A New Type Of Fluorescent Molecule That Recognizes Misfolded Proteins

Background: Neurodegenerative diseases,such as Alzheimer's disease,Parkinson's disease,amyotrophic lateral sclerosis,etc,are extremely harmful to human health.Early detection can prevent disease progression and achieve favorable prognosis.However,due to limited current detection methods,most diseases are not discovered until the onset of the disease,leading to the miss of the best treatment period.The cause of neurodegenerative diseases is not very clear,but studies have found that these diseases are usually accompanied by protein aggregation caused by protein misfolding.As long as these abnormal aggregations are detected,measures can be taken to prevent the disease from progressing before clinical manifestations occur.Therefore,the role of fluorescent molecules in this regard has begun to attract attentions.Green fluorescent protein is the first fluorescent protein found in the cell.Modifying green fluorescent protein can make it specifically label certain substances in the cell.Modification of its chromogenic skeleton to generate various specific fluorescent molecules is currently the more popular method.Objective: Synthesis of a new molecular dye that recognizes misfolded proteins to help clinical diagnosis and prevention of diseases caused by protein misfolding.Methods: 1.Chemical method to synthesize chromophore skeleton of green fluorescent protein.2 Design a suitable structure and modify the periphery of the skeleton.3.The glycerol lysate,the fluorescence properties of the synthesized molecules are measured,and the appropriate excitation wavelength was selected.4.Centrifuge and dilute the solution to obtain a solution of egg white protein.Using the controlled variable method to measure the appropriate concentration of egg white protein and the concentration of small molecules added.5.10 kinds of small molecules were mixed with egg white protein and heated separately,measure the fluorescence absorption and intensity change,and select suitable molecules.6.The plasmid induces E.coli to produce SOD1(V31A)protein,and extracts and purifies the protein.7.Use the heating experiment of egg white protein and SOD1(V31A)protein to screen and evaluate the best product molecule.8.Breast cancer 231 cells were treated with drugs,artificially induced protein misfolding,and the selected molecules and commercially available dye molecules were added.In the case of live cells and fixed cells,confocal photographs were taken to observe the results.9.Ordinary fluorescence microscope for imaging and observation results.Results: 1.The heating centrifugation experiment of egg white protein and the heating experiment of mixed chelating agent of SOD1(V31A)proved that the molecule of this experiment is indeed bound to the misfolded protein 2.In the heating experiment of egg white protein,this experiment proved that the fluorescence substance 80 # which was substituted by tert butyl propionate had better fluorescence intensity and multiple change of intensity.3.In the heating experiment of SOD1(V31A)protein,we found that the most suitable fluorescence effect was found in 80 #molecule,which was substituted by tert butyl propionate 4.In the medicated cells,we observed that the addition of different drugs can cause the aggregation of different proteins.The product molecules designed and synthesized in this experiment bind to these proteins and generate a fluorescent signal.5.In this experiment,we observed that the target molecules we synthesized can be imaged in both live and fixed cells,while commercially available molecules cannot stain live cells.6.Even when using ordinary fluorescence microscope,obvious phenomena can be observed.The molecules synthesized in this experiment have no strict requirements on the instrument.Conclusion: 1.The 80 # molecule synthesized in this experiment can bind to misfolded proteins and generate strong fluorescence signals and signal changes.2.The molecules synthesized in this study can be directly imaged in living cells,and the operation is simple.3.The products of this experiment do not have strict requirements on the imaging equipment,and ordinary fluorescence microscopes can observe more obvious phenomena.