SNAP-tag Targeted Fluorescent Labeling Of Proteins And Biological Application

Protein specific labeling technologies have been widely used in biological researches.Protein-tag methods labeled with small molecules have some advantages for labeling such as flexibility,specificity,stability and diversity of molecular structures and functions.They have attracted more and more attentions for specific tracing of proteins real-time in living cells.However,the potential biological applications of protein-tag methods still need to develop.To develop different protein target labeling systems and achieve specific labeling with novel functional fluorescent molecules are of great significance for the biological researches.In this research,various subcellular proteins were employed for construction of target labeling system based on SNAP-tag technology.Some fluorescent dyes and probes were applied for protein target biological detection and proteins dynamics analysis.(1)Subcellular protein specific detection of nitric oxide(NO)was achieved.A fluorescent probe TMR-NO-BG was used to obtain the specific covalent labeling of nucleus,mitochondria targeted and diffusely expressed SNAP-tag through the 06-benzylguanine(BG).The "o-phenylenediamine-locked" rhodamine spirolactam(TMR-NO)was used for highly selective(limit of detection 67.3 nM)and sensitive sensing of NO.Endogenous subcellular NO produced by immune stress in macrophage and apoptotic cells induced by etoposide were successful detected by this method.The strategy provides a tool for researches on subcellular biological function of NO.(2)Subcellular protein specific detection of micro-viscosity was achieved.A viscosity-sensitive fluorescent rotor BDP-V BG was used to obtain the specific labeling of SNAP-tag through the BG group.The fluorescence of rotor was enhanced for 53-fold after react with protein.The protein labeled probe BDP-V-SNAP was used for localized micro-viscosity sensing by two-photon excited fluorescent lifetime imaging.The probe was applicable for the measurement of different subcellular protein localized micro-viscosity(123-145 cP).The different changes of local micro-viscosity of histone H2B in apoptosis induced by three drugs were also characterized.The strategy should be useful for detecting of various proteins specific local micro-viscosity and dynamics.(3)Subcellular protein specific two-photon fluorescent imaging was achieved.Three naphthalimide-BG derivatives TNI-BG,QNI-BG and ONI-BG were used to obtain the specific labeling of the purified and bacterial SNAP-tag.The protein labeling in eukaryocyte cells demonstrated that the ONI-BG with a good two-photon absorption cross section(128 GM)can be used for specific labeling and two-photon excited fluorescent imaging of different subcellular protein fused SNAP-tag.The strategy provides method for the specific labeling of protein with two-photon fluorescent imaging technology.(4)A fluorescent ratiometric imaging system was constructed based on the tandem enhanced green fluorescent protein(EGFP)and SNAP-tag,which can be pulse labeling with a specific TMR-BG.The tandem EGFP-SNAP tag was applied to different subcellular targeted system such as peroxisome signal peptide PTS1,autophagy-related protein LC3B and miclear histone H2B.The ratiometric imaging was achieved by the comparison of red fluorescence(from pulse labeled TMR)to green fluorescence(EGFP).The strategy was applicable for ratiometric imaging and mialysis of peroxisome turnover and dynamics of autophagy process.The strategy was also used for ratiometric imaging and analysis of the regulation of histone H2B turnover under different conditions.