Cell Regulation Research Based On Functional Nuclear Acids Chimeric Receptor

Functional nuclear acids,including aptamers,DNAzymes and aptazymes,are nuclear acid molecules with functions beyond genetic storage,such as the specific recognition and highly efficient catalysis.They are obtained through in vitro selection and could specifically recognize target molecules for performing corresponding functions.Meanwhile,they have good biocompatibility,structure stability and rational sequence design.So,in most cases,they are used as artificial molecular recognition module,applied in the fields such as biosensing,biomimetic catalysis and bionanotechnology.As natural molecular recognition modules,receptors on cell membrane is one of the sensors that play an essential role on sensing signals from external environment.Generally,cells sense signals through the special interactions between receptors and ligands,activating the downstream signal pathway and leading to corresponding behaviors.Artificial receptors modification to control cell behaviors is of great importance in biomedicine and basic research field.As the number of natural receptor ligand pairs is very limited,modern biomedicine urgently requires that the cell membrane surface receptors own the ability to recognize new ligands.However,the related research is still in the primary stage.However,traditional receptors modification methods need genetic engineering techniques,which are limited by the difficulty,complexity and inefficiency of genetic modification.As artificial molecular recognition receptors,functional nuclear acids could endue cell receptors the ability to recognize specific target through non-covalent binding.Moreover,combining the programmable dynamic assembly characters of nuclear acids nanodevice,the regulation of receptor activation will be realized.Utilizing the molecular tools of functional nuclear acids,the specificities of receptors could be regulated by a controlled way without the need of any genetic modifications.In this research,we designed the bifunctional nucleic acids to modify the receptor to develop chimeric aptamer receptors.These chimeric aptamer receptors can recognize new orthogonal ligands and enable the cells to respond to these new ligands.This bifunctional nucleic acid contains two modules,the ligand recognition module and the receptor binding module.The recognition module uses aptamers or DNAzymes to recognize different molecules,while the receptor binding module is the receptor specific aptamer.Based on the dimerization induced activation,an important way of receptor activation,many new ligands molecules,including macromolecules,small molecules and ions,successfully activated the targeting cell signaling transduction pathway through the chimeric aptamer receptors,and further precisely regulated cell motility.Since different aptamers,which can specifically recognize different targets,can be obtained through in vitro selection,our method could endue receptors with the ability to recognize various ligands,ranging from small molecules to biomacromolecules,and even cells,which will have extensive promising prospects in applications.The main research contents are as follows:?1?Biomacromolecules-responding chimeric aptamer receptors were developed to regulate cell behaviors.Here,thrombin and c-Met are selected as model ligand and receptor,respectively.The natural ligand of c-Met is HGF,while thrombin is the orthogonal molecule for c-Met.Through the design of bifunctional nucleic acid,the thrombin-specific c-Met chimeric aptamer receptors were constructed.Our experiments demonstrated that thrombin could not only be recognized by c-Met chimeric aptamer receptor,but also induce the dimerization of c-Met.After dimerization,c-Met was phosphorylated and the related pathways were activated.The phosphorylation of two key downstream proteins,protein kinase B?Akt?and signal transducer and activator of transcription?STAT3?would induce the remodeling of actin,resulting in cell migration.The migration behavior of the chimeric aptamer receptor modified cells was detected after the addition of thrombin.We further replaced the thrombin aptamer with PDGF aptamer,realizing the cells to respond to PDGF.Hence,the design of biomacromolecules-responding chimeric aptamer receptor shows good universality,providing a universal method to satisfy the demand of biomedicine.?2?An aptamer based small molecule-responding chimeric aptamer receptor was developed to regulate cell behaviors.Here,a bifunctional nucleic acid-based DNA nanodevice was designed to construct the ATP-responding chimeric aptamer receptor?AR?.The experiments demonstrated that ATP could induce the dynamic DNA strand displacement on the cell membrane,resulting in the DNA-mediated chemically induced dimerization?D-CID?and phosphorylation of c-Met,followed by activation of the downstream signal pathways,and cell migration.Using microfluidic device that generate ATP gradient,not only golgi polarization,but also cell migration to the ATP gradient were observed in AR modified cells.This system could recruit cells to migrate towards the desired region,exhibiting the prospects for applying small molecules to cell therapy.?3?DNAzyme based small molecule-responding chimeric aptamer receptors were developed to regulate cell behaviors.Here,a DNAzyme sequence in the ligand recognition module was designed in the bifunctional nucleic acid to construct the L-histidine?L-his?-responding chimeric aptamer receptor?HR?.We demonstrated that L-his could induce cells to generate chemotaxis after modification the cells with HR.When the L-his DNAzyme was replaced by Zn²⁺DNAzyme?ZR?,the c-Met pathway could also be activated by Zn²⁺.The cells also exhibited chemotaxis and directed migration behavior in the Zn²⁺gradient.Based on the results above mentioned,a multicell microfluidic system was constructed,where the cells were modified with AR,HR,and ZR,respectively.It was found that input of the ligand molecule?s?could selectively control the cell behaviors,providing potential method to regulate cell behavior in tissue samples.?4?A sensitive,label-free and time-resolved luminescent aptasensor for proteins detection was developed based on the DNA-enhanced time-resolved luminescence of Tb³⁺and graphene oxide?GO?.We found that DNA could significantly enhance the long-lived emission of Tb³⁺.When the DNA–Tb³⁺complex was adsorbed onto GO,the luminescence of DNA-Tb³⁺was effectively quenched.The target protein combined with an aptamer to form a protein/DNA complex,where the 3D structure inhibited the efficient adsorption of DNA onto GO,and restrained the luminescence of DNA-Tb³⁺emission.Thrombin and a 29-mer anti-thrombin aptamer were employed as a model analyte and a recognition element.There is a good linear relationship between the luminescence of the aptamer-Tb³⁺complex and the thrombin concentrations from 1 to100 nM,and a low detection limit of 0.8 nM.Since the time-resolved luminescence can eliminate the unspecific background fluorescence,the proposed aptasensor has been successfully applied in complicated biological samples for thrombin detection.This novel strategy presents a potential universal method for detection of other molecules.