Synthesis And Application Of Fluorescent Probe Based On Traditional Nucleic Acid Dye Thiazole Orange

G-quadruplex（G4）is an atypical nucleic acid secondary structure formed by the self-assembly of oligonucleotide sequences rich in tandem guanine（G）.At present,it was confirmed the existence of G-quadruplexes in human cells,and the formation sequences of G-quadruplexes are widely distributed in important regulatory regions of the genome,such as telomeres,proto-oncogene promoter regions,etc.So G-quadruplexes play an important role in many important biological processes,such as DNA transcription,reverse transcription,DNA replication,telomere extension,chromatin remodeling,and inhibition of cancer cell proliferation.At present,the G-quadruplex has become an important target for cancer treatment and anti-cancer drug design.Small molecule ligands and probes that can specifically recognize G-quadruplexes are closely related to the diagnosis of cancer and the development of anti-cancer drugs.There is a highly negative anion channel located in the center of the G-quadruplex.Many metal cations can enter the anion channel to further stabilize the G-quadruplex structure,and the same oligonucleotide sequence can self-assemble into different G-quadruplex structures under the induction of different metal cations.Because the G-quadruplex structure is easily affected by the external environment（metal ions,etc.）,it is widely used to construct various biosensors.The construction of these sensors often requires a probe that is sensitive to G4 conformational changes.Therefore,the development of G-quadruplex-specific ligands and probes is very important.Small-molecule fluorescent probes are widely used for the specific detection and imaging of G-quadruplexes because of their high sensitivity,convenience,and high selectivity.At present,many small-molecule fluorescent probes that can specifically recognize G-quadruplexes have been developed and reported.Thiazole Orange（TO）is a class of classic hemicyanine nucleic acid dyes.It has almost no fluorescence signal due to molecular internal rotation and hydrophobic aggregation in water.After binding to ds DNA or G-quadruplex,the fluorescence signal of TO is significantly enhanced due to the restriction of molecular internal rotation and de-aggregation,achieving a"light-up"fluorescence response to ds DNA or G-quadruplex.Since both G-quadruplex and double-stranded DNA can cause significant signal enhancement,TO is not selective for G-quadruplex,which limits the further application of TO in G4 analysis and detection and related fields.In addition,TO’s green light emission（530 nm）and small Stokes shift（20 nm）are also not conducive to its further application in cell imaging.To construct a fluorescent probe with higher selectivity to G-quadruplex and better fluorescence performance by structural modification of TO is an effective method to solve the above problems.Therefore,in this paper,three new thiazole orange derivatives（TOVJ,TO-TPA,CDs-TO）were synthesized through structural modification of the traditional small-molecule fluorescent nucleic acid dye Thiazole Orange.The specific work content is as follows:（1）A new type of thiazole orange styrene derivative TOVJ with D-π-A structure was designed and synthesized by introducing 9-vinyljulonidine into the ortho position of 1-methylquinoline in the thiazole orange molecule.Due to fluorescence quenching caused by intermolecular aggregation and intramolectional rotation,TOVJ has almost no fluorescence signal alone in aqueous solution,and only weakly enhanced fluorescence signal after interaction with single-stranded DNA or double-stranded DNA.However,when TOVJ binds with G-quadruplex,especially antiparallel G4,The fluorescence signal was significantly enhanced（maximum 2742 times）,Stokes shift reached 198 nm,and the maximum emission peak was located at 694 nm,indicating that TOVJ has a very high selectivity for G4,especially antiflat G4,compared with other DNA topologies.TOVJ showed high sensitivity to antiparallel human telomeres G4,with detection limits of Hum24 and 22AG Na⁺as low as 164p M and 231 p M,respectively.Cell co-location experiments showed that TOVJ could enter living cells,and the Pearson correlation coefficient with Mito-Tracker Red was0.89,indicating that TOVJ could target mitochondria.The results of DNase or RNase treatment showed that TOVJ could interact with RNA in the nucleus,and the fluorescence spectra of TOVJ interacting with RNA of different conformations showed that TOVJ showed high selectivity for G4 RNA compared with other topological RNA（ss RNA/ds RNA）.This indicates that the probe has the potential to be used for fluorescence analysis of intracellular RNA G4.（2）A thiazole orange styrene derivative with triphenylamine（TPA）structure was constructed by introducing a styrene group with TPA structure into the o-position of 1-methylquinoline in thiazole orange.Triphenylamine is a strong electron-donating group with a propeller structure.On the one hand,the introduction of triphenylamine constructs a conjugated molecule with D-π-A structure,resulting in the red shift of its maximum fluorescence emission peak to 725 nm and Stokes shift to 225 nm.On the other hand,it inhibits the intermolecularπ-πstacking.Thus,TO-TPA exhibits AIE properties in an aqueous solution.The results of competitive equilibrium dialysis showed that TO-TPA showed the greatest binding affinity and selectivity for parallel G4 compared with other DNA topologies.Co-location experiments showed that TO-TPA could enter living cells and localize in mitochondria.The cytotoxicity test results showed that the cytotoxicity of TO-TPA was significantly lower than that of TO,which is conducive to its further application in vivo.（3）Thiazole orange（TO）was covalently linked to the surface of CDs to construct a ratio-type nano-fluorescence probe（CDs-TO）with dual fluorescence emission signals.The fluorescence emission signal（535 nm）of the TO part of the probe was significantly enhanced after binding to parallel G4,and the enhancement was not obvious in the presence of antiparallel G4.The fluorescence emission signal（455 nm）of the CDs part was not affected by G4.The aptamer PS2.M formed a parallel G4 structure under the induction of K⁺.In the presence of parallel PS2.M,the fluorescence emission intensity of CDs-TO at 535 nm was significantly enhanced.However,since the binding ability of Pb²⁺to G4 was much higher than that of K⁺,with the addition of Pb²⁺,the parallel G4 structure originally stabilized by K⁺was gradually induced to form an antiparallel G4 structure,causing CDs-TO to fall off from G4.The fluorescence signal at 535 nm gradually decreased.Based on the above principles,the CDs-TO and the aptamer PS2.M were mixed to construct a ratiometric fluorescence sensing platform for Pb²⁺detection,which achieved a highly sensitive detection of Pb²⁺with a detection limit of 9.647 nM.