Molecular Evolution of Multifunctional DNA Nanoparticles

多功能DNA纳米颗粒的分子进化

• 批准号: 8472338
• 负责人: BRADLEY T MESSMER
• 金额: $ 18.14万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8472338
• 关键词: Address; Adopted; Affinity; Alkynes; Antibodies; Antigens; Area; Avidity; Azides; Binding; Biotin; Breast Cancer Cell; Breeding; Cancer cell line; Cell Line; Cell Separation; Cell surface; Cells; Chemistry; Complex; DNA; DNA Binding; Data; Diversity Library; Elements; Epithelial; Flow Cytometry; Formaldehyde; Goals; Histology; Human; Image; K-562; Label; Libraries; Ligands; Liquid substance; Malignant Neoplasms; Malignant neoplasm of pancreas; Methodology; Methods; Microscopy; Molecular; Molecular Evolution; Molecular Models; Molecular Target; Mus; Nature; Normal Cell; Normal tissue morphology; Nucleotides; Oligonucleotides; Pancreas; Peptide aptamers; Peripheral Blood Mononuclear Cell; Post-Translational Protein Processing; Precipitation; Property; Proteins; Publishing; Randomized; Reagent; Single-Stranded DNA; Solid; Staining method; Stains; Structure; Surface; Suspension substance; Suspensions; Technology; Tissue Microarray; Tissues; Transplantation; anticancer research; cancer cell; cancer therapy; circulating cancer cell; clinical application; combinatorial; crosslink; in vivo; innovation; interest; iron oxide; leukemia; magnetic cell separation; molecular modeling; monomer; nanoparticle; novel; pancreatic cancer cells; particle; research study; tumor

DESCRIPTION (provided by applicant): We have developed a DNA nanoparticle library technology for the selection of cell binding DNA nanoparticles. Rolling circle amplification (RCA) of circular oligonucleotide templates containing randomized nucleotides produces libraries of single stranded DNA nanoparticles that can be screened for cell binding properties. The main goal of this project is to create multimodal DNA nanoparticles that specifically bind to cancer cells. The particles will be "bred" by a novel iterative selection and re-assortment method to create modular DNA nanoparticles that contain multiple distinct recognition elements within a single particle. This project addresses a significant challenge in many areas of cancer research and treatment, mainly the lack of cancer cell specific binding agents. Our DNA nanoparticles differ from other affinity reagents in that there is intrinsic multivalent display of the modules, allowing avidity to compensate for low monovalent affinity. The modular nature of the particle template construction allows multiple distinct recognition elements to be assembled into a single molecular entity. Furthermore, the combinatorial selection method allows the optimal particle to be evolved in the same molecular context in which it will be used. Collectively, the unique features of our DNA nanoparticle libraries represent a novel paradigm for cell affinity reagents that replaces high affinity binding to one or two defined molecular targets with a diverse landscape of high avidity interactions. The specific aims for this application are: Aim 1. Validate and optimize combinatorial selection methodology for multi- module particles. We have identified single module particles that bind to a mouse pancreatic cancer cell line. We will optimize the multi-module selection strategy with this line and confirm on two human pancreatic lines (MiaPaCa-2 and Panc-1) as well as a leukemia line (K-562) to demonstrate the feasibility against both solid and liquid tumor types. Aim 2. Demonstrate cancer specific cell binding of selected particles. Three applications will be addressed: histology, flow cytometry, and cell capture. Fluorescently labeled particles will used on tissue arrays for fluorescent microscopy and on suspension cells for flow cytometry. Particles tagged with biotin or iron oxide will used for magnetic cell separation. Aim 3. Identify the molecular targets of the cancer cell specific DNA nanoparticles. Two approaches will be used. In the first, we will perform co- precipitation experiments after crosslinking biotinylated DNA nanoparticles to the cell surface molecules. In the second, we will use "click" chemistry between azide or alkyne tagged particles to specifically react with cells that are either indiscriminately labeled with the partner click chemistry or with cells that contain the partner chemistry in specific protein modifications (e.g., farnesylated proteins).

描述（由申请人提供）：我们开发了一种DNA纳米颗粒库技术，用于选择细胞结合DNA纳米颗粒。包含随机核苷酸的圆形寡核苷酸模板的滚动圆扩增（RCA）产生单链DNA纳米颗粒的文库，可以筛选以获得细胞结合特性。该项目的主要目标是创建特异性结合癌细胞的多模式DNA纳米颗粒。这些颗粒将通过一种新型的迭代选择和重新分配方法“繁殖”，以创建模块化DNA纳米颗粒，这些纳米颗粒包含单个粒子内包含多个不同识别元件的模块化。该项目在许多领域的癌症研究和治疗领域都解决了重大挑战，主要是缺乏癌细胞特异性结合剂。我们的DNA纳米颗粒与其他亲和力试剂不同，因为模块内固有的多价显示，从而可以补偿低单价亲和力。粒子模板结构的模块化性质允许将多个不同的识别元素组装到单个分子实体中。此外，组合选择方法允许在使用它的相同分子环境中进化最佳粒子。总体而言，我们的DNA纳米颗粒库的独特特征代表了细胞亲和力试剂的新型范式，该试剂替代了高亲和力结合到一个或两个定义的分子靶标的高亲和力相互作用的景观。该应用程序的具体目的是：目标1。验证和优化多模块粒子的组合选择方法。我们已经确定了与小鼠胰腺癌细胞系结合的单个模块颗粒。我们将使用此系列优化多模块选择策略，并在两条人类胰腺（Miapaca-2和Panc-1）以及白血病系（K-562）上确认，以证明针对固体和液体肿瘤类型的可行性。 AIM 2。证明选定颗粒的癌症特异性细胞结合。将解决三种应用：组织学，流式细胞仪和细胞捕获。荧光标记的颗粒将用于组织阵列，用于荧光显微镜和悬浮细胞进行流式细胞仪。用生物素或氧化铁标记的颗粒将用于磁细胞分离。目标3。确定癌细胞特异性DNA纳米颗粒的分子靶标。将使用两种方法。首先，我们将在将生物素化的DNA纳米颗粒交联后进行共同沉淀实验。在第二个中，我们将使用叠氮化物或炔烃标记颗粒之间的“点击”化学反应与与伴侣单击化学的不分毒标记的细胞，或与包含特定蛋白质修饰的伴侣化学的细胞（例如，Farnesylated蛋白质）。