Construction Of Novel G-Quadruplex Fluorescent Probes Based On Fluorescent Protein Chromophores

Nucleic acid,a basic substance of life,are responsible for recording and transmitting the genetic information of organisms.G-quadruplexes（G4s）are formed from guanine-rich nucleic acid sequences by stacking of Hoogsteen bonded G-quartets.As a non-canonical nucleic acid secondary structure in the genome,G4s is widely distributed and involved in a variety of important physiological processes,such as regulation of transcription and translation,maintenance of genome stability and telomere homeostasis.Therefore,the development of tools to identify G4s is of great significance for exploring G4-related biological information.Among these tools,Organic fluorescent probes have attracted widespread attention due to characteristics of excellent photophysical properties and easy modification.It has become a significant research direction to design reasonable modification strategies for optimizing the binding capacity and spectral properties of probes.Currently,most studies have pursued the construction of red or near-infrared G4 probe by introducing an aromatic system into the core of the probe for deeper tissue imaging and avoiding the interference of biological autofluorescence.However,the enlarged aromatic conjugated structure usually makes the molecule prone to aggregation,limiting its water solubility,which is not conducive to further employment in biological systems.Considering binding ability,some research introduces a non-aromatic side chain into the core of the probe to effectively improve its binding affinity to G4s,but the effect on wavelength is limited.Our research is based on the intersection of analytical chemistry,organic chemistry and biochemistry,proposing a novel molecular modification strategy,designing a series of G4 fluorescent probes with excellent photophysical properties,and then realizing the sensitive detection of G4 structure and real-time imaging of cells.The specific research contents are as follows:（1）Design of novel modification strategy for constructing G4-specific red fluorescent probes.Green fluorescent protein chromophore（p-HBDI）is composed of phenol and imidazolidinone linked by methyl and vinyl bridge.Aiming at constructing G4 fluorescent probes based on chromophores,our research group previously reported a series of red and near-infrared G4 probes by introducing an additional aromatic ring at the C2 position of imidazolidinone,which would effectively enhance theπ-πstacking interaction between the chromophore and G-quartet.Based on those works,we proposed a novel modification strategy that introduces a non-aromatic side-chain module（nitrogen-dimethyl）at the imidazolidinone C2 in a conjugated extension manner.The introduction of this module is not only greatly enhanced the binding ability of the probe to G4s,but also successfully shifted the emission spectrum to the red region.Compared with the traditional Strategy that introduction of aromatic groups,G4 fluorescent probes obtained by this strategy exhibits higher fluorescence enhancement and stronger G4 binding ability.Fluorescence mechanism experiments,nuclear magnetic resonance spectra and molecular docking simulations show that the G4 probe NM-NMe₂ constructed by the novel modification strategy is binding to the3-terminal G-quartet of G4s via end-stacking mode,in which the nitrogen-dimethyl group at the C2 position of imidazolidinone counts matter for G4s.（2）Extending the novel modification strategy for construction G4 fluorescent probe library.We constructed a series of GFP chromophore analogs by modifying the benzene ring of chromophore,and further applied a novel modification strategy to obtain novel G4 probes NE-NMe₂,HN-NMe₂,JLND-NMe₂ and KZ-NMe₂.Compared with the original GFP chromophore,The novel G4 probes have 70-80 nm red-shifted in emission wavelength（560-630 nm）,large Stocks shift of 70-90 nm and enhanced G4binding ability.Among them,KZ-NMe₂ has a high signal-to-background response to G4s,up to 140 times（KZ-NMe₂/PU22）,and the fluorescence quantum yield is as high as 0.81,which has the potential to be used in detection and labeling.In addition,JLND-NMe₂has parallel G4 topology selectivity,which provides a powerful tool for G4conformation recognition.The successful construction of a series of high-quality G4probes with excellent photophysical properties confirms that the new modification strategy has good scalability and excellent modification ability,and is expected to be applied in more organic fluorescent probes for exploring G4-related biological research.（3）Visualization of intracellular G4s by G4 fluorescent probe based on novel modification strategy.In view of the excellent spectral properties and G4 binding ability of the above novel G4 probes,we further explored the cell imaging performance of NM-NMe₂,JLND-NMe₂ and the control probe NM-BNM constructed by traditional protocol.Cytotoxicity experiments confirmed that the novel G4 probe has excellent biocompatibility.The circular dichroism analysis showed that the novel G4 probe exhibit no inductivity and stabilizing effect on G4 structure,and thus couldn’t perturb the endogenous G4 folding dynamics,which was helpful for monitoring the native state of intracellular G4s.The cell imaging results showed that NM-NMe₂ exhibited bright red fluorescence in the nucleolus,while the control molecule NM-BNM only exhibited weak fluorescence bright spots in the cytoplasm,so NM-NMe₂ showed High-contrast imaging results compared to NM-BNM.Enzyme cleavage experiments,ligand competition experiments and Urea denaturation experiments confirmed that NM-NMe₂ and JLND-NMe₂ showed the characteristics of specific recognition of nucleolar G4s,which is expected to be used as nucleolar G4 probes to realize intracellular G4 monitoring.