Aptamer-based Detection And Manipulation Of Protein Activity

Protein is an important biological macromolecule and the main undertaker of life activities.Protein participates in many biological processes,such as responding to external stimuli,maintaining homeostasis of the body,transmitting information,and catalyzing biochemical reactions.The activity of protein is specifically and dynamically regulated by cells.Dysregulation might cause diseases.Therefore,dynamic monitoring of protein activity is significant to understand various biological processes.Meanwhile,most fluorescent probes focus on the downstream product of protein activity,which possible lead to conflicting results.Genetic encoded biosensors allow the real-time monitoring of protein activity though,they involve a time-consuming and complicated preparation procedure.In order to ensure that the life activities of the organism can proceed in an orderly manner,the biochemical reactions and biomolecules in the organism must be precisely regulated.Protein activity can be regulated in many ways.For example,small molecule compounds can activate,inhibit and stabilize protein activity though,their applications are limited by low specificity.Optogenetic method has attracted a broad interest,but is often cumbersome and complicated and involved the modification of the protein.The development of easy-to-operate and reversible protein activity regulation methods will be beneficial.Aptamers are single-stranded nucleic acids obtained by an in vitro selection strategy termed systematic evolution technology of ligands with exponential enrichment(SELEX)on the basis of their specific recognition of target molecules.Aptamers have several advantages including easy chemical synthesis,easy modification,high designability,and good programmability,thus holding great potential for the detection and regulation of protein activity.In order to solve the above-mentioned problems,this thesis constructed a universal protein activity detection and regulation platform based on the excellent molecular recognition performance of nucleic acid aptamers,and successfully realized the real-time monitoring of the activation of endogenous proteins in living cells.At the same time,taking advantage of the easy modification of aptamers,we constructed a light-activated regulation probe and realized protein activity inhibition with high spatiotemporal resolution.The main contents are as follows:In the second chapter,we constructed an aptamer-based nanoprobe,and investigated its synthesis conditions and preparation methods.Subsequently,we tested the detection performance of the probe in the buffer solution,and demonstrated its potential to detect active proteins.In addition,the probe has the advantages of simple synthesis,fast response speed,and high detection accuracy.It is expected to provide a powerful analysis tool for protein activity detection.In the third chapter,we mainly introduced the endocytosis of the aptamer nanoprobe,including endocytosis kinetics,and lysosomal escape ability.We selected protein NF-?B as a detection targeting in living cells.We successfully used the nanoprobe to detect the protein activity in living cells.The ability of aptamer nanoprobes to detect intracellular protein activity was evaluated from multiple aspects.We also proved the specificity of our nanoprobe by comparing it with random sequence group.Besides,the pros and cons of the nanoprobe are also explored in comparison to the traditional immunofluorescence method.In the fourth chapter;we introduced light control on the nanoprobe for photo-responsive protein regulation.The function of aptamer was restored after specific light illumination,accompanied with the inhibition of the protein activity.In addition,the subcellular localization of the activated protein in the cell was investigated by immunofluorescence,which proved that the nano-platform could modulate the spatial positioning of the target protein.Consequently,we have successfully constructed a light-controllable nanoplatform for regulation of protein activity.