Cytosolic Access and Instability of DNA nanoparticles

项目总结/摘要：许多候选疗法，如CRISPR-Cas9和基因治疗， 沉默需要将功能性核酸有效递送到细胞胞质溶胶和细胞核。不幸的是， 这种疗法目前缺乏适当的输送机制，从而妨碍了它们的广泛应用。自我 组装的脱氧核糖核酸（DNA）纳米颗粒显示出潜在的最小细胞毒性 在癌症和其他体外和体内模型中的治疗载体。虽然有证据表明DNA 基于纳米颗粒的药物载体可以通过内吞作用被哺乳动物细胞摄取，这是未知的。 这些DNA纳米颗粒如何克服内吞作用引发的降解， 细胞质，并且一旦到达那里，就可以可控地保持稳定性。科学解释了 它们的行为和控制它们在细胞胞质溶胶中稳定性的机制将有可能使大胆的 工程治疗输送系统的进展。为此，拟议的工作有两个总体目标， 科学收益回报1，诱导内体逃逸和增强DNA的胞质可及性 纳米粒子的钙在其组装过程中的整合。收益2，确定 在不同类型的细胞胞质溶胶中的DNA纳米颗粒的分解和稳定机制。 创新技术将是PI培训计划的重点，并将实施以实现 项目目标，即多步Förster共振能量转移光谱，用于高分辨率 跟踪细胞内的DNA纳米颗粒和体外细胞显微注射，从而能够研究这些 纳米颗粒直接在胞质环境中。首先，基于DNA折纸的纳米管将被测试， 在补充钙缓冲液中的结构稳定性。此后，纳米管将用作载体， 将功能性RNA分子递送至代表性的荧光蛋白表达细胞，并检查 因为其胞质可达性和在经历胞吞作用后的蛋白质调节中的功效。二是小 (20具有分支结构和非规范核酸的DNA纳米颗粒将被嵌入 用多步FRET报告子测量结构完整性。这些DNA纳米粒子将 显微注射到活细胞胞质区，并测定其断裂。最后，胞质稳定性 这些DNA纳米颗粒将与不同类型的哺乳动物细胞相关， 变异性（肿瘤，免疫和其他细胞类型），以映射结构多样的DNA的作用 纳米粒子在靶向不同生理细胞中的作用。PI还将接受严格的培训 分析体外研究，实验室管理和多产的赠款写作过程，这将有助于 向独立研究项目的过渡该项目的成果将为 开发更生物相容的递送系统，特别是用于功能性核酸治疗剂 在细胞质中至关重要。

PROJECT SUMMARY/ABSTRACT: A number of candidate therapies such as CRISPR-Cas9 and gene silencing require the efficient delivery of functional nucleic acids to the cell cytosol and nucleus. Unfortunately, such therapies currently lack proper delivery mechanisms, precluding their widespread applicability. Self- assembled deoxyribonucleic acid (DNA) nanoparticles have shown potential as minimally cytotoxic therapeutic carriers in cancerous and other in vitro and in vivo models. While evidence suggests that DNA nanoparticles-based drug carriers can be taken up by mammalian cells via endocytosis, it is unknown how these DNA nanoparticles can overcome the fate of endocytosis-triggered degradation to reach the cytosol and, once there, can controllably maintain stability. With the enabling science explaining their behavior and mechanisms of controlling their stability in the cell cytosol it will be possible to make bold advances in engineering therapeutic delivery systems. To that end, the proposed work has two overarching scientific payoffs. Payoff 1, induce endosomal escape and enhanced cytosolic accessibility of DNA nanoparticles by the integration of calcium in their assembly process. Payoff 2, identify the rate of breakdown and mechanisms of stabilization of DNA nanoparticles in different types of cell cytosols. Innovative technologies will be the foci of the PI's training program and will be implemented to achieve the project goals, namely, multi-step Förster resonance energy transfer spectroscopy for high-resolution tracking of DNA nanoparticle inside the cell and in vitro cell microinjections enabling study of these nanoparticles directly in the cytosolic environment. First, a DNA origami based nanotube will be tested for structural stability in calcium-supplemented buffer. Thereafter, the nanotube will be used as a carrier for the delivery of functional RNA molecules to representative fluorescent protein-expressing cells and checked for its cytosolic reachability and efficacy in protein regulation after undergoing endocytosis. Second, small (20 nm) DNA nanoparticles with branched architecture and non-canonical nucleic acids will be embedded with multi-step FRET reporters for measuring structural integrity. These DNA nanoparticles will be microinjected into live cells cytosolic region and their breakage be determined. Last, the cytosolic stability of these DNA nanoparticles will be correlated with different types of mammalian cells with known cytosolic variability (tumor, immune, and other cell types) in order to map the role of structurally diverse DNA nanoparticles in targeting cells with different physiologies. The PI will also receive training in rigorous analysis of in vitro research, lab management, and the prolific grant writing process, which will facilitate their transition to an independent research program. Outcomes of this project will pave the way towards developing more bio-compatible delivery systems, specifically for functional nucleic acid therapeutic agents that are vital in the cell cytosol.