Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures
Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures
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DOI：
10.1016/j.ymeth.2014.02.020
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发表时间：
2014-05-15
期刊：
影响因子：
4.8
通讯作者：
Wong, Wesley P.
中科院分区：
文献类型：
--
作者：
Koussa, Mounir A.;Sotomayor, Marcos;Wong, Wesley P.
DNA纳米技术的最新方法是通过使用可编程的自下而上的自组装来创建复杂的纳米结构。然而，仅由DNA组成的结构在其作用于其他生物分子的能力方面受到限制。另一方面，蛋白质在生物材料上执行各种功能，但对自组装过程的直接控制仍然是一个挑战。虽然DNA-蛋白质杂交体具有提供两全其美的潜力，但它们可能难以创造出许多将蛋白质连接到DNA的常规技术，从而使蛋白质功能失调。在这里，我们提出了一个基于分选酶的协议共价偶联蛋白质的DNA与蛋白质功能的干扰最小。为了实现这一点，我们开发了一个两步过程。首先，使用点击化学将小的合成肽生物正交地和共价地偶联到DNA寡核苷酸。接着，在蛋白相容性条件下使用酶分选酶将DNA-肽嵌合体共价连接至目的蛋白。我们的协议允许简单的耦合和纯化的功能DNA-蛋白质杂交。我们使用这种技术来形成带有钙粘蛋白-23和原钙粘蛋白-15蛋白片段的寡核苷酸。在并入线性M13支架后，这些蛋白质-DNA杂交体充当二元纳米开关的门。概述的协议是可靠的和模块化的，促进了寡核苷酸和蛋白质库的构建，这些寡核苷酸和蛋白质可以组合形成功能性DNA-蛋白质纳米结构。这些结构将使一类新的功能性纳米结构成为可能，可用于治疗和工业过程。(C)由Elsevier Inc.出版。这是一个在CC BY-NC-ND许可下的开放获取文章
Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes. (C) 2014 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license