Thursday, November 21, 2024
11/21/2024
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).
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
