Programmable DNA Nanostructures as Biomedical and Structural Scaffolds

项目摘要/总结 DNA纳米技术为建筑结构提供近原子控制，并精确定位客人 分子如抗体、荧光团和配体，使它们潜在地可用于许多 生物应用，例如生物传感、药物递送、细胞调节和生物成像。但有 仍然有许多挑战需要解决的DNA纳米技术，以充分发挥其潜力， 应用.在这个建议中，我们集中在DNA纳米技术的两个主要发展领域，以解决 这些挑战：（1）创建用于治疗多系统疾病的稳健的多功能药物递送平台， 和（2）设计3D DNA晶体作为用于X射线结构测定的支架并表征这种 3D晶格使用新技术的连续飞秒X射线晶体学（SFX）。 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的多环 化合物（MPC）对多器官系统的作用并使用肌强直增强候选药物筛选 1型营养不良（DM 1）作为试验床疾病。我们的工作将提供这些小沟可量化的负荷 结合药物并彻底验证从细胞培养到临床前DM 1小鼠的药物递送效率 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DNA基序， 组装成具有不同腔体大小的3D DNA晶体，允许容纳不同大小的客人， 纳米颗粒到蛋白质。我们将通过编程晶体触点来提高晶体的分辨率， 结合化学修饰，并使用三链体展示蛋白质的大分子支架 使用PNA接头形成作为系链和肽的寡核苷酸（TFO）。我们将开发方法， 使用SFX对这些DNA基序的微晶进行结构分析，其中使用高密度聚乙烯（HDPE）收集衍射数据。 强度X射线自由电子激光器，消除了对大单晶，冷冻和辐射的需要 与传统晶体学相关的损伤。 这项拟议的研究超出了单一疾病或健康问题，使这项工作非常适合 R35最大化研究者研究奖（MIRA）在NIGMS。从长远来看，我认为我们的 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），并获得难以结晶的晶体学信息 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).