Mechanical Modulation of Cell Migrations by DNA Nanoassemblies

摘要在这个项目中，我们将利用自组装DNA纳米结构的机械特性， 调节细胞迁移。细胞迁移代表了几乎所有生物功能的基本过程， 多细胞生物如果细胞迁移没有得到充分的调节，许多疾病就会发生。我们将 通过机械干扰跨膜细胞的侧向聚集或去聚集来调节细胞迁移 整合素受体，其是在配体（例如，RGD）与整合素受体结合之后的机械转导过程。 整联蛋白，其特征在于整联蛋白与对细胞运动重要的肌动蛋白纤维的结合。 我们将制造DNA折纸纳米组件，DNA纳米弹簧，用于这种调节。通过将离散的 当DNA螺旋束的延伸模板中的墩，DNA折纸模板将弯曲成螺旋的线圈。 当相邻的墩通过墩之间形成的紧凑的DNA连接体聚集在一起时，纳米弹簧。我们计划 使用这些DNA纳米弹簧作为纳米测力计， 力的集群和去集群的整合素受体，分别在细胞膜上。担任 力计，我们将首先确定光镊中纳米弹簧的弹簧常数。然后我们将 使用FRET染料对评估纳米弹簧在外力下的延伸变化。通过乘法 根据胡克定律，我们将揭示纳米弹簧所经历的力。 为了调节细胞迁移，我们将通过以下方式调整锚定在DNA纳米弹簧上的RGD分子的间距： 纳米弹簧在环境线索下的卷曲和展开。由于RGD可以与细胞上的整合素结合 在膜上，纳米弹簧上的RGD的间隔允许聚集/去聚集整合素受体。上 方法，我们将使用DNA i基序作为DNA纳米弹簧中的化学（pH）响应性接头。微酸性褶皱 连接子中的i基序，其卷曲纳米弹簧并缩短RGD与簇整联蛋白的间距。这样的 因此，纳米弹簧将抑制癌细胞在微酸性细胞外基质中的迁移。在pH 7或以上时， 纳米弹簧在双链体DNA在接头区域中形成后盘绕。当互补寡核苷酸 去除接头中的一条双链体，纳米弹簧展开，这延长了RGD 间距。这将促进细胞的迁移，如人类巨噬细胞，发挥重要作用，以愈合 皮肤伤口皮肤表面也允许使用的光没有深穿透局部应用 纳米弹簧对于这种光-机械调制，我们将在半导体中引入光敏偶氮苯基团。 DNA纳米弹簧的连接体。使用具有不同波长的光，偶氮苯发生顺/反 异构化这改变了相邻墩之间的DNA接头长度，通过卷曲或卷曲改变了RGD间距。 解开DNA纳米弹簧我们已经成功地证明了这种策略在细胞中的可行性 传播和迁移测定。在该提案中，我们将使用这些试验来测试纳米弹簧的效果， 不同的机械性能（目标1）对HeLa细胞（目标2）和人巨噬细胞（目标3）迁移的影响。

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).