Mechanical Modulation of Cell Migrations by DNA Nanoassemblies

DNA 纳米组件对细胞迁移的机械调节

• 批准号: 10659333
• 负责人: Hanbin Mao
• 金额: $ 34.16万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659333
• 关键词: 3-Dimensional; Abnormal Cell; Acidity; Acids; Actins; Address; Affinity; Autoimmune Diseases; Binding; Biological Assay; Biological Process; Blood Vessels; Cell Communication; Cell membrane; Cell physiology; Cells; Chemicals; Color; Competitive Binding; Complementary DNA; Cues; Cytoskeletal Proteins; DNA; Digestion; Disease; Distal; Dyes; Elements; Embryonic Development; Ensure; Environment; Enzymes; Equilibrium; Extracellular Matrix; Fiber; Fluorescence Resonance Energy Transfer; Focal Adhesions; Genetic; Growth; Hela Cells; Human; Image; Immune response; In Situ; Integrin Binding; Integrins; Isomerism; Knowledge; Lateral; Laws; Length; Ligand Binding; Ligands; Light; Linker DNA; Macrophage; Measurement; Measures; Mechanics; Membrane; Methods; Migration Assay; Monitor; Morphology; Motion; Nanostructures; Neoplasm Metastasis; Oligonucleotides; Organism; Pathway interactions; Penetration; Photons; Play; Process; Reporting; Role; Signal Transduction; Single-Stranded DNA; Skin; Skin wound healing; Specificity; Structure; Surface; Testing; Topical application; With laterality; alkalinity; azobenzene; biomaterial compatibility; cancer cell; cell motility; density; design; experience; fabrication; fighting; fluorophore; innovation; invention; laser tweezer; mechanical force; mechanical properties; mechanotransduction; migration; nano-objects; nanoassembly; nanodevice; nanometer; nanoscale; optic tweezer; photostimulus; receptor; self assembly; side effect; single molecule; skin wound; three dimensional structure; tool; tumor; wound healing

Summary. In this project, we will exploit mechanical properties of self-assembled DNA nanostructures to modulate cell migrations. Cell migration represents a fundamental process in biological functions of almost all multicellular organisms. Numerous diseases occur if cell migrations are not adequately regulated. We will modulate the cell migration by mechanically interfering with lateral clustering or declustering of transmembrane integrin receptors, which is a mechano-transduction process following the binding of the ligand (e.g. RGD) to the integrin and characterized by the association of the integrin to the actin fibers important for cell motions. We will fabricate DNA origami nanoassemblies, DNA nanosprings, for this modulation. By placing discrete piers in an extended template of DNA helix bundles, the DNA origami template will bend into coils of a nanospring when adjacent piers are brought together by compact DNA linkers formed between piers. We plan to use these DNA nanosprings as nanometer force gauges to monitor both compressive and tensile lateral forces for the clustering and declustering of integrin receptors, respectively, on cell membranes. To serve as force gauges, we will first determine the spring constant of nanosprings in optical tweezers. We will then evaluate the extension change of the nanospring under external force using FRET dye pairs. By multiplication of these two variables according to the Hooke’s law, we will reveal the force experienced by the nanospring. To modulate cell migration, we will adjust the spacing of RGD molecules anchored on DNA nanosprings by coiling and uncoiling of nanosprings under environmental cues. Since RGD can bind to the integrin on a cell membrane, the spacing of RGD on nanosprings allows to cluster/de-cluster integrin receptors. In the first approach, we will use DNA i-motif as a chemo(pH)-responsive linker in the DNA nanospring. Slight acidity folds i-motif in the linker, which coils the nanospring and shortens spacing of RGD to cluster integrins. Such a nanospring will therefore inhibit migrations of cancer cells in slightly acidic extracellular matrix. At or above pH7, nanosprings are coiled after duplex DNA is formed in the linker region. When complementary oligonucleotides are applied to remove one of the duplex strands in the linker, the nanospring is uncoiled, which elongates RGD spacing. This will promote migrations of cells such as human macrophages that play important roles to heal skin wounds. The skin surface also permits the use of the light without deep penetration on topically applied nanosprings. For this opto-mechanical modulation, we will incorporate light-sensitive azobenzene groups in the linkers of DNA nanosprings. Using the light with different wavelengths, the azobenzene undergoes cis/trans isomerization. This varies DNA linker length between neighboring piers, changing RGD spacing via coiling or uncoiling the DNA nanospring. We have successfully demonstrated the feasibility of this strategy in the cell spread and migration assays. In the proposal, we will use these assays to test the effects of nanosprings with different mechanical properties (Aim 1) on migrations of HeLa cells (Aim 2) and human macrophages (Aim 3).

概括。在这个项目中，我们将利用自组装DNA纳米结构的机械性能 调节细胞迁移。细胞迁移代表了几乎所有的生物学功能的基本过程 多细胞生物。如果细胞迁移不适当调节，就会发生许多疾病。我们将 通过机械干扰跨膜的横向聚类或解释来调节细胞迁移 整联蛋白受体，这是配体（例如RGD）与 整联蛋白的特征是整联蛋白与肌动蛋白纤维的关联对细胞运动很重要。 我们将针对此调节制造DNA折纸纳米夹，DNA纳米弹簧。通过放置离散 DNA螺旋束的扩展模板中的码头，DNA折纸模板将弯曲成一个线圈 当墩之间形成的紧凑型DNA接头将相邻的码头聚集在一起时，纳米弹簧。我们计划 使用这些DNA纳米串作为纳米力量测量值，以监测压缩和拉伸的侧面 整联蛋白受体在细胞膜上分别用于聚类和重聚物的力。作为 力量表，我们将首先确定光学镊子中纳米弹簧的弹簧常数。然后我们会 使用FRET染料对评估在外力下纳米弹簧的延伸变化。通过乘法 在根据胡克定律的这两个变量中，我们将揭示纳米弹簧所经历的力量。 为了调节细胞迁移，我们将调整通过通过 在环境线索下盘绕和解开纳米弹簧。由于RGD可以与细胞上的整联蛋白结合 膜，RGD在纳米弹簧上的间距允许聚集/簇整合素受体。在第一个 方法，我们将使用DNA I-MOTIF作为DNA纳米弹簧中的化学（pH）响应连接器。轻微的酸度折叠 链接器中的i-motif，该链接将RGD的纳米弹簧和缩短为群集整合素。这样的 因此，纳米弹簧将抑制略微酸性细胞外基质中癌细胞的迁移。在PH7上或以上， 在连接器区域形成双链DNA后，纳米弹簧盘绕。当完成寡核苷酸时 应用于卸下链接器中的双链链之一，纳米弹簧未覆盖，它会延长RGD 间距。这将促进细胞的迁移，例如人类巨噬细胞，这些巨噬细胞起着重要作用以治愈 皮肤伤口。皮肤表面还允许在局部施加的情况下使用光 纳米弹簧。对于这种选择机械调制，我们将在 DNA纳米弹簧的接头。使用具有不同波长的光，偶氮苯经历顺式/trans 异构化。这种多样的DNA接头长度在相邻墩之间，通过盘绕或 解开DNA纳米弹簧。我们已经成功证明了该策略在细胞中的可行性 传播和迁移分析。在提案中，我们将使用这些测定法测试纳米弹簧的影响 对HeLa细胞迁移（AIM 2）和人类巨噬细胞（AIM 3）的不同机械性能（AIM 1）。