Programmable DNA Nanostructures as Biomedical and Structural Scaffolds

项目摘要/摘要 DNA纳米技术为建筑结构提供了近原子控制，并对客人进行了精确的定位 抗体、荧光团和配体等分子，使它们在许多领域具有潜在用途 生物传感、药物输送、细胞调制和生物成像等生物应用。然而，有一些 DNA纳米技术要充分发挥其实用潜力，仍有许多挑战需要解决 申请。在这项提案中，我们将重点关注DNA纳米技术的两个主要发展领域 这些挑战：(1)创建一个强大的、多功能的药物输送平台，用于治疗多系统疾病， 以及(2)设计3D DNA晶体作为X射线结构测定和表征的支架 利用系列飞秒X射线结晶学(SFX)新技术的3D晶格。 在药物传递方面，我们将开发dna多面体作为药物载体，用于传递一类新的修饰多环化合物。 化合物(MPC)对多器官系统的作用及利用肌紧张素加强药物候选筛选 营养不良1型(DM1)是一种试验性疾病。我们的工作将为这些小凹槽提供可量化的载荷 结合药物和彻底验证从细胞培养到临床前DM1小鼠的药物传递效率 模型，建立细胞内化，缺乏毒性和免疫反应，细胞和疾病特异性 载药DNA纳米结构的靶向性、生物利用度和生物分布。 为了开发DNA纳米结构作为结构支架，我们将设计和构建 组装成具有不同腔大小的3D DNA晶体，允许接待不同大小的客人，范围从 从纳米颗粒到蛋白质。我们将通过编程晶体触点和提高晶体的分辨率 结合化学修饰并使用三链演示蛋白质的大分子支架 形成寡核苷酸(TFOS)作为系绳和使用PNA连接物的多肽。我们将开发出增长的方法 这些DNA基序的微晶用于使用SFX进行结构分析，其中衍射数据是使用High 强度高的X射线自由电子激光，消除了大单晶、冷冻和辐射的需要 与传统结晶学相关的损伤。 这项拟议的研究超越了单一的疾病或健康问题，使这项工作非常适合 R35 NIGMS最大限度地提高调查人员研究奖(MIRA)。从长远来看，我设想我们的 使用DNA纳米结构的模块化平台技术可以被其他实验室用于不同的疾病 治疗和药物筛选(药物输送)，并获得难以结晶的结晶学信息 分子(大分子支架)。

PROJECT ABSTRACT/SUMMARY DNA nanotechnology offers near-atomic control for building structures, with precise positioning of guest molecules such as antibodies, fluorophores and ligands that make them potentially useful in a number of biological applications such as biosensing, drug delivery, cell modulation and bioimaging. However, there are still many challenges that need to be addressed for DNA nanotechnology to reach its full potential for practical applications. In this proposal, we focus on two main areas of development in DNA nanotechnology to address these challenges: (1) Creating a robust, multifunctional drug delivery platform for treating multisystemic diseases, and (2) designing 3D DNA crystals as scaffolds for X-ray structure determination and characterization of such 3D lattices using the new technique of Serial Femtosecond X-ray Crystallography (SFX). For drug delivery, we will develop DNA polyhedra as drug carriers for delivering a new class of modified polycyclic compounds (MPCs) to multiple organ systems and enhancing drug candidate screening using myotonic dystrophy type 1 (DM1) as a testbed disease. Our work will provide quantifiable loading of these minor groove binding drugs and thorough validation of drug delivery efficiency from cell culture to preclinical DM1 mouse models, establishing cell internalization, lack of toxicity and immune response, cell- and disease-specific targeting, bioavailability and biodistribution of the drug-loaded DNA nanostructures. For developing DNA nanostructures as structural scaffolds, we will design and construct DNA motifs that assemble into 3D DNA crystals with different cavity sizes that allow hosting guests of different sizes ranging from nanoparticles to proteins. We will improve resolution of the crystals by programming crystal contacts and incorporating chemical modifications and demonstrate macromolecular scaffolding of proteins using triplex forming oligonucleotides (TFOs) as tethers and peptides using PNA linkers. We will develop methods to grow microcrystals of these DNA motifs for structural analysis using SFX, where diffraction data is collected using high intensity X-ray free-electron lasers, that eliminate the need for large single crystals, freezing, and radiation damage associated with traditional crystallography. This proposed research extends beyond a single disease or health issue, making this work well-suited for the R35 Maximizing Investigators’ Research Award (MIRA) at the NIGMS. In the long-term, I envision that our modular, platform technology using DNA nanostructures can be adopted by other labs for different disease treatments and drug screening (drug delivery) and to obtain crystallographic information of hard-to-crystallize molecules (macromolecular scaffolds).