Photoluminescence Imaging Of Newly Synthesized Proteins In Living Cells Based On Bioorthogonal Reaction

Some important biological processes,such as cell growth,differentiation and metabolism,are regulated by protein synthesis.Mutation in newly synthesized proteins may cause serious diseases,such as cancers.Therefore,it is essential to develop a suitable method to observe the dynamic changes of the newly synthesized proteins in living cells.Bioorthogonal chemistry has emerged as essential tools to visualize specific biomolecules such as proteins,nucleic acids,glycans,lipids and protein posttranslational modifications.The most widely used bioorthogonal reaction is Cu(I)-catalyzed azide–alkyne cycloaddition(CuAAC),known as “click reaction”.During the past ten years,click reaction has made great progress in the imaging of newly synthesized proteins in fixed cells.However,the dynamic imaging of the new proteins in living cells is still facing many problems.In this study,we constructed a new method for the detection and analysis of newly synthesized proteins,and we want to establish new methods and develop new probes suitable for living cell imaging.The main contents of the study include three parts.1.We coupled noncanonical amino acid tagging with click reaction to label the newly synthesized proteins in normal cells and cancerous cells.We studied the influence of six different drugs on the protein synthesis.The results showed the differences of protein synthesis rate and spatial distribution in normal cells and cancerous cells,as well as their different respondings to anticarcinogen stimulation.This approach could help us to understand the growth of proteins and distinguish cancerous cells from normal cells,which would benefit the diagnosis and monitoring of cancer,as well as evaluating the curative effect of drugs.Based on this strategy,more significant biological process about newly synthesized biomolecules would be studied in-depth.2.Designing probes for real-time imaging of dynamic processes in living cells is a continual challenge.We successfully designed and synthesized a novel bioorthogonal probe of Ir-alkyne complex and achieved the dynamic imaging of the newly synthesized proteins in living cells by PLIM.This probe offers benefits of desirable deep-red to NIR emission,long stokes shift,excellent cell penetration,low cytotoxicity and good resistance to photobleaching.The large lifetime shifts of 414 ns based on click reaction has been successfully applied to discriminate the reacted probes from unreacted probes in living cells for the first time to eliminate the background interference without any washing steps.Importantly,the PLIM would be a promising method for the dynamic detection of newly synthesized proteins in living cells.Based on the unique properties of the Iridium complexes,it is expected to be used for more important issues in living cells.3.Based on the unique properties of the Iridium complexes and the significant advantages of PLIM,we develop a series of Iridium luminescent complexes as microsecond-scale viscosity probes for lifetime imaging in living cancer cells.Based on the good properties of the Iridium complexes,we realized the imaging detection of microviscosity in living cells,compared the changes of viscosity in normal cells and cancer cells stimulated by drugs,and dynamically detected the changes of intracellular viscosity in the process of cell apoptosis.Currently,we are expanding more other applications of this PLIM strategy to take full advantage of this kind of Iridium probes.