Study On Application Of SNAP-tag Fluorogenic Probe And Intracellular Protein Labeling

Protein is an important part of the cell and it is the main bearer of life activities.The type of protein is various,but many proteins that are unknown.Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique in living cells.Protein labeling plays an increasingly important role for the real-time visualizing and manipulating proteins in living cells.Numerous technologies to label proteins with chemical probes have been validated,mainly including genetically encoded fluorescent proteins(FPs),site-specific incorporation of non-natural amino acids and self-labeling protein tags.However,the application of FPs in vivo is sometimes limited by their relatively large size and the single fluorescence spectrum.Non-natural amino acid technology suffers from complex pre-genetically modified.Therefore,self-labeled protein labeling technology has been widely used to study the localization and dynamic function of proteins in living cells.To date,various protein tags have been developed to study proteins in living systems.SNAP-tag is the most prominent fusion tags,which specially reacts with O6-benzylguanine(BG)derivatives.Various fluorescent probes have been designed to rapidly and irreversibly react with the SNAP-tag,which make it widely application in drug monitoring,protein-protein interactions,fluorescent sensor and super-resolution microscopy.While these probes are very useful and well established,the washout of the unreacted probes is required before the imaging.Besides the tedious and time-consuming process,thiswashout requirement may potentially limit their application in real-time monitoring of molecular events such as receptor-ligand binding,endocytosis,trafficking and so on.In addition,the complete washout is difficult because of the accumulation of the synthetic probes in various organelles.Thus,to overcome this limitation,new fluorogenic probes with turn-on effect have been created.Using the SNAP-tag protein labeling technique,a novel fluorescent probe was used to label the protein in the body,and the distribution of the protein in the cell was monitored in real time under the condition of no-wash.Based on this,this paper mainly does the following words:Firstly,based on the environmental sensitivity and quenching mechanism of fluorophores,we designed and synthesized a series of fluorescent probes BGAN-R(R = 2C? 8C?12C?DM)based on 1,8-naphthalimide fluorophores.The results showed that BGAN-2C could react with SNAP-tag in a rapid and specific manner,and the fluorescence intensity was significantly enhanced by fluorescence detection and kinetic detection.Toxicity test indicated that the probe is nontoxic to the cells.So it is possible to rapidly label intracellular specific proteins.Subsequently,BGAN-DPA probes were designed and synthesized based on the photo-induced electron transfer mechanism.The results showed that BGAN-DPA could react with SNAP-tag in a rapid and specific manner,and the fluorescence intensity was significantly enhanced by fluorescence detection and kinetic detection.And the protein-probe complexes have a specific response to copper ions.Finally,BGAN-DPA was applied to the biomechanical study of HEK 293 cells.The labeling of intracellular proteins and he detection of copper ions have been successfully achieved without a wash-out procedure.In conclusion,we present the design and application of a1,8-naphthalimide-derived fluorogenic probe for labeling SNAP-tag fusion proteins and the detection of copper in living cells that does not require any washing procedure.