Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK.
Physik-Department, Technische Universität München, James-Frank-Str. 1, Garching, Germany.
Sci Rep. 2019 Sep 25;9(1):13820. doi: 10.1038/s41598-019-49843-1.
Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible.
准确且稳定地将 DNA 分子特异性地附着到蛋白质上，是许多单分子力谱技术的要求。最常用的方法仍然依赖于马来酰亚胺化学，涉及感兴趣的蛋白质中的半胱氨酸残基。因此，研究通常集中在没有半胱氨酸的模型蛋白或只有少数几个可以删除的半胱氨酸的模型蛋白上，这样就可以在特定位置引入半胱氨酸。然而，许多蛋白质，特别是真核生物中的蛋白质，含有太多的半胱氨酸，不适合这种策略，因此非常需要新的和广泛适用的方法来进行特异性连接。在这里，我们提出了用于制作力谱实验所需的 DNA-蛋白质偶联物的生物正交方法。非天然氨基酸被特异性地引入，并与带有相应修饰的 DNA 寡核苷酸连接，以进行应变促进的叠氮-炔环加成（SPAAC）或逆电子需求 Diels-Alder（IE-DA）反应。我们还表明 SPAAC 与以前发表的基于肽的附着方法兼容。通过将可用工具包扩展到基于生物正交反应的无标记方法，我们希望能够使研究人员能够研究比目前更广泛的蛋白质的力学性质。
