、和熱穩定性的性質，可作為生物探針(bio-probe)。奈米鑽石亦為一個良好的運輸載體，可在生物體中運輸特定的生物分子。綠色螢光蛋白質為一種螢光發光效率極高的蛋白質，當受到藍光或UV波段的電磁波激發時，會在發出強烈的綠色螢光，此一特色，讓綠色螢光蛋白質可作為良好的分子信標。藉由奈米鑽石與綠色螢光蛋白質結合，可作為具有強烈綠色螢光的運輸載體。P53蛋白質為一種腫瘤抑制蛋白質，由TP53基因編碼而成。P53蛋白質藉由與DNA結合調控控生物體內之種種生理機制，包括修復受損的DNA、啟動細胞凋零的路徑、轉錄因子等。 本研究中，我們利用化學修飾的方式，將p53蛋白質與奈米鑽石-綠色螢光蛋白質系統結合，以建立蛋白質-去氧核醣核酸交互作用的檢測平台。此一複合物能保留各分子的特性，亦可辨認特定的啟動子序列，且此一交互作用可直接被綠色螢光觀察亦能透過本實驗室的微拉曼系統在體內與體外觀察。

It is highly desired to develop a rapid and sensitive detection system for analyzing protein-DNA interaction. For developing such detection system, it is important to select appropriate protein carrier and good indicator. Nanodiamond (ND) is one of the biocompatible nanomaterials with large tunable surface for chemical modification. It possesses unique mechanical, spectroscopy, and thermal conducting properties. It is an excellent molecular vehicle to deliver specific molecules in biological system. The green fluorescent protein (GFP) is a protein that emits strong green fluorescence when it is excited by ultra-violet to blue light. It makes GFP a good indicator. By combining ND-GFP, a visible biocompatible delivery system will be developed. p53 is a tumor suppressor protein encoded by the TP53 gene. p53 plays an important role in apoptosis, genomic stability, and inhibition of angiogenesis by interacting with specific DNA sequence of promoter of related genes. In this study, a p53 functionalized ND-GFP DNA detecting probe will be developed. This complex can recognize the specific DNA sequence of promoter and the intermolecular interactions can be monitored directly by fluorescence and Raman spectroscopy both in vivo and in vitro.