Wilkinson Andrew A, Jagu Elodie, Ubych Krystian, Coulthard Steven, Rushton Ashleigh E, Kennefick Jack, Su Qiang, Neely Robert K, Fernandez-Trillo Paco
School of Chemistry, University of Birmingham, Edgbaston, Birmingham, U.K., B15 2TT.
ACS Cent Sci. 2020 Apr 22;6(4):525-534. doi: 10.1021/acscentsci.9b01023. Epub 2020 Mar 27.
Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selectively , , and chemical functionality to a biomolecule, DNA in this case. Our method combines the precision and robustness of methyltransferase-directed labeling with the reversibility of acyl hydrazones and the efficiency of click chemistry. Underpinning the method is a new -adenosyl-l-methionine derivative to site-selectively label DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional are conjugated via the azide and can be when needed at the acyl hydrazone via exchange with hydroxyl amine. The formed hydrazide-labeled DNA is a versatile intermediate that can be either to reset the original chemical handle or covalently reacted with a . This ability to , , , and DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.
当前的生物共轭方法依赖于引入稳定的连接子，这些连接子缺乏进行连续功能化所需的通用性。然而，连续操作在化学生物学中的需求日益增加，因为它们可以支持对同一样本进行多次分析，从而更全面地了解细胞行为。在这里，我们提出了一种新方法，用于对生物分子（在本文中为DNA）进行位点选择性地连接、断开和重新连接化学官能团。我们的方法将甲基转移酶导向标记的精确性和稳健性与酰腙的可逆性以及点击化学的高效性结合起来。该方法的基础是一种新的S-腺苷-L-甲硫氨酸衍生物，用于用含有酰腙连接子和末端叠氮化物的双功能化学手柄对DNA进行位点选择性标记。功能性基团通过叠氮化物进行共轭，并且在需要时可以通过与羟胺交换在酰腙处进行断开。形成的酰肼标记的DNA是一种通用中间体，可以被还原以重置原始化学手柄，或者与一种试剂发生共价反应。这种对DNA进行连接、断开、重新连接和共轭的能力被用于在DNA上依次引入两种荧光染料。最后，我们通过开发一种使用磁珠对标记的DNA进行分选的方案，并随后对分选后的DNA样本进行扩增以进行进一步分析，展示了该方法的潜力。所提出的方法为位点选择性生物共轭开辟了新途径，并且应该为化学生物学中需要对同一样本进行连续功能化的综合方法提供支持。
