活细胞内基于Diels-Alder反应的量子点标记蛋白质研究

Protein labeling with quantum dots in live cells based on Diels-Alder reactions

目的建立一种基于Diels-Alder环加成反应用于活细胞内量子点(quantum dots,QDs)标记蛋白质的新方法。方法反式环辛烯(trans-cyclooctene 2,TCO2)修饰的类转录激活因子效应物(transcription activator-like effectors,TALE)蛋白与偶联四嗪(tetrazine 1,Tz1)的红色量子点QD625在Vero活细胞内特异性结合,形成量子点荧光标记的蛋白TALE-QD625,通过激光共聚焦显微镜观察TALE-QD625在Vero细胞内的荧光信号分布。结果基于Tz1和TCO2的Diels-Alder环加成反应在Vero活细胞内将QD625标记至TALE蛋白,并随TALE进入细胞核内,在核内观察到红色的QDs荧光信号,成功建立活细胞内量子点标记蛋白方法。结论通过Diels-Alder环加成反应首次实现活细胞内量子点标记蛋白,为蛋白单分子成像提供一种新的标记方法。

Objective To establish a new method of labeling a protein with quantum dots（ QDs） within live cells via the Diels-Alder cycloaddition. Methods Transcription activator-like effectors（ TALE） decorated with trans-cyclooctene TCO2 and tetrazine 1（ Tz1）-conjugated QD625 were combined specially in the live Vero cells to yield a QD625-labelled TALE（ TALE-QD625）. The distribution of fluorescent signal for TALE-QD625 in Vero cells were observed through confocal microscopy. Results The QD625 was conjugated with TALE based on the Tz1 and TCO2 Diels-Alder cycloaddition labeling system and subsequently entered the cell nucleus. Red fluorescent signal of TALE-QD625 was observed in the nucleus of Vero cells. Conclusion It is the first time that QDs have been used to specially label a protein via the Diels-Alder cycloaddition within live cells,which will provide a new labeling method for single protein molecule imaging.