Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.
Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.
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DOI：
10.1021/jacs.6b08733
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发表时间：
2016-11-02
影响因子：
15
通讯作者：
Hang HC
中科院分区：
文献类型：
--
作者：
Peng T;Hang HC
在过去的几年里，荧光蛋白(如GFP)被广泛用于显示重组蛋白在活细胞中的表达和定位。虽然功能强大，但荧光蛋白质标签因其相对较大的尺寸和对蛋白质功能的潜在干扰而受到限制。或者，利用生物正交化学的小分子有机荧光团对蛋白质进行定点标记，可能会提供一种更精确、更少干扰的方法。这种方法包括通过遗传密码扩展将非天然氨基酸(UAA)特定地结合到蛋白质中，然后在活细胞中用小的有机荧光团进行生物正交化学标记。虽然这种方法已被用于标记细胞外蛋白质用于活细胞成像研究，但活细胞内蛋白质的定点生物正交标记和荧光成像仍然具有挑战性。在这里，我们系统地评估了非对映异构体纯生物正交性UAA与四嗪功能化荧光团的反相电子需求Diels-Alder环加成反应(IEDAC)在哺乳动物细胞中用于活细胞标记和成像的位置特异性掺入。我们的研究表明，反式-环辛基-2-烯-赖氨酸(2‘-aTCOK)的轴向非对映异构体(2’-aTCOK)在活的哺乳动物细胞中提供了高效和特异的单取代四嗪荧光团生物正交标记，这使我们能够首次成像IFITM3的细胞内定位和实时动态转运，IFITM3是一种只有137个氨基酸的小分子膜相关蛋白。我们优化的UAA掺入和生物正交标记条件也使其他细胞内蛋白质的高效定点荧光标记用于哺乳动物细胞的活体细胞成像研究。
Over the past years, fluorescent proteins (e.g., GFP) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large size and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse–electron–demand Diels–Alder cycloaddition (IEDAC) reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine (2’-aTCOK) robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine-fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.
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影响因子：
15
作者：
Halo TL;Appelbaum J;Hobert EM;Balkin DM;Schepartz A
通讯作者：
Schepartz A
影响因子：
15
作者：
Blackman, Melissa L.;Royzen, Maksim;Fox, Joseph M.
通讯作者：
Fox, Joseph M.
DOI：
10.1002/anie.201403349
发表时间：
2014-09-15
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Erdmann RS;Takakura H;Thompson AD;Rivera-Molina F;Allgeyer ES;Bewersdorf J;Toomre D;Schepartz A
通讯作者：
Schepartz A
影响因子：
16.6
作者：
Carlson, Jonathan C. T.;Meimetis, Labros G.;Hilderbrand, Scott A.;Weissleder, Ralph
通讯作者：
Weissleder, Ralph
影响因子：
46.9
作者：
Fernandez-Suarez, Marta;Baruah, Hemanta;Ting, Alice Y.
通讯作者：
Ting, Alice Y.