Construction Of G-quadruplex Red Fluorescent Probes For Monitoring Of The Cellular Membrane Microenvironment

Cellular microenvironment is the local surrounding with which cells communicate by processing various biological signals,and by contributing their own impacts to this environment.Cell membrane microenvironment is closely related to various cellular physiology processes including cell adhesion,signaling,proliferation and differentiation,and thus real-time visualization of the interactions of cells with the local cellular environment can afford a better insight of physiological and pathological processes.Conventional assays,such as flow cytometry,enzyme-linked immunosorbent assay?ELISA?,immunostaining and polymerase chain reaction,usually require stepwise staining,washing or complicated manipulation prior to analysis.Alternative methods using chemical probes or nanoparticles that measure markers under"static"conditions,making them inappropriate for real-time detection in a dynamic manner.In recent years,small molecule fluorescent probes as powerful molecular tools have made significant progress in the field of cell membrane imaging,but they often suffered from shortcomings such as poor water solubility,easy internalization into the cytoplasm,and local accumulation of fluorophores.In summary,it is still a major challenge for monitoring and tracking biomolecules on cell membrane surfaces at high spatial and temporal resolutions.Nucleic acids,as genetic material of organisms,can fold to form a variety of secondary structures,such as duplexes,stem-loop structures,triplets,and G-quadruplex?G4?.G4 is a high-order structure formed by G-rich DNA or RNA in tandem repeat guanine.Four guanines form an annular plane through the Hoogsteen bond,and a plurality of such planar structures are chelated by metal ions to form G4.Based on this particular topology,G4 can achieve molecular therapeutics,diagnostics,and sensing by combining with different classes of targets,including enzymes,proteins,and small molecules.Among them,the fluorescence of some small molecule G4 probes is significantly enhanced after binding with G4,providing a new molecular tool for bioimaging and sensing.G4 can be obtained by commercial DNA solid phase synthesis and has excellent design ability,hydrophilicity and biocompatibility.The small molecule G4 probe has the characteristics of simple synthesis and easy structural modification.In this research,by binding with the advantages of G4 and small molecule fluorescent probes,cell membrane sensors based on G4/G4 probe complexes were constructed to solve the problems encountered in current cell membrane imaging.Firstly,inspired by the natural red fluorescent protein?RFP?luminescence mechanism,we designed a series of RFP chromophore analogues and found that they could bind with G4 for emitting red fluorescence,which was the first time for RFP mimick artificially.These DNA mimics of RFP exhibited attractive photophysical properties,and could be used for imaging of membrane proteins in live cells and deep tissue.Secondly,we introduced the reaction sites of SO₂ derivatives and NO into the core structure of the benzothiazole analogues of G4 probe,and combined with functional DNA to construct a ratio-type cell membrane sensor for in situ probing cellular extrusion process of endogenous signaling molecules,including SO₂derivatives and NO.The detailed research content of this paper are as follows:?1?Constructing RFP mimics based on G4/small molecules and studying their fluorescence characteristics.Based on the core structure of the native RFP chromophore,we synthesized six RFP chromophore analogues.Their own fluorescence was extremely weak,and increased greatly by the G4 encapsulating,which was the first time for mimicking RFP artificially.The RFP mimics?G4/RFP chromophore analogues?have maximum fluorescence emission wavelengthes of583-668 nm acrossing red and far red spectral regions,which perfectly match the existing RFP palette.They have excellent photophysical and chemical properties such as high quantum yield,large Stokes shift,excellent photobleaching resistance and two-photon fluorescence compared to natural RFP.Among them,the DFHBFSI-based RFP mimic has a quantum yield of 0.39 and a Stokes shift of 93 nm.In addition,RFP chromophore analogues,as special G4 probes,are selective for parallel G4 topologies and could be used for discrimination of parallel,anti-parallel and hybrid parallel G4topologies.This work not only points the way for the development of new topologically selective G4 probes,but also provides a new guiding for the development of a new generation of FP?fluorescent protein?simulation systems,including red,near-infrared and two-photon fluorescence.?2?Fluorescence emission mechanism of the RFP mimics and their applications in cell membrane protein imaging.Density functional theory?DFT?calculations indicated that the extremely low quantum yield of RFP chromophore analogues is due to the distorted intramolecular charge transfer?TICT?of the excited state molecules.Molecular dynamics?MD?simulations showed that the binding of RFP chromophore analogues with G4 greatly limited the TICT-induced chromophore-free radiation transition,resulting in a substantial increase in fluorescence emission.Based on the specific recognition of the target molecule by the aptamer,we further use d the RFP mimics for bioimaging of the cell membrane protein PTK7.Compared with the fluorescent protein tags,RFP mimics does not require complex genetic engineering.In addition,RFP mimics were resistant to photobleaching and could provide long-term real-time monitoring of live cell membrane proteins.Moreover,RFP mimics had good two-photon properties and could be used for deep tissue imaging.The experimental results showed that the tissue penetration depth was over 80?m.It can be used as a potential toolkit for biomedicalimaging and clinical diagnosis through integration of DNA RFP mimics with functional nucleic acids.?3?Construction of a ratiometric sensor based on G4/respon sive G4 probe.We introduced a reactive C=C double bond into the benzothiazole core for constructing a G4 probe 1a,which was capable of undergoing a Michael addition reaction with the SO₂ derivatives.Both 1a and the addition product were non-fluorescent,so 1a alone could not be used for detection of the SO₂ derivatives.The fluorescence was enhanced by 722 times at the maximum fluorescence emission wavelength?619 nm?after 1a combinding with G4 to form 1a-G4 complex.After addition of HSO₃^-to the complex,the fluorescence of the 1a-G4 complex decreased by 87.6%in 10 min,so that the1a-G4 complex could be a fluorescent probe for the SO₂ derivative sensing.We labeled a fluorescent molecule JOE as a donor at the G4 terminus and constructed a FRET sensor for selectively responding to HSO₃^-with a detection limit of 0.45?M.Using a similar strategy,we synthesized a G4 probe 2a and constructed a corresponding FRET sensor for NO measurement in micromolar concentration.This strategy of constructing sensors based on the combination of stimuli-responsive cofactors and functionalized DNA greatly expands the effective range of DNA probes.?4?Applications of the modular sensors for monitoring cell microenvironment.Based on the research content?3?,we developed a modular cell membrane sensor that could be used to monitor real-time dynamic changes of target molecules in cell membrane microenvironment.The sensor contain two components,a functional DNA motif and a synthetic stimuli-reactive cofactor.The functionalized DNA module provide a large?-planar structural scaffold?G4?,a FRET donor,and an positioning unit of cell membrane,and the stimuli-responsive cofactor motif provide a signal recognition unit and can bind with the G4.The fluorescence characteristics changed after the cofactor reacting with the signal molecule,with the FRET signal changes at the same time.In this paper,cholesterol was used as the inserting unit,and the sensors responding with SO₂ derivatives and NO were immobilized on the surface of HepG2 and Raw264.7 cells respectively,and the in situ monitoring of SO₂ derivatives and NO efflux process in living cells were achieved.Benefiting from the advantages of modular design,ratiometric cell membrane sensors for responding to multiple signal molecules can be constructed by designing responsive cofactors and coordinating the spectra of donor fluorophores.Therefore,the modular strategy developed here combines the advantages of nucleic acid molecules and small molecule probe s,providing a common method for constructing ratiometric cell membrane sensors.We envision that this modular sensor may represent a new strategy for"smart probe"design and provide potentially powerful tools for versatile applications in biomedical imaging and clinical diagnostics.