Molecular Evolution of Multifunctional DNA Nanoparticles

描述(申请人提供)：我们开发了一种DNA纳米粒文库技术，用于选择细胞结合的DNA纳米粒。包含随机核苷酸的环状寡核苷酸模板的滚环扩增(RCA)产生单链DNA纳米粒的文库，可以筛选细胞结合特性。该项目的主要目标是创造与癌细胞特异结合的多模式DNA纳米颗粒。这些颗粒将通过一种新的迭代选择和重新分类的方法来“繁殖”，以创建模块化的DNA纳米颗粒，这种纳米颗粒在单个颗粒中包含多个不同的识别元件。该项目解决了癌症研究和治疗的许多领域中的重大挑战，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试剂的不同之处在于，有内在的多价模块展示，允许亲和力来补偿低单价亲和力。颗粒模板结构的模块化性质允许将多个不同的识别元件组装成单个分子实体。此外，组合选择方法允许在使用该粒子的相同分子环境中进化最佳粒子。总而言之，我们DNA纳米粒子库的独特功能代表了细胞亲和试剂的新范例，它用一系列高亲和力相互作用取代了与一个或两个已定义分子靶标的高亲和力结合。这一应用的具体目标是：目标1.验证和优化多模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模块颗粒。我们将利用这一系优化多模块选择策略，并在两个人胰腺系(MiaPaCa-2和PANC-1)以及一个白血病系(K-562)上进行验证，以证明其针对固体和液体肿瘤类型的可行性。目的2.展示所选颗粒与癌症特异性细胞的结合。将涉及三个应用：组织学、流式细胞术和细胞捕获。荧光标记的颗粒将用于荧光显微镜的组织阵列和用于流式细胞术的悬浮细胞。标记了生物素或氧化铁的颗粒将用于磁性细胞分离。目的3.确定肿瘤细胞特异性DNA纳米粒的分子靶点。将使用两种方法。首先，我们将在生物素化的DNA纳米颗粒与细胞表面分子交联后进行共沉淀实验。在第二个实验中，我们将使用叠氮化物或炔标记颗粒之间的“点击”化学来与不分青红皂白地被伙伴点击化学标记的细胞或与在特定蛋白质修饰中包含伙伴化学的细胞(例如，法尼化蛋白质)发生特异性反应。

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).