Novel FRET Biosensors Based On Reactive Protein-tagged Ligands

Enzymes are biological macromolecules with high specificity and catalytic function for substrates produced by living cells,and their essence is protein or RNA.Enzymes not only maintain the normal metabolic functions of organisms,but also serve as biomarkers to diagnose many diseases.Due to the complex environment in organisms and the different abundances of various enzymes,it is of great significance to develop sensitive methods for the specific detection of enzyme activity.Fluorescent sensors have been widely applied in biology,medicine,pharmacology and other fields because of their high sensitivity and good biocompatibility.Among them,fluorescent proteins-based F?rster resonance energy transfer(FRET)ratiometric sensors are suited for imaging in complex matrix environments such as cells.However,protein engineering techniques are needed to re-optimize the domain of the sensors to detect different targets.Combined with the advantages of fluorescent protein sensors and the modular idea of small molecule probe modification,this thesis developed a FRET sensor without protein molecule transformation.Because of the non-covalent and reversible binding of the Fluorescence-Activating and absorption-Shifting Tag(FAST)to the ligand molecule,as well as the features of easy engineering of 4-hydroxy-3,5-dimethoxybenzylidene rhodanine(DOM)molecule,we used it as an acceptor for FRET pairs.In order to satisfy the conditions for constructing a FRET sensor,we selected the green fluorescent protein(GFP)whose emission spectrum overlaps with the FAST:DOM excitation spectrum and has a high quantum yield as the FRET donor.Through rational design and simple modification of small molecules,sensitive detection of different enzymes was achieved.The key finds are as follows:1.In vitro expression and FRET efficiency of GFP-FAST.In this section,the GFP-FAST recombinant plasmid was successfully constructed by homologous recombination technology,and the expression conditions of the fusion protein in vitro were optimized.By varying the temperature,the sensors were found to provide stable FRET efficiencies at 25-42°C.Changing the solution p H impacts FRET efficiency,mainly because the acidic environment inhibits the deprotonation of DOM molecule and affects its binding to FAST.Comparing GFP-FAST sensors with different ligation lengths,it was found that 12 amino acids were used to connect GFP and FAST resulted in optimal FRET efficiency between the two proteins.By K_D value measurement,it is found that the fusion of GFP slightly reduces the affinity between FAST and DOM.2.Ratiometric detection of alkaline phosphatase(ALP).By modifying a phosphate ester group on the hydroxyl group of DOM not only hinders the binding of DOM to FAST,but also explicitly initiates the energy transfer between GFP and FAST under the action of alkaline phosphatase.Through a series of conditions optimization,there was a good linear relationship between the sensor ratio signal and the concentration in the range of 5-60 U/L,and the detection limit was 0.71 U/L.Finally,GFP-FAST was used to detect alkaline phosphatase activity in the lysates of four cancer cells,and the results indicated that the sensor has the potential to detect ALP activity in complex biological samples,laying the foundation for cell imaging in the future.3.FRET sensor for tyrosinase.Initially,we continued the molecular design idea of detecting alkaline phosphatase by modifying the m-hydroxyphenol substituent on the DOM molecule as a tyrosinase recognition site.However,fluorescence spectra showed that after TYR catalyzed substituent leaving to release DOM,it would continue to react with the 4-hydroxy-3,5-dimethoxybenzylidene group of DOM molecules,which was also verified by UV-Vis absorption spectra.So far,we have found a new recognition moiety for specific detection of tyrosinase activity.Unlike the classic substrate,in this moiety,the ortho positions of the phenolic hydroxyl group are occupied by two methoxy groups.Interestingly,TYR maintained catalyst activity towards it.TYR was subsequently detected using GFP-FAST sensor,with a good linear relationship between concentration and ratiometric signal in 2-15 U/m L,with a detection limit of 0.54 U/m L.The sensor constructed based on the fluorescent protein GFP and the protein-tagged FAST provides a new idea for designing protein FRET pairs.The experimental results show that the sensor is suitable for detecting a variety of enzymes,which is expected to be developed into a general-purpose intracellular imaging tool.