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. ? ? ?
| Park, Sunho; Hamad-Schifferli, Kimberly (2010) Nanoscale interfaces to biology. Curr Opin Chem Biol 14:616-22 |
| Park, Sunho; Hamad-Schifferli, Kimberly (2010) Enhancement of in vitro translation by gold nanoparticle--DNA conjugates. ACS Nano 4:2555-60 |
