Development Of S-Adenosyl-Lmethionine (SAM) Fluorescent Probes

S-adenosyl-L-methionine(SAM),a major donor of methyl groups in all organisms,participates in various metabolic processes and regulates the expression of genes.In recent years,more and more studies have shown that SAM is closely linked with epigenetic modification and the occurrence of a variety of diseases,and maintaining normal SAM levels is essential for many biological processes.Currently,SAM is mostly detected by high-performance liquid chromatography(HPLC),liquid chromatograph-mass spectrometer(LC-MS),enzyme linked immunosorbent assay(ELISA).It is difficult to detect the changes of SAM in the whole cellular metabolic regulation process,and precisely understand the specific regulatory mechanism of SAM.To study the specific effects of dynamic changes of SAM on organisms,it is necessary to develop a probe that can monitor the dynamic changes of SAM in living cells in real-time.In recent years,gene-encoded fluorescent probes relying on fluorescence resonance energy transfer(FRET)are favored by researchers due to their high sensitivity and low error,and have been widely used to detect a variety of signal molecules and metabolites,such as ATP,Ca2+,glycans,amino acids,ROS,and hormones,making a significant contribution to research in related fields.Therefore,we developed a fluorescent probe based on FRET principle,which can monitor the changes of SAM in cells in real time.Firstly,we screened five SAM-binding proteins as probe reporter proteins by searching SAM-binding proteins and analyzing and comparing the structures of SAM-binding proteins.Based on the binding mechanism of each protein to SAM,six probe vectors were designed and constructed,named SFP-1,SFP-2,SFP-3,SFP-4,SFP-5,and SFP-6,respectively.After preliminary screening in Escherichia coli(E.coli),we obtained two probes(SFP-5 and SFP-6)that responded to SAM.Furthermore,comparing the two probes in E.coli in terms of time and concentration of SAM addition and substrate specificity,SFP-6 was found to have better SAM response performance.Then,we constructed the eukaryotic expression vector of SFP-6 probe,and found that SFP-6 has a good expression in many kinds of mammalian cells.The SFP-6 probe was verified could detect changes of SAM concentration in HEK293 T cells.A cell line HEK293T-SFP-6 was constructed to stably express the probe using the Piggy Bac transposon system.By detecting the proliferation activity of wild type HEK293 T and HEK293T-SFP-6 cells,it was demonstrated that the stable expression of SFP-6 probe had less cytotoxicity.We constructed the overexpression vector of SAM synthase MAT2A(p PBCAG-MAT2A-Puro)and the MAT2 A interference vectors(p LKO.1-sh RNA1-MAT2A-puro and p LKO.1-sh RNA2-MAT2A-puro),and then transfected them into HEK293T-SFP-6 cells respectively,to detect the MAT2 A transcription level,protein level and SAM level on Day 1,Day 2,and Day 3 after transfection.By using Confocal Laser Scanning Microscope to photograph the fluorescence of cells on Day 3 and calculating and analyzing its FRET ratio value,it was verified that the SFP-6 probe could indicate the change of SAM concentration in overexpression or interference system with SAM synthetase MAT2 A.Finally,we cultured HEK293T-SFP-6 cells for 3 days using a culture system containing different concentrations of methionine.The fluorescence results of the probe showed that low concentration methionine significantly reduced the intracellular SAM content,and high concentration methionine also caused a slight decrease of intracellular SAM concentration,which was consistent with our mass spectrometric results.It is proved that SFP-6 probe can indicate the change of intracellular SAM concentration caused by different concentrations of methionine.After that,the key enzymes in the methionine metabolic cycle were analyzed,and MAT2 A,MAT2B,and SUV39H1 had high expression levels at low concentration methionine,while the expression of MAT2 A,MAT2B,SUV39H1,SAHH,and MTR genes were all down-regulated at high concentration methionine.In summary,this study developed a fluorescent probe that can monitor the SAM concentration in real time,which provides a monitoring means for studying the concentration changes of SAM in gene expression regulation and many metabolic processes,and has guiding significance for understanding the mechanism of SAM in biological processes.