Protein Oriented Ligation on Nanoparticles Exploiting O6-Alkylguanine-DNA Transferase (SNAP) Genetically Encoded Fusion
Protein Oriented Ligation on Nanoparticles Exploiting O6-Alkylguanine-DNA Transferase (SNAP) Genetically Encoded Fusion
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DOI：
10.1002/smll.201102284
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发表时间：
2012-05-21
期刊：
影响因子：
13.3
通讯作者：
Prosperi, Davide
中科院分区：
文献类型：
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作者：
Colombo, Miriam;Mazzucchelli, Serena;Prosperi, Davide
结合可检测的光和磁信号的发射和集中的靶向作用的多功能纳米颗粒（MFN）的使用在癌症诊断中引起了广泛的兴趣[1-3]。靶向癌细胞的选择性是最重要的，并且通常利用对特定细胞膜受体具有高亲和力的生物分子修饰MFN来实现[4-6]。设计用归巢肽和蛋白质官能化以优化分子识别的MFN的最大挑战之一在于精细控制纳米颗粒表面上的配体取向的可能性[7，8]。迄今为止，已经遵循三种主要方法来达到这一目标：（1）具有对小肽具有高亲和力的结构域的天然蛋白质可以通过在MFN上的配体固定来捕获[8];（2）含有被通常用于蛋白质纯化的小分子或复合物识别的亲和标记的蛋白质可以被遗传修饰以引入对蛋白质固定特异的识别序列[9，10];和（3）位点特异性缀合可通过化学选择性连接发生[11]。蛋白质在平面上的共价固定有更长的历史，主要用于生物传感目的[12]。一个优雅的策略涉及使用融合蛋白，该融合蛋白含有能够与锚定在固体表面的自杀抑制剂发生不可逆交叉反应的小酶（通常为20-30 kDa）。实例包括人α 6-羟基鸟嘌呤-DNA烷基转移酶（SNAP-标签）[13]、卤代烷烃脱卤酶（Halo-标签）[14]和丝氨酸酯酶[15]的突变体。这种方法具有几个优点：（1）固定在表面上的配体是一种小分子;（2）在生理条件下，它与酶的结合迅速发生;（3）它是高度特异性的，基本上是不可逆的;和（4）所有这些结合系统都涉及单价识别配偶体，其克服了通常与其它常规配体对一起发生的交联效应，例如生物素/抗生物素蛋白[16]。原则上，SNAP可以克服在使用流行的EDC/NHS方法时总是发生的关键问题，即，非特异性连接的形成，导致蛋白质的随机定向连接（EDC = 1-乙基-3-[3-二甲氨基丙基]碳二亚胺盐酸盐; NHS = N-羟基琥珀酰亚胺）。
The use of multifunctional nanoparticles (MFN) combining the emission of detectable optical and magnetic signals and a focused targeting action is attracting broad interest in cancer diagnostics [1–3]. The selectivity in targeting cancer cells is of primary importance and is usually achieved exploiting the modification of MFN with biomolecules endowed with high affinity for specific cell membrane receptors [4–6]. One of the greatest challenges in designing MFN functionalized with homing peptides and proteins to optimize molecular recognition resides in the possibility to finely control the ligand orientation on the nanoparticle surface [7, 8]. So far, three main approaches have been followed to reach this goal: (1) native proteins having domains with high affinity for small peptides can be captured via ligand immobilization on MFN [8]; (2) proteins containing affinity tags recognized by small molecules or complexes commonly utilized for protein purification can be genetically modified in order to introduce a recognition sequence specific for protein immobilization [9, 10]; and (3) sitespecific conjugation can occur via chemoselective ligation [11]. The covalent immobilization of proteins on flat surfaces has a longer story, mainly for biosensing purposes [12]. An elegant strategy involves the use of fusion proteins containing a small (typically 20-30 kDa) enzyme capable of irreversibly cross-reacting with a suicide inhibitor anchored to the solid surface. Examples include mutants of human 06-ahkylguanine-DNA alkyltransferase (SNAP-tag) [13], haloalkane dehalogenase (Halo-tag) [14], and a serine esterase [15]. This approach presents several advantages: (1) the ligand to be immobilized on the surface is a small molecule; (2) its binding to the enzyme occurs quickly under physiological conditions; (3) it is highly specific and essentially irreversible; and (4) all these binding systems involve monovalent recognition partners, which overcomes the crosslinking effects that usually occur with other conventional ligand pairs, such as biotin/avidin [16]. In principle, SNAP may overcome a crucial problem that invariably occurs when using the popular EDC/NHS method, i.e., the formation of nonspecific linkages, which results in a randomly oriented ligation of the protein (EDC = 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride; NHS = N-hydroxysuccinimide).