Functionalized DNA Self-assemblies For Chemical Regulation Of Cell Signaling And Communications

Cell chemical communication plays an essential role in programming higher-order biological behavior in multicellular organisms.The strategy for precisely regulated cell communication currently remains challenging in cell engineering.In cell chemical communication,the sender cells express transmembrane or secret soluble ligands to propagate information to the receiver cells,which can transduce receptor-mediated signaling to regulate cellular functions.During cell communication,the patterns of the ligand-receptor interaction determine the strength and duration of transmembrane signaling,which affect downstream signaling pathways and cellular behaviors.Exploring and perturbing the ligand-receptor interaction will facilitate our understanding of cell communication.Recent advances in synthetic biology have made it possible to engineer mammalian cells or artificial cells to achieve synthetic control over cell communication.However,most studies on engineered cell communication mainly focus on the communications within artificial cell populations or the crosstalk between mammalian cells.Therefore,there is an urgent need to develop chemical biology strategies to precisely modulate ligand-receptor interactions and chemical communication between artificial and living cells.Such strategies would bridge the information gap between inanimate and living forms of matter,deepening our understanding of the basic processes of cellular life.In this thesis,we harnessDNA nanotechnology and functional nucleic acids to construct functional DNA assemblies to precisely regulate cell signaling or chemical communication.To engineer the ligand-receptor interface,we develop the DOTA platform capable of precisely defining receptor oligomerization with prescribed valence,spacing and stoichiometry,realizing artificial control over cell signaling and response.For controllable communication between protocells and living cells,we fabricate DNA-assembled protocells that can respond to external stimuli,realizing the information perception,transmission and programming signaling and function of mammalian cells.The details are as follows:(1)DNA origami-enabled precisely regulated ligand-receptor interaction.In this chapter,we harnessDNA nanotechnology to develop a novel DOTA nanoplatform(mDOTA)capable of precisely organizing the valence and spatial distribution of RTK monomers to systematically explore and control RTK signaling.In mDOTA architecture,the mDOTA can equip m ALs to directly arrange receptor monomers to form oligomers,which presents an artificial oligomerization not existing in natural RTK activation.Moreover,the well-addressability of DOTA allows for the customization of oligomers with defined nanoscale distribution by adjusting interligand spacing in mDOTAs.We demonstrate that the multivalent effect and nanoscale spatial distribution of receptor monomers cooperatively regulate RTK activation.This study provides a general approach for exploring the relationship between spatial distribution and receptor activation to better understand the key parameters of receptor signaling and precisely customize receptor oligomerization to regulate receptor signaling.(2)Spatially reprograming activated receptor-dimers using DNA origami.Based on the DNA nanoplatform developed in the previous chapter,we further developed a DOTA nanoplatform(dDOTA)capable of precisely organizing the oligomer of the receptor dimer to systematically investigate and control receptor signaling and cellular responses.In the dDOTA architecture,dDOTA can be flexibly functionalized with divalent aptamer(d AL)to generate an oligomer composed of pre-organized receptor dimers,and we have proved that the multivalency further cooperatively elevates the signaling strength beyond that induced by natural RTK-dimerization.Moreover,dDOTA can define the nanoscale spacing of d AL for the customization of oligomers with defined nanoscale distribution.We demonstrate that multivalent effect and nanoscale spatial distribution cooperatively regulate receptor activation to elicit differential intracellular signaling and transcriptional response.Accordingly,we prove that dDOTA can be rationally engineered to switch cellular phenotype from epithelial to mesenchymal stage,associated with altered cellular behavior.This approach provides a general strategy to study receptor activation mechanisms and accurately control cellular functions,which is expected to promote regenerative medicine and tumor immunotherapy.(3)Artificial regulation of cell chemical communication by DNA protocell.In this chapter,we synthesize highly programmable DNA protocells by liquid-liquid phase separation of specific single-stranded DNA(ssDNA).The synthesized protocells can sense and transmit information,regulating mammalian cell behaviors.We integrate DNAzyme into the DNA shell of protocells as the responsive module,endowing the protocells with ion-responsiveness.Further,we embed DNA aptamer-based artificial ligands into DNA protocells as signaling molecules.In the presence of external stimuli,DNA protocells can deliver DNA messages to mammalian cells to promote receptormediated behaviors in a user-defined manner.In addition,multicellular aggregates can be artificially controlled using protocell-cell communication in the multicell mixture.Therefore,the protocell-enabled cell communication strategy will contribute to a better understanding and precise manipulation of the fundamental processes of cell communication.(4)Orthogonal control of cell-cell communications via the DNA protocell.Based on the method to synthesize DNA protocells in the previous chapter,we further engineer photo-responsive protocells,expanding the toolkit for regulating protocellcell communication.Light-responsive protocells are constructed by incorporating the AuNRs in DNA hybridization shells as the light-responsive module.Under NIR irradiation,the protocells respond to light and release DNA-based signaling molecules,which as the ligands to activate receptors for signal transduction in mammalian cells,regulating cellular behavior.Additionally,we demonstrate that ion-responsiveness and light-responsiveness can be integrated for orthogonally controlled release of cell signal transduction and cell behavior in multicellular populations.Our study provides an engineerable platform capable of orthogonal regulating protocell-mammalian communication,shedding light on precisely regulated cell-based approaches for future diagnostic and therapeutic applications.