权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular Desighn of DNA-Encapsulating Nano-Particle Capable of Nuclear Targeting

能够核靶向的 DNA 封装纳米粒子的分子设计

基本信息

批准号：
16300166
负责人：
NAKANISHI Mahito
金额：
$ 9.89万
依托单位：
National Institute of Advanced Industrial Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

项目摘要

Gene transfer and expression in mammalian cells should become a technology required in advanced medical application in future. Virus vectors are favored in current medical application as they have their intrinsic machinery to deriver the genetic materials across two major barriers, the cell membrane and the nuclear membrane. However, safer non-viral vectors are desired in many clinical applications. Performance of non-viral vectors has been improved through enhancing the transport across the cell membrane. However, no technology realizing the transport of large DNA molecules across the nuclear membrane has been established yet. In this project, I investigate the conditions allowing the efficient nuclear transport of the DNA encapsulating nano-sized particles. For this purpose, I employ bacteriophage Lambda as a model. Various peptides with nuclear transport activity(nuclear transport signal, NLS) based on T antigen of SV40 were displayed on the surface of the head of the phage particle by fusing them to D protein, one of two major capsid proteins. All of these NLS peptide assisted the nuclear transport of the D proteins, but only a few could assist the transport of nano sized particles. By using the nuclear transport of the phage particles injecting into the cytoplasm by microinjection as an Index, I succeeded to optimize the signals so that up to 5% of the particles could be transported into the nucleus actively through the nuclear pore complex(NPC). On the other hand, I also found that the NLS-mediated particle disruption in the cytoplasm, possibly through the proteasome, limited the efficiency of the nuclear transport. This is the first evidence that the NLS peptide derived from SV40 T antigen, used most popularly in the research of nuclear transport, may not necessarily be suited for transport of large particles.

在哺乳动物细胞中的基因转移和表达将成为未来先进医学应用所需的技术。病毒载体在当前的医学应用中受到青睐，因为它们具有其内在的机制来跨越两个主要屏障（细胞膜和核膜）获得遗传物质。然而，在许多临床应用中需要更安全的非病毒载体。非病毒载体的性能已经通过增强跨细胞膜的转运而得到改善。然而，尚未建立实现大DNA分子跨核膜运输的技术。在这个项目中，我调查的条件，使有效的核运输的DNA封装纳米尺寸的颗粒。为此，我采用噬菌体λ作为模型。通过将基于SV 40的T抗原的具有核转运活性的各种肽（核转运信号，NLS）与两种主要衣壳蛋白之一的D蛋白融合，将它们展示在噬菌体颗粒的头部表面上。所有这些NLS肽都能促进D蛋白的核转运，但只有少数能促进纳米颗粒的核转运。通过使用通过显微注射注射到细胞质中的噬菌体颗粒的核转运作为指标，我成功地优化了信号，使得高达5%的颗粒可以通过核孔复合物（NPC）主动转运到细胞核中。另一方面，我还发现，NLS介导的颗粒在细胞质中的破坏，可能通过蛋白酶体，限制了核运输的效率。这是第一个证据表明，在核转运研究中最常用的来自SV 40 T抗原的NLS肽可能不一定适合于大颗粒的转运。