Cytosolic Access and Instability of DNA nanoparticles

DNA 纳米颗粒的细胞质进入和不稳定性

• 批准号: 10400170
• 负责人: Divita Mathur
• 金额: $ 9.29万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2022-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400170
• 关键词: Address; Animal Model; Architecture; Award; Behavior; Behavioral Mechanisms; Biological Models; Biological Sciences; Biomedical Engineering; Buffers; CRISPR/Cas technology; Calcium; Cancerous; Cell Culture Techniques; Cell Nucleus; Cells; Confocal Microscopy; Cytosol; DNA; Dictyoptera; Dose; Drug Carriers; Endocytosis; Engineering; Environment; Family suidae; Fibroblasts; Fluorescence Resonance Energy Transfer; Gene Silencing; Genes; Goals; Grant; Hypoxia; Immune; In Vitro; Kinetics; Learning; Ligands; Lysosomes; Mammalian Cell; Maps; Measures; Mentorship; Messenger RNA; Microinjections; Mus; Nanotubes; National Institute of Biomedical Imaging and Bioengineering; Nucleic Acids; Outcome; Pharmacology; Physiology; Process; Property; Protein Biosynthesis; Proteins; RNA; Regulation; Reporter; Research; Resolution; Role; Science; Series; Site; Small Interfering RNA; Spectrum Analysis; Structure; System; Technical Expertise; Testing; Therapeutic; Therapeutic Agents; Training; Training Programs; Transfection; Transformed Cell Line; Translations; Untranslated RNA; Vesicle; Work; Writing; base; biomaterial compatibility; cell type; cytotoxic; delivery vehicle; gene therapy; improved; in vitro Model; in vivo Model; innovative technologies; nanoparticle; neoplastic cell; nuclease; nucleic acid-based therapeutics; particle; physical science; programs; protein expression; receptor mediated endocytosis; recruit; self assembly; targeted treatment; therapeutic nanoparticles; tool; tumor; uptake

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.

项目概要/摘要：CRISPR-Cas9 和基因治疗等多种候选疗法 沉默需要将功能性核酸有效递送至细胞质和细胞核。 目前此类疗法缺乏适当的传递机制，妨碍了其广泛的适用性。 组装的脱氧核糖核酸（DNA）纳米颗粒已显示出具有最低细胞毒性的潜力 而有证据表明，癌症和其他体外和体内模型中的治疗载体。 基于纳米粒子的药物载体可以通过胞吞作用被哺乳动物细胞摄取，目前尚不清楚 这些 DNA 纳米颗粒如何克服内吞作用引发的降解的命运，以达到 细胞质中，一旦到达，就可以通过科学的解释来可控地保持稳定性。 它们的行为和控制它们在细胞质中稳定性的机制将有可能使大胆 为此，拟议的工作有两个总体目标。 科学回报 1，诱导内体逃逸并增强 DNA 的胞质可及性。 纳米粒子通过在其组装过程中整合钙来确定 2 的比率。 DNA 纳米颗粒在不同类型细胞胞浆中的分解和稳定机制。 创新技术将成为 PI 培训计划的重点，并将被实施以实现 项目目标，即用于高分辨率的多步福斯特共振能量转移光谱 跟踪细胞内的 DNA 纳米粒子和体外细胞显微注射，从而能够研究这些 首先，将测试基于 DNA 折纸的纳米管。 此后，纳米管将用作补钙缓冲液中的结构稳定性。 将功能性 RNA 分子递送至代表性荧光蛋白表达细胞并进行检查 其在胞吞作用后的胞浆可达性和蛋白质调节功效第二，小。 将嵌入具有分支结构和非规范核酸的 (20 nm) DNA 纳米颗粒 与多步骤 FRET 生产者一起测量这些 DNA 纳米颗粒的结构完整性。 显微注射到活细胞胞质区域并测定其破裂最后，确定胞质稳定性。 这些 DNA 纳米粒子将与具有已知胞质的不同类型的哺乳动物细胞相关联 变异性（肿瘤、免疫和其他细胞类型），以绘制结构多样化 DNA 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资助写作过程，这将有助于 他们向独立研究计划的转变将为该项目铺平道路。 开发更具生物相容性的递送系统，特别是功能性核酸治疗剂 在细胞质中至关重要。

项目成果

Tuning between Quenching and Energy Transfer in DNA-Templated Heterodimer Aggregates.

DNA 模板异二聚体聚集体中淬灭和能量转移之间的调节。

• 发表时间: 2022-03-31
• 作者: Chowdhury, Azhad U;Díaz, Sebastián A;Huff, Jonathan S;Barclay, Matthew S;Chiriboga, Matthew;Ellis, Gregory A;Mathur, Divita;Patten, Lance K;Sup, Aaron;Hallstrom, Natalya;Cunningham, Paul D;Lee, Jeunghoon;Davis, Paul H;Turner, Daniel B;Yurke
• 通讯作者: Yurke

Rapid DNA origami nanostructure detection and classification using the YOLOv5 deep convolutional neural network.

使用 YOLOv5 深度卷积神经网络进行快速 DNA 折纸纳米结构检测和分类。

• 发表时间: 2022-03-09
• 影响因子: 4.6
• 作者: Chiriboga, Matthew;Green, Christopher M;Hastman, David A;Mathur, Divita;Wei, Qi;Díaz, Sebastían A;Medintz, Igor L;Veneziano, Remi
• 通讯作者: Veneziano, Remi

Understanding Self-Assembled Pseudoisocyanine Dye Aggregates in DNA Nanostructures and Their Exciton Relay Transfer Capabilities.

了解 DNA 纳米结构中的自组装伪异花青染料聚集体及其激子中继转移能力。

• DOI: 10.1021/acs.jpcb.1c09048
• 发表时间: 2022-01-13
• 期刊: The journal of physical chemistry. B
• 作者: Chiriboga M;Diaz SA;Mathur D;Hastman DA;Melinger JS;Veneziano R;Medintz IL
• 通讯作者: Medintz IL

Understanding Förster Resonance Energy Transfer in the Sheet Regime with DNA Brick-Based Dye Networks.

使用基于 DNA 砖的染料网络了解片状体系中的 Förster 共振能量转移。

• DOI: 10.1021/acsnano.1c05871
• 发表时间: 2021-10-26
• 期刊: ACS nano
• 影响因子: 17.1
• 作者: Mathur D;Samanta A;Ancona MG;Díaz SA;Kim Y;Melinger JS;Goldman ER;Sadowski JP;Ong LL;Yin P;Medintz IL
• 通讯作者: Medintz IL

Determining the Cytosolic Stability of Small DNA Nanostructures In Cellula.

确定细胞中小 DNA 纳米结构的胞质稳定性。

• DOI: 10.1021/acs.nanolett.2c00917
• 发表时间: 2022-06-22
• 期刊: Nano letters
• 影响因子: 10.8
• 作者: Mathur, Divita;Rogers, Katherine E.;Diaz, Sebastian A.;Muroski, Megan E.;Klein, William P.;Nag, Okhil K.;Lee, Kwahun;Field, Lauren D.;Delehanty, James B.;Medintz, Igor L.
• 通讯作者: Medintz, Igor L.