日本京都大学Michiyuki Matsuda团队开发出在活体双光子（2P）光遗传学中，荧光共振能量转移（FRET）辅助的黄素蛋白的光活化技术。相关论文2019年9月9日在线发表在《自然—方法学》上。

研究人员介绍，现有的光学二聚体以优化信号传导为途径，但它们在体内的用途有限，部分原因是它们在2P激发下的低效活化。

为了克服这一问题，研究人员开发了FRET辅助光活化技术，或称为FRAPA。在2P激发下，mTagBFP2有效地吸收能量并将能量转移到CRY2的发色团。基于结构引导工程，开发了具有40％FRET效率的嵌合蛋白，并命名为2P激活的CRY2或2paCRY2。2paCRY2用于开发名为2paRAF的RAF1激活系统。在表达2paRAF的三维培养细胞中，细胞外信号调节激酶（ERK）在单细胞分辨率下通过2P激发被有效激活。在表达2paRAF的小鼠的表皮细胞中也完成了ERK的光激活。他们进一步开发了mTFP1融合的LOV结构域，其表现出对2P激发的有效响应。总的来说，FRAPA将为体内单细胞光学信号传导途径控制铺平道路。

附：英文原文

Title: FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics

Author: Tomoaki Kinjo, Kenta Terai, Shoichiro Horita, Norimichi Nomura, Kenta Sumiyama, Kaori Togashi, So Iwata, Michiyuki Matsuda

Issue&Volume: 2019-09-09

Abstract: Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Frster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo. Fusion to fluorescent proteins enables efficient two-photon activation of blue-light-controlled optical dimerizers via FRET. FRET-assisted photoactivation was used to study extracellular signal-regulated kinase activation in 3D epithelial cysts, organoids and living mice.

DOI: 10.1038/s41592-019-0541-5

Source:https://www.nature.com/articles/s41592-019-0541-5