Nucleic acid probes based on covalent coupling of aggregation-induced emission agents and phase-separated peptides and their applications

Abstract

The present invention provides a nucleic acid probe and application based on the covalent coupling of an aggregation-induced luminescence agent and a phase separation peptide. Among them, the phase separation peptide is composed of a nucleic acid binding domain of a repeated amino acid sequence connected in series. After the nucleic acid probe is combined with the nucleic acid, a liquid condensate with a size between 100nm-100μm and significantly enhanced fluorescence can be formed. The fluorescence signal intensity of the liquid condensate is positively correlated with the nucleic acid concentration in the environment, and the fluorescence signal can change dynamically and reversibly with the change of the nucleic acid concentration in the environment. The present invention proposes a method for constructing a fluorescent probe based on the supramolecular condensation phase change of a phase separation peptide, which improves the fluidity, dispersibility and real-time dynamic change ability of the fluorescence signal of the probe, and can be used for continuous monitoring of nucleic acids.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid probes, and in particular relates to a nucleic acid probe based on covalent coupling of an aggregation-induced luminescence agent and a phase separation peptide and an application thereof. Background Art

[0002] Aggregation-induced emission (AIE) effect refers to the phenomenon that some organic luminescent agents exhibit weak fluorescence in a dispersed state and enhanced fluorescence in an aggregated state. AIE luminescent agents are widely used as bioimaging agents, drug carriers, and molecular probes for medical applications such as photodynamic therapy and photothermal therapy of tumors. However, most existing AIE luminescent agents have the problem of poor hydrophilicity. It is a common practice in this field to use polyethylene glycol PEG or hydrophilic peptides to modify the surface of AIE luminescent agents and then improve their dispersibility in aqueous solution. However, the modified AIE luminescent agents often show a decrease in luminescence efficiency, or solid nanoparticles are precipitated after aggregation, resulting in a significant decrease in solubility and dispersibility.

[0003] Coacervation (or "liquid-liquid phase separation") refers to the phenomenon that biological macromolecules such as proteins precipitate from aqueous solutions after being assembled with each other, and finally form water / water emulsions. Coacervation is widely present in the metabolic activities of cells. For example, membraneless liquid organelles, nucleoli, pressure bodies, etc. all rely on the coacervation between "phase separation proteins" and RNA to form coacervates, which then regulate various cellular functions such as biochemical reaction rates, transmit cell signals, and regulate gene expression. Phase-separating peptides are new disordered protein structures or disordered polypeptide fragments discovered in recent years. They are composed of polar amino acids with simple repeating sequences, glycine, and a small amount of aromatic amino acids. After the phase-separating peptides bind to RNA, coacervates can be formed with a size between 100 nanometers and 100 microns. Different from traditional solid micro-nano particles, coacervates exist in the form of micro-nanoscale liquid phases, and have good water solubility, dispersion stability, and sensitive environmental responsiveness.

[0004] The prior art uses amphiphilic polypeptides to modify AIE luminescent agents as nucleic acid probes. The amphiphilic polypeptide molecules that modify the AIE luminescent agent contain a hydrophobic tail and a hydrophilic head. When nucleic acids (RNA or DNA) are present in the environment, this type of probe mainly relies on the electrostatic interaction between the polypeptide probe and the nucleic acid, as well as the hydrophilic and hydrophobic forces between the probes to complete the assembly of aggregates to form nanoscale solid particles. The solid particles enhance the fluorescence of the AIE luminescent agent.

[0005] Reference: https: / / pubs.acs.org / doi / full / 10.1021 / acs.accounts.9b00348

[0006] Xia F, Wu J, Wu X, et al. Modular design of peptide-or DNA-modifiedAIEgen probes for biosensing applications. Accounts of chemical research, 2019, 52(11): 3064-3074.

[0007] Traditional amphiphilic peptide-modified AIE probes rely on the hydrophilic and hydrophobic forces between probe molecules to complete aggregate assembly, which leads to the formation of solid particles after the probes are enriched in cell lysosomes.

[0008] On the one hand, it is difficult for the solid particles to escape through the lysosomes and enter the cell nucleus, resulting in low efficiency of the probe in detecting nucleic acids in the cell nucleus.

[0009] On the other hand, the solid particles reflect that the binding between the probe and the nucleic acid is irreversible. When the nucleic acid concentration in the environment changes, the probe that has participated in the formation of solid particles cannot track and reflect the dynamic changes of the nucleic acid concentration in the environment in real time. Summary of the invention

[0010] The present invention aims to address the problem in the prior art that AIE-type nucleic acid probes form solid aggregated particles after combining with the nucleic acid to be tested, resulting in reduced dispersibility and penetration of the particles into biological tissues, or the inability to monitor changes in intracellular nucleic acid concentrations in real time and dynamically. The purpose of the present invention is to provide a nucleic acid probe or nucleic acid carrier with strong penetration that forms droplet-state condensates after combining with RNA.

[0011] To achieve the above object, the solution of the present invention is:

[0012] The AIE luminescent agent is covalently coupled to the phase separation peptide, and the phase separation peptide undergoes a condensation phase transition after binding to the substrate, and then produces emulsion droplets to achieve high fluidity of the probe, reversibility of binding to the substrate, and enhancement of its own luminescence signal. The phase separation peptide is composed of repeating amino acids with a relatively simple sequence, and its sequence structure contains a nucleic acid binding domain, such as the amino acid sequence (RGG)n, (RGGYS)n, (RRASL)n, etc. Where n is the number of repeating units, and the value is between 2-10. The phase separation peptide is a hydrophilic short peptide, and the common proportion of polar amino acids and glycine should be higher than 50%. The AIE luminescent agent used refers to a luminescent agent with aggregation-induced emission effect.

[0013] Preferably, the phase-separated peptide surface in the nucleic acid probe generally exhibits a positive charge.

[0014] Preferably, there is a strong cation-π conjugation effect between the phase-separated peptide in the nucleic acid probe and the AIE luminescent unit.

[0015] Preferably, the AIE luminescent agent in the nucleic acid probe is tetraphenylethylene (TPE) and its chemically modified products.

[0016] When the probe combines with the nucleic acid to be detected, a droplet-shaped luminescent aggregate with a size between 100nm and 100μm is formed. The droplet-shaped luminescent aggregate can flow or break under external shear stress to form extremely tiny "water-in-water" emulsion droplets.

[0017] The intensity of the fluorescent signal of the nucleic acid probe is positively correlated with the concentration of nucleic acid (RNA or DNA) in the environment.

[0018] When the concentration of nucleic acids in the environment changes, the fluorescence signal can undergo dynamic and reversible changes.

[0019] A nucleic acid probe based on covalent coupling of an AIE luminescent agent and a phase-separated peptide as described above is used as an intracellular RNA developer in tumor cell imaging.

[0020] An application of a nucleic acid probe based on covalent coupling of an AIE luminescent agent and a phase-separated peptide as described above in a fluorescently indicated RNA vaccine vector and an mRNA drug vector.

[0021] Specifically, the "AIE luminescent agent-phase separation peptide covalent conjugate" of the present invention is used as a probe for monitoring nucleic acid (RNA or DNA), or as a fluorescent protein carrier carrying nucleic acid, and is used for RNA vaccine vectors, mRNA drug carriers, etc. with fluorescence indication.

[0022] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0023] The fluorescent probe of the present invention forms a droplet-shaped complex after combining with nucleic acid, which better maintains the fluidity, dispersibility and membrane penetration ability of the probe. The fluorescence intensity of the droplet-shaped complex can produce real-time dynamic changes with the change of nucleic acid concentration in the environment, and can be used for continuous monitoring of nucleic acids; its combination mode is dynamic reversible combination, which can be used for real-time and continuous monitoring of nucleic acid concentration in the environment.

[0024] The present invention couples a phase separation peptide with an AIE unit to synthesize a nucleic acid molecule probe, and utilizes the condensation phase change of the phase separation peptide to enhance the fluorescence signal of the AIE luminescent unit. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1a) Fluorescence microscope photo shows that the phase separation peptide probe (bright color marker) combines with RNA (gray color marker) to form an emulsion-like luminescent condensate; b) The condensate exhibits the high fluidity characteristic of emulsion droplet fusion; C) When the RNA concentration in the solution environment changes continuously, the phase state of the condensate changes accordingly. It appears droplet-like at low RNA concentration and gel-like at high RNA concentration. When the RNA concentration in the environment decreases again, the droplet-like condensate re-forms; the scale bar represents 20μm.

[0026] Figure 2 Schematic diagram of obtaining the fluorescence intensity of the aggregate.

[0027] Figure 3 Cell imaging under fluorescence microscopy (a) shows the commercial dye ethidium bromide, b) shows TPE-(RRASL)2, and c) shows that the two can be completely co-localized). Implementation

[0028] The present invention provides a nucleic acid probe based on covalent coupling of an AIE luminescent agent and a phase separation peptide and an application thereof.

[0029] 1. Preparation of probe

[0030] AIE luminogens were coupled to phase-separated peptides using solid-state synthesis.

[0031] Take (RRASL)2 modified AIE-type luminescent agent tetraphenylethylene (TPE) as an example:

[0032] First, the amino acid was coupled to the resin: 2-chlororesin (1 g, 1 mmol / g) was used as the solid phase substrate and swelled in dichloromethane for 30 min. After swelling, Fmoc-protected leucine (424 mg, 1.2 mmol), diisopropylethylamine (155 mg, 1.2 mmol) and diisopropylcarbodiimide (379 mg, 3 mmol) were mixed with 20 mL of dimethyl sulfoxide for 1 h. After the coupling reaction, methanol (1 mL) was added to the above mixture to stop the reaction for 10 min. The obtained leucine-labeled resin was washed twice with dimethyl sulfoxide, and then the Fmoc protecting group of the amino acid was removed by soaking the resin in 20% (v / v) piperidine / dimethyl sulfoxide solution for 15 min and then washing it twice with dimethyl sulfoxide. Whether the Fmoc amino acid was successfully bound to the resin was checked by ninhydrin. This step was repeated until the target phase-separated peptide was obtained.

[0033] After the amino acids are connected, the AIE-type luminescent agent tetraphenylethylene (TPE) is finally connected.

[0034] Cleavage of peptide from resin: The peptide-bound resin was dried by vacuum filtration and then transferred to a 50 mL centrifuge tube. Subsequently, the resin was soaked in a mixture of trifluoroacetic acid / water / triisopropylsilane at a volume ratio of 95:2.5:2.5 for 2 h. After vacuum soaking, the resin peptide was precipitated in ice-cold ether. The peptide / ether mixture was centrifuged at 5000 × g for 10 min, and the crude peptide was collected and washed three times with ice-cold ether.

[0035] 2. AIE-phase-separated peptide conjugates and RNA form condensate droplets

[0036] Mixing the AIE-phase-separated peptide conjugate with RNA solution can produce droplet-like aggregates. Figure 1 As shown in the figure, the aggregates exhibit the fusion properties of emulsion droplets. As the RNA concentration in the environment increases, the aggregates become more viscous and present a soft gel state; when the RNA concentration in the environment decreases, the aggregates reappear in a droplet state. The fluorescence signal at different RNA concentrations is detected using a fluorescence photometer or an enzyme marker, and the fluorescence intensity of the aggregates is obtained as shown in Figure 2 shown.

[0037] 3. Intracellular RNA Imaging

[0038] Cell culture method: Gallbladder cancer SCG-996 cell line was thawed and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. All cells were passaged to the third generation before testing cell viability and RNA imaging.

[0039] Intracellular nucleic acid imaging experiment: cells were seeded into 96-well plates at a density of 5000 cells per well, TPE-phase separation peptide-coupled probes were added to the cell culture and incubated for 24 h. The culture medium was then removed and the samples were washed twice with phosphate buffer.

[0040] SGC-996 cells were suspended in serum-free RPMI 1640 medium at a seeding density of 50,000 / mL. TPE-(RRASL)n was dissolved in the medium at a concentration above the minimum aggregation concentration. The concentration of TPE-(RRASL)2 used for intracellular RNA imaging was 0.2 mM.

[0041] After TPE-(RRASL)2 and ethidium bromide (EtBr, dilution ratio 1:2000) were co-cultured with SGC-996 cells for 2 hours, the cells were imaged using a fluorescence microscope. As shown in Figure 3. In Figure a), the fluorescence enhancement occurred only after the TPE-(RRASL)2 probe bound to the intracellular RNA, and there was no obvious noise signal outside the cell. In Figure b), the non-specific binding of the commercial dye ethidium bromide to the extracellular matrix resulted in the presence of noise signals outside the cell.

[0042] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A nucleic acid probe based on covalent coupling of tetraphenylethylene and phase separation peptide, characterized in that: The phase separation peptide is composed of a nucleic acid binding domain of a repeated cationic amino acid sequence in series, and its specific sequence includes (RGG) n , (RGGYS) n、 (RRASL) n , the value of n is between 2-10.

2. The nucleic acid probe based on covalent coupling of tetraphenylethylene and phase separation peptide according to claim 1, characterized in that: After the nucleic acid probe is combined with the nucleic acid, a light-emitting aggregate in the form of an emulsion droplet with a size between 100 nm and 100 μm is formed.

3. The nucleic acid probe based on covalent coupling of tetraphenylethylene and phase separation peptide according to claim 1, characterized in that: The fluorescence enhancement mechanism of the nucleic acid probe depends on the aggregation state of the phase-separated peptide when it forms an aggregate, and its fluorescence intensity is positively correlated with the nucleic acid concentration in the environment.

4. The nucleic acid probe based on covalent coupling of tetraphenylethylene and phase separation peptide according to claim 3, characterized in that: When the concentration of nucleic acids in the environment changes, the fluorescence signal can undergo dynamic and reversible changes.

5. A use of the nucleic acid probe based on covalent coupling of tetraphenylethylene and phase separation peptide as claimed in claim 1 as an intracellular RNA developer in tumor cell imaging.

6. Use of the nucleic acid probe based on covalent coupling of tetraphenylethylene and phase separation peptide as claimed in claim 1 in RNA vaccine vectors and mRNA drug vectors with fluorescence indication.

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