Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular Targeting
Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular Targeting
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DOI：
10.1021/acs.bioconjchem.9b00490
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发表时间：
2019-09-01
影响因子：
4.7
通讯作者：
de Greef, Tom F. A.
中科院分区：
文献类型：
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作者：
Cremers, Glenn A. O.;Rosier, Bas J. H. M.;de Greef, Tom F. A.
抗体的特异性和DNA纳米技术的可编程性的结合为科学界提供了一个强大的工具，使用基于荧光的读出方法标记和明确区分大量的亚细胞靶点。尽管一抗可商购获得用于大类靶标，但缺乏用于该亲和试剂的一般化学计量位点选择性DNA标记策略。在这里，我们提出了一个通用的，位点选择性的缀合方法，使用一个小的光交叉标记蛋白G适配器，允许标记不同的宿主物种的抗体与控制数量的短寡核苷酸（ODNs）。重要的是，我们说明了这种缀合方法可以在小规模上直接对市售的一抗进行，并且对牛血清白蛋白没有交叉反应性。此外，我们提出了一个通用的实验室兼容的策略来纯化DNA标记的抗体而不丧失功能。蛋白G-ODN标记的一抗的应用通过采用三种公知的使用荧光读出检测亚细胞靶标的方法来证明，包括流式细胞术、DNA-PAINT和dSTORM。因此，这项工作建立了一个通用和有效的平台，用于合成独特的ODN-抗体缀合物的库，促进更广泛地使用基于DNA的可编程标签进行多重标记，以纳米精度识别亚细胞特征，并提高我们对细胞结构和功能的理解。
The combination of the specificity of antibodies and the programmability of DNA nanotechnology has provided the scientific community with a powerful tool to label and unambiguously distinguish a large number of subcellular targets using fluorescence-based read-out methods. Whereas primary antibodies are commercially available for a large class of targets, a general stoichiometric site-selective DNA labeling strategy for this affinity reagent is lacking. Here we present a universal, site selective conjugation method using a small photo-cross-linkable protein G adaptor that allows labeling of antibodies of different host species with a controlled number of short oligonucleotides (ODNs). Importantly, we illustrate that this conjugation method can be directly performed on commercially available primary antibodies on a small scale and without cross-reactivity towards bovine serum albumin. In addition, we present a general benchtop-compatible strategy to purify DNA-labeled antibodies without a loss of function. The application of protein G-ODN-labeled primary antibodies is demonstrated by employing three well-known methods for detecting subcellular targets using fluorescence read-out, including flow cytometry, DNA-PAINT, and dSTORM. This work thus establishes a general and efficient platform for the synthesis of a library of unique ODN-antibody conjugates, facilitating the broader use of DNA-based programmable tags for multiplexed labeling to identify subcellular features with nanometer precision and improving our understanding of cellular structure and function.