Le Duc H T, Hu He, Commandeur Ulrich, Steinmetz Nicole F
Department of Biomedical Engineering, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106, USA.
Department of Molecular Biotechnology, RWTH-Aachen University, Aachen 52064, Germany.
J Struct Biol. 2017 Dec;200(3):360-368. doi: 10.1016/j.jsb.2017.06.006. Epub 2017 Jun 21.
Potato virus X (PVX), a type member of the plant virus potexvirus group, offers a unique nanotechnology platform based on its high aspect ratio and flexible filamentous shape. The PVX platform has already been engineered and studied for its uses in imaging, drug delivery, and immunotherapies. While genetic engineering procedures are well established for PVX, there is limited information about chemical conjugation strategies for functionalizing PVX, partly due to the lack of structural information of PVX at high resolution. To overcome these challenges, we built a structural model of the PVX particle based on the available structures from pepino mosaic virus (PepMV), a close cousin of PVX. Using the model and a series of chemical conjugation experiments, we identified and probed the addressability of cysteine side chains. Chemical reactivity of cysteines was confirmed using Michael-addition and thiol-selective probes, including fluorescent dyes and biotin tags. LC/MS/MS was used to map Cys 121 as having the highest selectivity for modification. Finally, building on the availability of two reactive groups, the newly identified Cys and previously established Lys side chains, we prepared multifunctional PVX nanoparticles by conjugating Gd-DOTA for magnetic resonance imaging (MRI) to lysines and fluorescent dyes for optical imaging to cysteines. The resulting functionalized nanofilament could have applications in dual-modal optical-MRI imaging applications. These results further extend the understanding of the chemical properties of PVX and enable development of novel multifunctional platforms in bio/nanotechnology.
马铃薯X病毒（PVX）是植物病毒马铃薯X病毒属的典型成员，因其高纵横比和灵活的丝状形状提供了一个独特的纳米技术平台。PVX平台已被设计并研究用于成像、药物递送和免疫疗法。虽然PVX的基因工程程序已经很成熟，但关于PVX功能化的化学偶联策略的信息有限，部分原因是缺乏高分辨率的PVX结构信息。为了克服这些挑战，我们基于与PVX亲缘关系较近的佩普ino花叶病毒（PepMV）的现有结构构建了PVX颗粒的结构模型。利用该模型和一系列化学偶联实验，我们鉴定并探究了半胱氨酸侧链的可寻址性。使用迈克尔加成和硫醇选择性探针（包括荧光染料和生物素标签）证实了半胱氨酸的化学反应性。液相色谱串联质谱（LC/MS/MS）用于确定Cys 121对修饰具有最高的选择性。最后，基于新鉴定的半胱氨酸和先前确定的赖氨酸侧链这两个反应性基团的可用性，我们通过将用于磁共振成像（MRI）的钆-二乙三胺五乙酸（Gd-DOTA）与赖氨酸偶联，以及将用于光学成像的荧光染料与半胱氨酸偶联，制备了多功能PVX纳米颗粒。所得的功能化纳米丝可应用于双模态光学-MRI成像。这些结果进一步扩展了对PVX化学性质的理解，并有助于在生物/纳米技术中开发新型多功能平台。
