Construction And Bioimaging Of G-quadruplex Probes Based On Fluorescent Protein Chromophores

G-quadruplex（G4）is a noncanonical nucleic acid secondary structure formed by a guanine-rich sequence,which contains two or more G-quartets stabilized by Hoogsteen hydrogen bonding.The G4 structures are polymorphic and can be classified as parallel,anti-parallel and mixed topologies based on the orientation of the strands.G4 motifs in living organisms play important roles in a variety of biological processes,such as DNA replication,regulation of gene expression and stability and telomere biology.As the biological functions of G4are intensively studied,the visualization,as well as precise detection of G4structures and the dynamic changes in living cells and in vivo are important to elucidate the relationship between different G4 structures and functions in complex biological systems.A large number of fluorescent probes with excellent properties have been developed for the visualization of intracellular G4,establishing the basis for studying the distribution and function of G4 in living cells.G4 nucleic acids in organisms are a mixture of multiple G4 topologies,and the dynamics of G4conformations are closely related to their functions.Most current G4 probes respond to all G4 conformations and cannot achieve precise recognition of the specific topology or monogenic G4 sequence,especially in physiological environments where multiple types of G4 coexist.Therefore,the construction of probes that accurately identify the specific type of G4 structures is of great importance to study their related biological functions.Fluorescent protein chromophores have been widely used in the fields of aptamer and G4 nucleic acid imaging due to their excellent fluorescence properties.In this study,utilizing the fluorescent protein chromophore as the parent core structure and aiming to improve the selectivity and accuracy of the probes for G4 recognition,we constructed a series of G4 probes with excellent performance through molecular restructuring and modification,which can achieve precise recognition of the specific G4 topology or monogenic G4 sequence.The contents of th is research are as follows:（1）Construction of the parallel G-quadruplex probe and application for imaging in cells.Inspired by current strategies for the modification of fluorescent protein chromophores,we constructed the probe with the ability to specifically recognize parallel G4 by introducing the methyl group into the backbone of the fluorescent protein chromophore imidazolidinone.In addition,the probe possesses excellent properties such as low background fluorescence,emission wavelength redshift,and enhanced viscosity sensitivity.T he decrease in the rotational energy barrier of the probe after the methyl modification validated the principle of background reduction,and the theoretical calculations revealed the mechanism of the molecular emission redshift.This methyl modification strategy can be applied to a variety of fluorescent protein chromophore derivatives,and the spectroscopic results show that the constructed probes all can selectively bind parallel G4,providing an effective solution for the construction of probes that specifically recognize parallel G4.Furthermore,the ligand competition,the ¹H NMR s p ect ra,an d m ol ecu l a r d o ck i n g ex p eri m en t s o f th e T M B I⁺w it h G4 n u cl ei c aci d s rev eal ed t h e i nt eract i o n an d b i n d i n g p att ern o f t h e p ro b e wi t h p aral l el G4 was en d-s t ack i n g.Finally,the TMBI⁺probe was successfully applied to the imaging of RNA G4in living cells,possessing the potential to study intracellular G4 biological functions.（2）Construction of the dual-emission parallel G-quadruplex probe with an internal reference and investigation of its properties.Based on the rational design and modification of the green fluorescent protein chromophore structure,we have constructed a ratiometric G4 probe with an internal reference,NHCou I,by fusing the green fluorescent protein chromophore backbone with coumarin6H.The ratiometric G4 probe with an internal reference exhibited a green emission of constant intensity（490 nm）and a red fluorescence emission（613nm）that specifically responded to G4.The successful construction of the G4topological switch demonstrated that the probe can specifically respond to parallel G4.Additionally,the probe enabled rapid dual-color visualization of parallel G4.The ratiometric signal shielded the ratiometric probe from interference with its concentration and the viscosity disturbances in major subcellular organelles（50-130 c P）,allowing the probe to achieve the absolute intensity-independent,viscosity-independent,local probe concentration-independent,and excitation laser power-independent signal readout for the accurate identification of parallel G4.Theoretical calculations revealed the luminescence mechanism of the ratiometric G4 probe.Finally,we have applied the molecular design approach to the rhodanine backbone,demonstrating the versatility of this strategy and providing a new solution for the construction of functionalized G4 probes.（3）The probe with an internal reference for ratiometric signal transduction and precision imaging of G-quadruplex in cells.First,we utilized the ratiometric probe to construct the MMP-2 responsive PR_MMP-2 sensor,enabling highly accurate ratiometric detection of MMP-2.NHCou I showed strong resistance to photobleaching and weak cytotoxicity,confirming its good photophysical properties and biocompatibility.The ability of the probe to image DNA G4 in cells was demonstrated by enzymatic digestion,ligand competition,and immunofluorescence experiments.Additionally,imaging results of the ratiometric probe NHCou I and the classical single-emission G4 probe Th T at different concentrations in cells demonstrated the ability of the ratiometric probe to self-calibrate in concentration and to distinguish interfering signals.Based on the fragmentation properties of nucleic acids during apoptosis,the probe NHCou I was utilized to visualize the differences in G-quadruplex during apoptosis and ferroptosis for the first time,providing a novel perspective for studying nuclear changes during cell death and opening up a new avenue for high-fidelity and reliable analysis of G4 imaging studies.（4）Construction and imaging investigation of the fluorescent hyb ridization probe for the identification of monogenic G-quadruplex.We constructed a fluorescent hybridization probe Pu27T-NMNa PI that specifically recognized the target Pu27T sequence by connecting the G4 probe NMNa PI to the downstream adjacent sequence antisense strand of the target G4 strand via a click chemistry reaction.The oligonucleotide unit hybridized with the target G4 Pu27T sequence to provide a precise identification function and the G4 probe unit was used to light on the G4 sequence in this fluorescent hybridization probe.The results of fluorescence property studies showed that compared to the probe NMNa PI,the fluorescent hybridization probe Pu27T-NMNa PI enabled precise identification of the monogenic Pu27T sequence in the complex environment where multiple G4 structures coexisted,with the high affinity and low detection limit for the Pu27T sequence.The fluorescent hybridization probe Pu27T-NMNa PI successfully achieved the precise imaging of the monogenic Pu27T in the cellular environment,indicating that this probe holds great potential for application in the precise imaging research of G4 in organisms.