Construction And Application Of Multifunctional Membrane-targeted FRET Probe Based On Protein Nucleic Acid Chimera

Cell membrane is an important barrier for intercellular communication.Biomolecules such as proteases,receptors,gas signal molecules,ions,and cytokines initiate cell signaling directly or indirectly on cell membrane.Development of detection tools that can monitor moleculecell secretion,metabolism,and intercellular communication in situ and in real time will help us deepen our understanding of cellular physiology and pathological processes,and develop new therapeutic and diagnostic methods.Cellular microenvironment where the cell membrane locates has complex components with weak and rapid changes.Therefore,it is still challenging to quantitatively measure the molecules on the cell surface in real time.Fluorescence resonance energy transfer（FRET）-based sensor,as a“molecular ruler”with high sensitivity,is widely used to monitor micro events such as intracellular ion concentration,protease activity,protein interaction and conformational change.Fluorescent protein（FP）-based FRET sensors have the advantages of high sensitivity and biocompatibility,and can realize visual detection with high spatial and temporal resolution in cells.FP-based FRET sensors are mainly composed of protein-protein coupled FRET pairs,and sensing modules can usually be designed between fusion proteins.FRET pairs are strict for the spectral overlap of donor and acceptor and the spatial distance between them,and currently available FP-based FRET pairs with superior performance are still limited.Aptamer-based FRET sensors are easy to be synthesized and designed,but they require expensive fluorophore labelling.The new alternative strategy is to develop fluorescent nucleic acid,a complex composed of aptamer and their small molecule ligands.Protein-nucleic acid chimeric FRET pairs,which integrate the diversity of protein functions and the programmability of nucleic acids,can provide more ideas for the design of novel multifunctional FRET biosensors.In order to develop a protein-nucleic acid chimeric FRET probe,we chose the large Stokes-shifted fluorescent protein m Ametrine as the donor and the fluorescent nucleic acid complex G4（1a）as the acceptor.DCV,a HUH-endonulease domain which can specifically nick and covalently connect single-stranded DNA（ss DNA）,worked as the protein-nucleic acid chimeric connecting element.The advantage of the protein-nucleic acid chimeric FRET pair is that both the protein part and the nucleic acid part can be designed as detection elements.The specific interaction between G4（1a）and HSO₃^-can quench the acceptor’s fluorescence and turn off FRET.In addition,protease substrate sites can insert into the fusion proteins of the FRET probe.In presence of the target proteases can hydrolyze the substrate site and move the receptor away,resulting the FRET signal turn off.Further,fusion of the intercalated peptide MIP to the protein module enabled the probe inserting on cell membrane,and constructed a multifunctional FRET probe for analyzing the biomolecules on cell membrane.Specific research is as follows:（1）Construction and optimization of the protein-nucleic acid chimeric FRET pair.We constructed the recombinant plasmid p ET28a-mip-m Ametrine-dcv and obtained the recombinant protein MIP-m Ametrine-DCV after prokaryotic expression and purification.The functions of the recombinant protein,including the fluorescent properties of m Ametrine and the ability of DCV to recognize and link ss DNA,were confirmed.Accordingly,we constructed the protein-nucleic acid chimeric FRET pair,called MIP-m Ametrine-DCV-G4（1a）,where m Ametrine is the donor and G4（1a）is the acceptor.By optimizing the reaction conditions and the protein sequence,the FRET efficiency of MIP-m Ametrine（s）-DCV-G4（1a）was increased from 47%to 67%.（2）Protein-nucleic acid chimeric FRET probe for the detection of HSO₃^-on cell membrane.In living cells,endogenous SO₂ participates in the regulation mechanism of cardiovascular system,and is converted into bisulfite（HSO₃^-）after metabolism.Therefore,monitoring the changes of HSO₃^-on cell membrane helps us to understand the pathological mechanism of related diseases.The organic ligand 1a of the fluorescent nucleic acid complex G4（1a）is in an electron-deficient group that can react with the nucleophile HSO₃^-to quench its autofluorescence.Hence,we applied the FRET pair to construct a FRET probe for the detection of HSO₃^-.The experimental results show that the FRET probe can specifically respond to HSO₃^-with the decreased FRET efficiency,and the detection limit as low as 0.34μM.Meanwhile,the FRET probe is anchored to the cell membrane by MIP,realizing the detection of HSO₃^-on the cell membrane surface.Therefore,the probe expected to be further applied to in situ analysis of Endogenous HSO₃^-on the cell membrane,which provides a potential tool for constructing a sensing platform anchored to cell membranes.（3）Protein-nucleic acid chimeric FRET probe for detection of the Furin activity on cellular membrane.The cell membrane protease Furin is up-regulated in various cancer cells and has been considered a cancer marker.Hence,monitoring its activity is of great significance for the early diagnosis and prognosis of the related diseases.Considering the designability of the fusion protein,we insert the Furin substrate sequence into the linker between m Ametrine（s）and DCV,and constructed a FRET probe MIP-m Ametrine（s）-Furin-DCV-G4（1a）that can specifically respond to Furin.When Furin specifically recognizes and cleaves the substrate linker,the donor moves away from the acceptor.The ratio of F_donor/F_acceptor can reflect the activity of Furin with the detection limit as low as 6.7×10^-6 U/μL.Meanwhile,the FRET probe also realized monitoring of Furin activity in complex physiological environments such as cell lysates.Therefore,the probe expected to be further applied to in situ analysis of Furin activity on the cell membrane.It lays the foundation for the construction of a multifunctional cell membrane biomolecule detection platform for in situ analysis.