Molecular Evolution of Multifunctional DNA Nanoparticles

描述（由申请人提供）：我们开发了一种DNA纳米颗粒文库技术，用于选择细胞结合DNA纳米颗粒。含有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纳米颗粒文库，可以筛选细胞结合特性。这个项目的主要目标是创造多模态DNA纳米颗粒，专门与癌细胞结合。这些粒子将通过一种新的迭代选择和重新分类方法进行“繁殖”，以创建模块化的DNA纳米粒子，在单个粒子中包含多个不同的识别元素。该项目解决了许多癌症研究和治疗领域的重大挑战，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，模块具有内在的多价显示，允许亲和度补偿低单价亲和度。颗粒模板结构的模块化特性允许多个不同的识别元素被组装成一个单一的分子实体。此外，组合选择方法允许最优粒子在相同的分子环境中进化，它将被使用。总的来说，我们的DNA纳米粒子文库的独特特征代表了细胞亲和试剂的新范式，它取代了与一个或两个定义的分子靶标的高亲和结合，具有不同的高亲和相互作用景观。此应用程序的具体目标是：目标1。验证和优化多模粒子组合选择方法。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。我们将利用该细胞系优化多模块选择策略，并在两个人类胰腺细胞系（MiaPaCa-2和Panc-1）以及一个白血病细胞系（K-562）上进行验证，以证明其对固体和液体肿瘤类型的可行性。目标2。展示选定粒子的癌症特异性细胞结合。三个应用将解决：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流式细胞术的悬浮细胞。带有生物素或氧化铁标记的颗粒将用于磁性细胞分离。目标3。确定癌细胞特异性DNA纳米颗粒的分子靶标。将采用两种方法。首先，我们将在生物素化DNA纳米颗粒与细胞表面分子交联后进行共沉淀实验。在第二种方法中，我们将使用叠氮化物或炔标记颗粒之间的“点击”化学来特异性地与不加区分地标记有伴侣点击化学的细胞或在特定蛋白质修饰中包含伴侣化学的细胞（例如，法酰化蛋白质）发生反应。

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). PUBLIC HEALTH RELEVANCE: The main goal of this project is to create a new type of particle that can bind to cancer cells but no to normal cells. These particles, made out of DNA, can help understand the differences between cancer cells and normal. They can also be used to capture and observe cancer cells in clinical applications.