由於MGMT的含量多寡與烷基化藥物的耐藥性相關，可作為化學治療的重要指標及有效的預後標記。而醫院每天須檢測大量的細胞樣品，故開發一種快速又簡單的分析方法，以檢測在不同類型的細胞中MGMT之含量是有其必要性。在本研究中，我們開發一種新穎的MGMT蛋白之螢光增益型探針。以分子轉子CCVJ為螢光基底，修飾上MGMT假性受質O6-BG。當與MGMT蛋白作用後，探針轉移至MGMT蛋白的活性位點。由於蛋白質為巨大結構之立體障礙影響，使分子轉子單鍵旋轉的自由度下降，產生170倍螢光增益效果。將探針與含MGMT表達之Hela S3、MCF-7及HEK293細胞株作用後，可觀察到明顯的螢光訊號，而缺乏MGMT表達之CHO細胞株則無螢光訊號產生。我們所設計的螢光探針容易合成，並具有標記速率快且免清洗之細胞成像等優勢。我們相信此螢光增益型探針的設計可延伸運用於偵測其他轉移酶或結合蛋白，並能成功地運用在活體細胞成像。

MGMT protein, which has been associated with resistance to antitumor alkylation drugs for many patients, is a very useful prognostic marker to provide a guide for therapeutic decisions. Considering the large number of cellular samples that have to be handled at the hospital every day, it is thus important to develop a rapid and simple analytical method to distinguish MGMT activity in different types of cells. In this study, we describe the first MGMT fluorescence activation probe for the rapid no-wash imaging of MGMT in living cells. The probe consists of a specific MGMT suicide pseudosubstrate, O6-benzylguanine and a fluorescent molecular rotor CCVJ. In the presence of MGMT, the enzyme transfers CCVJ to the protein active site where the crowded surrounding restricts the bond rotation of the fluorescent molecular rotor to trigger fluorescence activation of about 170-fold. With this probe, bright fluorescence was observed for MGMT-positive, Hela S3, MCF-7 and HEK293 cells, while MGMT-deficient CHO cells displayed no fluorescence. This fluorescence activation probe design can also be extended for the detection of other transferases and binding proteins for which there are still no effective methods to image them in living cells.

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