Molecular Evolution of Multifunctional DNA Nanoparticles

描述（由申请人提供）：我们已经开发了用于选择细胞结合DNA纳米颗粒的DNA纳米颗粒文库技术。含有随机化核苷酸的环状寡核苷酸模板的滚环扩增（RCA）产生单链DNA纳米颗粒的文库，其可以针对细胞结合性质进行筛选。该项目的主要目标是创造特异性结合癌细胞的多模态DNA纳米颗粒。这些颗粒将通过一种新的迭代选择和重新分配方法进行“繁殖”，以创建在单个颗粒内包含多个不同识别元件的模块化DNA纳米颗粒。该项目解决了癌症研究和治疗许多领域的重大挑战，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，存在模块的固有多价展示，允许亲合力补偿低单价亲和力。颗粒模板结构的模块化性质允许多个不同的识别元件组装成单个分子实体。此外，组合选择方法允许最佳颗粒在其将被使用的相同分子背景下进化。总的来说，我们的DNA纳米颗粒库的独特功能代表了细胞亲和试剂的新范式，其用高亲合力相互作用的多样化景观取代了与一个或两个定义的分子靶标的高亲合力结合。本申请的具体目标是：目标1。对多模块粒子组合选择方法进行了研究和优化。我们已经鉴定了与小鼠胰腺癌细胞系结合的单模块颗粒。我们将使用该细胞系优化多模块选择策略，并在两个人胰腺细胞系（MiaPaCa-2和Panc-1）以及一个白血病细胞系（K-562）上进行确认，以证明针对实体和液体肿瘤类型的可行性。目标2.证明所选颗粒的癌症特异性细胞结合。三个应用程序将被解决：组织学，流式细胞术，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。用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纳米颗粒的分子靶点。将采用两种方法。首先，我们将在将生物素化的DNA纳米颗粒交联到细胞表面分子后进行共沉淀实验。在第二种方法中，我们将在叠氮化物或炔标记的颗粒之间使用“点击”化学，以与用伴侣点击化学不加区别地标记的细胞或与在特定蛋白质修饰中含有伴侣化学的细胞特异性反应（例如，法尼基化蛋白质）。

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).