Using nanoparticle-DNA to enhance antisense gene regulation

描述（由申请人提供）：反义作为一种基因沉默的方法引起了极大的兴趣。在反义中，与特定基因的mRNA序列互补的DNA短链可以与编码特定基因的mRNA结合，阻止蛋白质被核糖体合成。然而，关闭一个特定的基因并不是100%有效的，因为核糖体有时可以克服这个物理障碍。因此，增强核糖体的机械屏障可以提高反义效率。提议的工作将使用纳米颗粒连接到反义DNA，与mRNA结合并阻断翻译。由于它们的大小，纳米粒子有望成为核糖体的重要障碍。特别是，金纳米颗粒非常适合这一目的，因为它们可溶于水，并且可以与反义DNA化学连接。我们将化学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）来量化纳米颗粒- dna偶联物的大小。我们还将通过凝胶电泳、光谱学和DLS来量化它们与目标结合的能力。从这些实验中获得的信息将决定哪种纳米颗粒大小和DNA序列最适合阻断核糖体。然后，我们将使用这些不同的纳米颗粒- dna偶联物在体外抑制绿色荧光蛋白的翻译，并通过产物的荧光光谱定量翻译。我们将量化纳米颗粒-DNA的反义效率，并与普通DNA进行比较。这一应用将在短期内改善反义基因治疗。由于反义在多种疾病中被用于靶向基因，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，预计它将有助于使用与DNA相关的NPs的生物应用，包括传感、基因传递和智能生物机器的创建。

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.