Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
J Am Chem Soc. 2020 Apr 22;142(16):7245-7249. doi: 10.1021/jacs.9b11520. Epub 2020 Apr 10.
Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8 × 10 M s with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino acids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exceptionally high reaction rate of this ligation minimizes label concentration, allowing for substoichiometric eukaryotic protein labeling where the concentration of the label is less than the concentration of the protein. This approach offers unprecedented control over the composition and stability of the protein tag. We anticipate that this system will have a broad impact on labeling and imaging studies because it can be used where all generally orthogonal PylRS/tRNA pairs are employed.
生物分子在活真核细胞中的标记受到低组分稳定性和缓慢反应速率的限制。我们表明，蛋白质中遗传编码的四嗪氨基酸与 sTCO 试剂达到 8×10^M s^-1 的反应速率，使它们成为真核系统中最快的定点生物正交标记物。我们证明四嗪氨基酸在蛋白质上稳定，并且能够与 sTCO 试剂进行定量标记。这种连接的异常高反应速率最小化了标记物的浓度，从而允许亚化学计量的真核蛋白标记，其中标记物的浓度小于蛋白质的浓度。这种方法对蛋白质标签的组成和稳定性提供了前所未有的控制。我们预计，由于该系统可以在所有通常正交的 PylRS/tRNA 对都被使用的情况下使用，因此它将对标记和成像研究产生广泛的影响。
