Using nanoparticle-DNA to enhance antisense gene regulation

描述(申请人提供)：反义基因作为一种基因沉默方法引起了极大的兴趣。反之，具有与特定基因的信使核糖核酸互补的序列的短链DNA可以与编码特定基因的信使核糖核酸结合，从而阻止蛋白质被核糖体合成。然而，关闭特定基因并不是100%有效的，因为核糖体有时可以克服这一物理障碍。因此，增强核糖体的机械屏障将提高反义效率。这项拟议的工作将使用与反义DNA连接的纳米颗粒，这些纳米颗粒与mRNA结合并阻止翻译。由于纳米粒子的大小，预计它们将成为核糖体的重大障碍。特别是，金纳米颗粒非常适合于这一目的，因为它们可以溶于水，并且可以与反义DNA化学连接。我们将用化学方法合成不同大小的金纳米颗粒，并将它们与反义DNA连接。我们将通过凝胶电泳和动态光散射(DLS)对纳米颗粒-DNA偶联物的大小进行定量。我们还将通过凝胶电泳、光学光谱和DLS来量化它们与靶标结合的能力。来自这些实验的信息将确定哪些纳米颗粒大小和DNA序列最有利于阻断核糖体。然后，我们将使用这些不同的纳米颗粒-DNA结合物来抑制绿色荧光蛋白的体外翻译，并通过荧光光谱对产物的翻译进行量化。我们将量化纳米DNA的反义效率，并与普通DNA进行比较。这一应用将在短期内带来改善反义基因治疗的好处。由于反义基因被用来针对各种疾病的基因，从长远来看，它将对疾病的研究和理解产生积极的影响。此外，预计它将帮助使用与DNA相连的NPs的生物应用，包括传感、基因传递和创造智能生物机器。 项目简介：该项目将使用纳米颗粒来提高反义基因的效率，即关闭特定的基因。这项技术可以改进反义基因的使用，以靶向涉及疾病的基因。

DESCRIPTION (provided by applicant): Antisense has elicited enormous interest as a method for gene silencing. In antisense, short strands of DNA with sequences complementary to the mRNA of a particular gene can bind to mRNA encoding a particular gene, preventing the protein from being synthesized by the ribosome. However, shutting off a particular gene is not 100% efficient as the ribosome can sometimes overcome this physical barrier. Therefore, enhancing the mechanical barrier for the ribosome would improve antisense efficiency. The proposed work will use nanoparticles linked to antisense DNA that bind to mRNA and block translation. Because of their size, nanoparticles are expected to be significant obstacles for the ribosome. In particular, gold nanoparticles are highly suitable for this purpose because they are soluble in water and can be chemically linked to the antisense DNA. We will chemically synthesize gold nanoparticles of different sizes and link them to antisense DNA. We will quantify the size of nanoparticle-DNA conjugates by gel electrophoresis and dynamic light scattering (DLS). We will also quantify their ability to bind to a target by gel electrophoresis, optical spectroscopy, and DLS. Information from these experiments will determine which nanoparticle sizes and DNA sequences are best for blocking the ribosome. We will then use these different nanoparticle-DNA conjugates to inhibit translation of green fluorescent protein in vitro and quantify translation by fluorescence spectroscopy of the product. We will quantify the antisense efficiency of the nanoparticle-DNA and compare to plain DNA. This application will have the short term benefits of improving antisense gene therapy. Because antisense is used to target genes in a variety of diseases, it will have positive ramifications in the study and understanding of diseases in the longer term. In addition, it is anticipated that it will aid biological applications that use NPs linked to DNA, including sensing, gene delivery, and the creation of smart biological machines. Project Narrative: This project will use nanoparticles to increase the efficiency of antisense, the shutting off a specific gene. This technology can improve the use of antisense to target genes involved in diseases.