Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.
Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.
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DOI：
10.1021/ja302832g
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发表时间：
2012-06-27
影响因子：
15
通讯作者：
Chin, Jason W.
中科院分区：
文献类型：
--
作者：
Lang, Kathrin;Davis, Lloyd;Wallace, Stephen;Mahesh, Mohan;Cox, Daniel J.;Blackman, Melissa L.;Fox, Joseph M.;Chin, Jason W.
对于化学生物学家来说，用不同的探针快速、特定地标记蛋白质仍然是一个突出的挑战。酶介导的标记方法可能是快速的，但使用的是蛋白质或多肽融合，这会给研究中的蛋白质带来扰动，并可能限制可以标记的位置，而许多可以通过基因编码的“生物正交”反应太慢，无法在对许多生物过程研究有用的时间尺度上对蛋白质进行特定部位的定量标记。我们报道了双环[6.1.0]非-4-氧-9-基甲醇(BCN)与四嗪类化合物之间的荧光反应，该反应比许多生物正交反应快3-7个数量级。与包括反式-环辛烯和降冰片烯在内的应变烯烃与四嗪的反应不同，BCN-四嗪反应产生了单一的确定的立体化学产物。我们已经发现了氨酰-tRNA合成酶/tRNA对，它能有效地将含有BCN的氨基酸1和含有反式环辛烯的氨基酸2(它也能非常迅速地与四嗪反应)掺入在大肠杆菌和哺乳动物细胞中表达的蛋白质中。我们展示了在体外、在大肠杆菌和活的哺乳动物细胞中对含有1和2的蛋白质进行快速荧光标记。这些方法可能会扩展到动物体内的部位特异性蛋白质标记，我们预计它们将对标记和成像研究产生广泛的影响。
Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many “bioorthogonal” reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3–7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN–tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli, and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.
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影响因子：
15
作者：
Blackman, Melissa L.;Royzen, Maksim;Fox, Joseph M.
通讯作者：
Fox, Joseph M.
影响因子：
15
作者：
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影响因子：
3.2
作者：
McKay, Craig S.;Chigrinova, Mariya;Pezacki, John Paul
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Pezacki, John Paul
影响因子：
15
作者：
Seitchik, Jason L.;Peeler, Jennifer C.;Taylor, Michael T.;Blackman, Melissa L.;Rhoads, Timothy W.;Cooley, Richard B.;Refakis, Christian;Fox, Joseph M.;Mehl, Ryan A.
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Mehl, Ryan A.
影响因子：
16.6
作者：
Dommerholt, Jan;Schmidt, Samuel;Temming, Rinske;Hendriks, Linda J. A.;Rutjes, Floris P. J. T.;van Hest, Jan C. M.;Lefeber, Dirk J.;Friedl, Peter;van Delft, Floris L.
通讯作者：
van Delft, Floris L.