Institute of Applied Physics, TU Wien, Vienna, 1060, Austria.
Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, 1090, Austria.
ACS Nano. 2021 Sep 28;15(9):15057-15068. doi: 10.1021/acsnano.1c05411. Epub 2021 Aug 31.
DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry, and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as the protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to three proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral peptide nucleic acid (PNA) in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.
DNA 折纸结构为单分子生物分子的纳米级精确组织提供了灵活的支架。虽然它们在各种生物应用中越来越受欢迎，但在保持蛋白质功能的情况下，以确定的化学计量、高产量对其进行功能化仍然具有挑战性。在这项研究中，我们应用单分子荧光显微镜结合细胞生物学功能测定，系统地评估了 DNA 折纸结构的定点修饰的不同策略，重点是效率、化学计量和蛋白质功能。我们使用 T 细胞受体 (TCR) 的激活配体作为感兴趣的蛋白质，发现两种常用的方法在化学计量和蛋白质功能方面表现不佳。虽然使用四价野生型链霉亲和素 (streptavidin) 偶联生物素化的 TCR 配体的策略产生了多达三个蛋白质的 DNA 折纸结构的混合群体，但使用二价 (dSAv) 或 DNA 偶联的单价链霉亲和素 (mSAv) 允许特异性附着单个生物素化的 TCR 配体。最直接的修饰策略，即共价 DNA 偶联，导致配体效力降低了 3 倍，这可能是由于电荷介导的蛋白质功能受损。在配体缀合物中用带正电荷的肽核酸 (PNA) 替代 DNA 作为偶联策略，在我们的研究中表现出最佳的整体性能，因为它产生了最高的产量，没有多价 DNA 折纸结构，并完全保留了蛋白质功能。通过我们的研究，我们旨在为具有广泛生物应用的蛋白质提供具有化学计量定义的、定点修饰 DNA 折纸结构的指南。
