開發苄基鳥嘌呤化學探針應用於SNAP-tag蛋白結合

Development of Benzylguanine Chemical Probes for SNAP-tag Protein Binding

莊沛軒(Chuang, Pei Hsuan)

指導教授 ： 陳貴通

國立清華大學/理學院/化學系/碩士(2016年)

摘要

蛋白質在生物體內的位置、轉移與交互作用等，往往提供生物學研究重要資訊。對蛋白質標記螢光分子具有快速、靈敏度高、專一性結合且方便觀察的特點，是廣泛應用於生物體內的方法之一。
本論文選擇以SNAP-tag蛋白標記技術，利用SNAP-tag蛋白對O6-BG基團的專一辨識性且以共價鍵的方式鍵結，發展兩種類型螢光探針: (一)籠閉型苄基鳥嘌呤螢光探針 ；(二)尼羅染料螢光探針。第一類型探針在鳥嘌呤基團N-9位置修飾上辨識端，形成籠閉型式探針，期望藉由加入目標分析物切斷辨識端後，使裸露的鳥嘌呤基團會與SNAP-tag蛋白結合，而有螢光增益的現象，達到偵測目標分析物的。第二類型計畫是開發近紅外光尼羅染料螢光探針，近紅外螢光具有低生物背景訊號干擾、低光損傷害以及較好的生物組織穿透性等，利用對極性敏感螢光探針的特性，成功地開發了SNAP-tag蛋白標記之螢光探針，並應用在細胞標記影像中。

關鍵字

螢光探針 ； 蛋白標記

並列摘要

Protein functions and dynamics in living cells can provide important information in biological research and medical diagnosis. The uses of fluorescent probes to study proteins are advantageous, as they allow for fast, high sensitivity, high specificity and ease for observation. In this thesis, I have designed two novel fluorescent probes for SNAP-tag which is an important protein in chemical biology for specific and covalent reaction with probes consisting O6-benzylguanine moiety. The first probe is based on the caged-benzylguanine concept by chemical modification of benzylguanine N-9 position to prevent the reaction of benzylguanine and SNAP-tag. Upon uncaging with the target molecule, the fluorescent dye can then be transferred to the SNAP-tag or MGMT (benzylguanine wild type protein) to emit stronger fluorescence. In the second fluorescent probe, we have successfully developed two near-infrared fluorescent probes based on Nile red and Nile blue, respectively, for fluorescent turn-on labeling of SNAP-tag protein. We have successfully applied these two probes for no-was and highly organelle specific labeling of SNAP-tag proteins in living cells.

並列關鍵字

probe ； protein labeling

參考文獻

18. 劉淘愷,開發環境敏感螢光增益探針應用於蛋白質辨認與活體細胞蛋白標記,國立清華大學碩士論文,2014.

8. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64.

52. Yu, W. Y.; Wu, T. W.; Huang, C. L.; Chen, I. C.; Tan, K. T. Chem. Sci. 2016, 7, 301.

21. (a) Slavoff, S. A.; Chen, I.; Choi, Y.-A.; Ting, A. Y. J. Am. Chem. Soc. 2008, 130, 1160.

(b) Gautier, A.; Juillerat, A.; Heinis, C.; Corrêa, I. R. Jr.; Reis, I.; Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. Biol 2008, 15, 128.