Here, we report genetically encoded AviTag conjugating system for Channelrhodopsin-2(ChR2) in order to attach various nanostructures to the membrane protein in a cell type specific manner. First, AviTag peptide sequence is cloned to N-terminal site of ChR2 construct and expressed at the membrane of primary-cultured hippocampal neurons via lentiviral transduction. Second, with the help of BirA enzyme and ATP, biotin coated quantum dots (Qdots) and streptavidin (SAv) coated Qdots are successfully bound to AviTag sites at the membrane where ChR2 is located and confirmed by fluorescence imaging. Moreover, we synthesize biotinylated Traptavidin-DNA conjugate probes containing a desthio-biotin that has weaker affinity than a regular biotin, and successfully exchange them with pre-conjugated Biotin-AviTag-ChR2 site at the membrane of neuronal cells which can potentially solve the crosslinking issue of Avidin linked probes. Therefore, we expect the AviTag-ChR2 fusion platform to become a great tool for incorporating various nanostructures at the specific sites of a cellular membrane in order to overcome the limits of optogenetic opsins.

在这里，我们报告遗传编码的AviTag共轭系统的Channelrhodopsin-2（ChR2），以便以细胞类型特异性的方式将各种纳米结构附着到膜蛋白上。首先，将AviTag肽序列克隆到ChR2构建体的N末端位点，并通过慢病毒转导在原代培养的海马神经元膜上表达。其次，借助BirA酶和ATP，生物素包被的量子点（Qdot）和链霉亲和素（SAv）包被的Qdot成功地结合到ChR2所在膜的AviTag位点，并通过荧光成像确认。此外，我们合成了含有脱硫生物素的生物素化Traptavidin-DNA共轭探针，其亲和力比常规生物素弱，并成功地将它们与神经元细胞膜上预先偶联的Biotin-AviTag-ChR2位点进行交换，这有可能解决与抗生物素蛋白连接的探针的交联问题。因此，我们希望AviTag-ChR2融合平台将成为在细胞膜特定位点整合各种纳米结构的强大工具，从而克服光遗传视蛋白的局限性。