Design,synthesis And Bioimaging Of G-quadruplex Fluorescent Probes Based On Fluorescent Protein Chromophores

Nucleic acids are significant biological macromolecules,which store and transmit genetic information through replication,transcription,and translation.What’s more,nucleic acids are polymorphic and can fold into multiple secondary structures through Watson-Crick hydrogen bond and/or Hoogsteen hydrogen bond,including hairpin,triplexes,G-quadruplex(G4),and i-motif,etc.Among them,G4 is one kind of nucleic acid secondary structure with stacked multi-layer G-quartets formed by G-rich sequences.G4s widely exist in genomes and are thought to be involved in the regulation of diverse biological processes,such as DNA replication,transcription,and translation.Visualization of G4 is of great significance to clarify the structure and function of G4in complex biological systems.In recent years,research on organic fluorescent probes for specific recognition of G4 has achieved explosive growth due to the advantage of simple operation,easy modification,and good reproducibility.Various probes are designed through modifying the structural skeleton to meet different needs,which provide essential tools for real-time detection,in situ imaging,and physiological process tracing of G4.Slight variations of probes based on the same scaffold commonly result in different photophysical properties for G4 binding and cellular imaging manifestation.However,most reported module modification strategies still suffer from some issues,such as complex preparation processes and poor universality.Only a few near infrared G4fluorescent probes have been developed,and the interference of subcellular accumulation,moderate specificity,or poor photophysical properties prevents them from achieving G4 imaging in vivo.Owing to the excellent fluorescence properties of fluorescent protein(FP)chromophores,they have attracted particular interest and have been applied for lightening fluorescent aptamers and G4 structures.Using FP chromophores as core scaffolds,we designed and synthesized a series of G4 fluorescent probes according to the fluorescent principle of organic fluorescent molecules and chemical structure modification.These probes exhibit excellent photophysical properties,providing new solutions for bottleneck problems of G4 fluorescent dye.This research is the intersection of organic chemistry,analytical chemistry,biological science.(1)Construction of fluorescent probes for specific recognition of parallel G4 by modular modification.Inspired by the fluorophore of Ds Red,we developed a novel fluorophore 1c by introducing an oxime group into the GFP chromophore.Interestingly,subtle structural modulation resulted in its enhanced interaction binding to G4s and a significant bathochromic effect.What’s more,this probe exhibits excellent topological selectivity to parallel G4.Notably,due to the advantages of mild reaction conditions and simple synthesis steps,this strategy is further exploited for designing various FP chromophores and other G4 probe scaffolds(triphenylamine and triarylimidazole core).All the above modified fluorescent probes are capable of specifically recognizing parallel G4.Hence,we speculate that this modular modification strategy is promising for generalizing to a wide range of G4 fluorescent scaffolds.(2)Modular modified fluorescent probes for nucleic acid logic gate and intracellular G-quadruplex imaging.The ~1H-NMR experiment revealed that the probe3b binds with G4 via end-stacking mode.In addition,molecular docking results indicated there is a hydrogen bond between the oxime group and G4.Since the cations can regulate the conformation of PW17(a G4 sequence),we constructed two new logic gates based on the G4 conformation switch,which use the fluorescence signal of the probe 3b as the output.It provides a potent tool for the design of nucleic acid logic gates and label-free DNA logic nanoplatforms.We also demonstrate that our oxime-modified chromophore is a smart G4 dye competent for detection of the G4 structural switch in real-time.Finally,live-cell imaging suggested that 3b mainly stained the DNA G4 structure in nucleoli,providing a promising tool for exploring the biological function of intracellular G4.(3)Construction of infrared-emission G-quadruplex mimics of FPs(igMFPs).Based on our previous work,we propose a unique asymmetric donor-acceptor-acceptor(D-A-A’)framework of HBI-based chromophores with rationally designed electron-donating D moieties and additional electron-withdrawing A’moieties.The D-A-A’-configured design rationale enhances the photoinduced intramolecular charge transfer,leading to infrared-shifted spectra.Thus,we developed a series of near-infrared emission FP chromophores.Due to their conjugatedπ-ring system,the fluorescence of these chromophores(NIR-1,NIR-2,and NIR-3)can be elicited by specific interaction with G4.These G4/chromophore complexes,termed infrared G4 mimics of FPs(igMFPs),greatly expand the spectral palette to the near-infrared region with tunable emission maxima(664～705 nm),exhibiting tremendous potential in vivo imaging.Furthermore,igMFPs possess high quantum yields,large Stokes shift,and good photostability.The viscosity sensitivity results suggested that NIR-2 transformed more readily into a TICT state and required higher viscosity to restrict its rotation,leading to recognizing G4 with higher sensitivity and selectivity in a high-viscosity environment.Therefore,NIR-2 with high viscosity sensitivity is promising for high-contrast live-cell imaging.(4)Visualization of HCV G4 in living cells and in vivo via in-situ formation of igMFPs.Recent reports demonstrated a highly conserved G4 structure in the core genome of the hepatitis C virus.As a proof-of-concept application,we exploited HCV G4(termed CG2a)as the target to explore the imaging performance of igMFPs in cells and in vivo.NIR-2 is successfully applied to real-time monitoring of CG2a transcription kinetics in vitro and imaging of intracellular CG2a transfection via forming igMFPs.Furthermore,confocal imaging results proved that the selective recognition of the G4 structure in the HCV core gene by NIR-2 accounted for the formation of igMFPs,presenting a potential intrinsic fluorescent tag for tracking HCV RNA.Our igMFP system also could provided precise spatial information of HCV RNA genome,presenting a potent tool for real-time analysis of the subcellular distribution of viral RNA in living cells.Moreover,igMFPs are feasible for high contrast HCV RNA imaging in living animals bearing HCV RNA-presenting mini-organ.To our knowledge,this is the first time that FP mimics are employed for whole-body imaging to realize real-time visualization of endogenous G4 related to pathology in vivo.In addition,NIR-2 is also the first near-infrared G4 probe to achieve intracellular G4imaging in living animals.