FRET Sensor Based On Protein-nucleic Acid Chimera

As an efficient optical"molecular ruler",F(?)rster Re sonance Energy Transfer(FRET)has a wide range of applications in biological macromolecular interactions,immunoassays,nucleic acid detection,etc.The genetically encoded FRET sensor based on fluorescent protein has high sensitivity and non-invasive characteristics,which plays an important role in illustration how intracellular signaling is organized and regulated within cells,and achieves the visualization of signaling events with unprecedented temporal and spatial resolution.FRET requires a large overlap between donor emission and receptor absorption spectra,but fluorescent protein s that meet the demand is limited currently.Different combinations of chimera can combine the performance of a variety of materials,and we can achieve the purpose of complementing the advantages and disadvantages of each component through design,which not only enrich the functions of chimerism,but even bring new solutions to scientific research bottlenecks.In view of the rapid development of fluorescent protein mimetics,and the fluorescence activation of some fluorescent protein analogues after G4 encapsulation,the development of protein-nucleic acid chimeric FRET pairs is expected to further expand the construction of FRET sensors.Efficient,site-specific,and stable connection between proteins and nucleic acids is the key for the development of protein-nucleic acid chimeric FRET sensor.Herein,this paper intensively studied the function of duck circovirus protein(DCV),which belongs to the super family of HUH endonucleases.Due to its unique restriction endonuclease activity,ligase activity and protein nature,DCV is a very desirable module for covalent connection of target protein and specific nucleic acid sequence,through fusion expression to achieve the target protein.We designed this"protein-DCV-nucleic acid"modular protein-nucleic acid chimera as a FRET pair,in which the FRET donor was LSSm Orange,and the receptor was assembly product DNA(1a)of DNA and a small molecule dye(1a)synthesized in our previo us work.FRET pair based on protein-nucleic acid chimerism greatly expanded the choice of FRET donor and receptor.Cell membrane localization peptide and protease substrate peptide were further fused to the fusion protein through protein engineering technology,and the obtained sensor has the potential to realize in situ and real time detection of cell membrane specific protease activity,which further extended the application of such protein-nucleic acid-based FRET sensor.The specific works are as follows:1.Expression and functional analysis of DCV and LSSm Orange in vitro.By comparing the reaction efficiency between DCV and nucleic acid under different conditions,it was confirmed that a fast,stable and efficient covalent connection c ould occur between DCV and the specific DNA,and extension of the DNA at its 3?end did not affect its connection with DCV.LSSm Orange was cloned,expressed,and purified,and its fluorescent property was preliminarily studied.2.Construction and optimization of a FRET pair based on protein-nucleic acid chimera.The p ET28a-lssmorange-dcv recombinant plasmid was constructed and the LSSm Orange-DCV fusion protein was expressed and purified.Successful connection between LSSm Orange-DCV and DNA was achieved,and proved that fusion of other proteins at the N-terminal of DCV would not affect its activity.After addition of 1a,FRET signal can be detected from LSSm Orange-DCV-DNA(1a).By analyzing the three-dimensional structure of LSSm Orange,the C-terminal of LSSm Orange was truncated to reduce the distance between the donor and the receptor,and E_FRET of the FRET pair was raised from 18%to about 60%.3.FRET sensor based on the protein-nucleic acid chimera.According to our previous work,the electron-deficient structure of 1a can react with nucleophilic reagents.Here,we developed a new FRET sensing method based on the protein-nucleic acid chimera,which responded to HSO₃^-in a solution system with high specificity and high sensitivity.Meanwhile,membrane insertion peptide and proteolytic enzyme substrate sequence were introduced into the LSSm Orange?-DCV fusion protein.The resulting sensor can not only localize in the cell membrane,but also respond to the specific protease activity,and produce FRET signal.Our work lays a foundation for the design and application of the FRET sensing platfo rm based on protein-nucleic acid chimera.