Bernardinelli Giulio, Högberg Björn
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17 177 Stockholm, Sweden.
Nucleic Acids Res. 2017 Oct 13;45(18):e160. doi: 10.1093/nar/gkx707.
While proteins are highly biochemically competent, DNA offers the ability to program, both reactions and the assembly of nanostructures, with a control that is unprecedented by any other molecule. Their joining: DNA-protein conjugates - offer the ability to combine the programmability of DNA with the competence of proteins to form novel tools enabling exquisite molecular control and the highest biological activity in one structure. However, in order for tools like these to become viable for biological applications, their production must be scalable, and an entirely enzymatic process is one way to achieve this. Here, we present a step in this direction: enzymatic production of DNA-protein conjugates using a new self-labeling tag derived from a truncated VirD2 protein of Agrobacterium tumefaciens. Using our previously reported MOSIC method for enzymatic ssDNA oligo production, we outline a pipeline for protein-DNA conjugates without the need for any synthetic chemistry in a one-pot reaction. Further, we validate HER2 staining using a completely enzymatically produced probe, enable the decoration of cell membranes and control of genetic expression. Establishing a method where protein-DNA conjugates can be made entirely using biological or enzymatic processing, opens a path to harvest these structures directly from bacteria and ultimately in-vivo assembly.
虽然蛋白质具有高度的生化活性，但DNA能够对反应和纳米结构的组装进行编程，其控制能力是其他任何分子都前所未有的。它们的结合物——DNA-蛋白质缀合物，能够将DNA的可编程性与蛋白质的活性相结合，形成新的工具，在一个结构中实现精确的分子控制和最高的生物活性。然而，为了使这类工具在生物应用中可行，其生产必须具有可扩展性，而全酶促过程是实现这一目标的一种方法。在此，我们朝着这个方向迈出了一步：利用源自根癌农杆菌截短VirD2蛋白的新型自标记标签，酶促生产DNA-蛋白质缀合物。使用我们之前报道的用于酶促生产单链DNA寡核苷酸的MOSIC方法，我们概述了一种无需任何合成化学步骤的一锅法生产蛋白质-DNA缀合物的流程。此外，我们使用完全酶促生产的探针验证了HER2染色，实现了细胞膜的修饰和基因表达的控制。建立一种能够完全通过生物或酶促加工制备蛋白质-DNA缀合物的方法，为直接从细菌中获取这些结构并最终实现体内组装开辟了一条道路。
