Photocatalysis-based method for crosslinking and marking biomolecules in living animals

Abstract

The invention provides a method for crosslinking and marking biomolecules in living animals based on photocatalysis. According to the method, the hydrophilic up-conversion nano composite material and the photosensitizer are in contact with the biological sample to be detected, and the target position of the biological sample to be detected is subjected to near-infrared light irradiation, so that the RNA-RNA binding protein compound is formed in situ at the target position of the biological sample to be detected, and the biological sample is obtained. Especially in situ crosslinking of RNA and RNA binding proteins in an organism. On the basis of the cross-linking method, marking, enrichment and analysis of all kinds of RNA-RNA binding protein compounds and RNA binding proteins can be realized by further matching with a compound as shown in a formula 1, and the method has broad spectrum; meanwhile, pollution caused by cross-linking of protein and protein and cross-linking of DNA and protein is effectively avoided, and high specificity is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry, and in particular to a method for cross-linking and labeling biological molecules in living animals based on photocatalysis. Background Art

[0002] Biomacromolecules (mainly including proteins, nucleic acids and polysaccharides) are important basic substances for the structure and function of organisms. The concentration of biomacromolecules in cells is as high as 300g / L, accounting for 20%-40% of the total weight of cells. Interactions between biomacromolecules and between biomacromolecules and ligand small molecules are widely present in cells, and these interactions play a vital role in the life process. RNA is a biomacromolecule that is a carrier of genetic information in biological cells and some viruses and viroids. Some RNA can interact with specific biomacromolecule proteins (such as RNA binding proteins, RBPs) to form RNA-RNA binding protein complexes. RNA-RNA binding protein complexes play a key role in RNA processing, transportation, stability, translation regulation and RNA-mediated gene silencing, and regulate a variety of biological processes such as cell apoptosis, cell proliferation, cell differentiation and self-renewal. Therefore, its disorder has many effects on human diseases, including autoimmune diseases, cancer, diabetes and neurodegenerative diseases. Therefore, understanding and studying the interaction mechanism between proteins and RNA molecules helps to have a deeper understanding of life processes and provide theoretical support for the pathogenesis, treatment methods and drug development of diseases.

[0003] Methods for detecting the interaction between proteins and RNA molecules include, for example, various spectroscopy methods (ultraviolet visible absorption spectroscopy, infrared spectroscopy, Raman spectroscopy), resonance light scattering and surface plasmon resonance, fluorescence spectroscopy, circular dichroism, X-ray crystal diffraction, electrochemical methods, mass spectrometry, thermochemical methods, chromatography and electrophoresis techniques, scanning probe microscopy, equilibrium dialysis and ultracentrifugation techniques, etc. Studies on biomacromolecules are mainly conducted in vitro in dilute solutions (containing only water and a small amount of salt, with a concentration of less than 10 g / L) or cells, but cells cannot represent the heterogeneity and complexity present in actual specimens. The important biological properties of the interaction of biomolecules need to be analyzed at the level of the organism.

[0004] At present, it has been reported that ultrathin sections of mice are used to crosslink mouse liver proteins and RNA through short-wavelength ultraviolet crosslinking and formaldehyde crosslinking. However, during the sampling of ex vivo tissues, the samples themselves may change due to ischemia, animal sacrifice, slight damage during dissection and tissue collection, and possible inflammation. In this way, ex vivo tissues may not accurately show the interaction between RNA and protein. At the same time, short-wavelength ultraviolet irradiation may cause light loss in biological samples, and severe absorption and scattering of photons will cause them to decay rapidly in tissues, ultimately failing to achieve crosslinking of proteins and RNA in intact organs. Although formaldehyde can achieve crosslinking of proteins and RNA, it also brings contamination of protein-protein crosslinking, making the results lack specificity and the identification scale far less than expected. The above technical bottlenecks severely limit the application research of biomolecular crosslinking labeling at the organism level, including the evaluation of biomolecular crosslinking labeling in situ in living animals, which is also a common research difficulty currently faced. Summary of the invention

[0005] The present invention provides a method and product for in situ labeling and / or enrichment and / or analysis of RNA-RNA binding protein complexes and / or RNA binding proteins in biological samples.

[0006] The present invention also provides hydrophilic up-conversion nanocomposite materials and compounds involved in the above method and product.

[0007] In a first aspect, the present invention provides a method for in situ labeling and / or enrichment and / or analysis of RNA-RNA binding protein complexes and / or RNA binding proteins in a biological sample, comprising: contacting a hydrophilic upconversion nanocomposite material and a photosensitizer with a biological sample to be tested, and after the hydrophilic upconversion nanocomposite material and the photosensitizer enter a target position of the biological sample to be tested, irradiating the target position of the biological sample to be tested with near-infrared light, so as to promote in situ cross-linking between RNA and RNA-binding protein in the target position of the biological sample to be tested, thereby forming an RNA-RNA binding protein complex; Using the probe to label and / or enrich and / or analyze the RNA-RNA binding protein complex and / or RNA binding protein in the biological sample to be tested; The hydrophilic upconversion nanocomposite material comprises upconversion nanoparticles and a hydrophilic layer coated on the surface of the upconversion nanoparticles; the upconversion nanoparticles have a heterogeneous core-multi-shell structure, can absorb near-infrared light and emit at least ultraviolet light with a wavelength of 254 nm and visible light with a wavelength of 390 nm-780 nm, which is the same as the absorption spectrum of the photosensitizer.

[0008] In the above method, the biological sample can be an organism or at least one of an organ, tissue, or cell derived from an organism, that is, the above method can be carried out in vivo or in vitro. In the case of in vivo, the hydrophilic up-conversion nanocomposite and photosensitizer can be applied to an organism through various routes of administration, thereby contacting the cell, tissue, or organ of the organism in vivo. In the case of in vitro, the hydrophilic up-conversion nanocomposite and photosensitizer are contacted with an in vitro sample (e.g., a cell, tissue, or organ sample obtained from a living organism, a cultured cell, tissue, or organ sample).

[0009] Further, the organism refers to an individual with life, for example, an animal. Further, the animal can be a mammal, for example, bovine, equine, feline, canine, lagomorph, suid, camelid, rodent and primate, etc., including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, orangutans) and humans, preferably cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys and humans.

[0010] In the method described above, the upconversion nanoparticles absorb near-infrared light and convert it into ultraviolet light and visible light with shorter wavelengths. The wavelength of the near-infrared light absorbed by the upconversion nanoparticles is 780-1100nm; further, the near-infrared light is a near-infrared short wave with a wavelength of 808nm, which has a strong tissue penetration ability and is suitable for irradiation of biological samples. The wavelength of the ultraviolet light emitted by the upconversion nanoparticles is 254nm, and the ultraviolet light of 254nm coincides with the base absorption peak of RNA, so that the hydrophilic upconversion nanocomposite material and the base of RNA undergo a fluorescence resonance energy transfer (FRET) process, which promotes the cross-linking of the excited RNA base with the amino acid residues of the RNA binding protein, and forms a cross-linked product of RNA and RNA binding protein in situ in the biological sample, that is, RNA-RNA binding protein complex. The wavelength of the visible light emitted by the upconversion nanoparticles is 390nm-780nm, and the visible light has the same wavelength as the absorption spectrum of the photosensitizer, so that the hydrophilic upconversion nanocomposite material and the photosensitizer undergo a FRET process, prompting the excited photosensitizer to release singlet oxygen, which is convenient for subsequent labeling, enrichment and analysis of the RNA-RNA binding protein complex.

[0011] Further, based on the wavelength range of light absorption and light emission of the upconversion nanoparticles, the upconversion nanoparticles provided by the present invention are fluorides formed by rare earth elements and sodium elements; The rare earth element is selected from at least one of La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), Sc (scandium), and Y (yttrium).

[0012] Furthermore, the upconversion nanoparticles have a core-shell structure, which includes a core particle and a first layer, a second layer and a third layer sequentially coated on the surface of the core particle; wherein the core particle is doped with rare earth elements Gd, Yb and Tm, the first layer is doped with rare earth elements Y and Yb, the second layer is doped with rare earth elements Gd, Yb and Nd, and the third layer is doped with rare earth element Gd.

[0013] Furthermore, in the core particles, the molar ratio of Gd, Yb and Tm is (48-50): (49-50): (1-2). In the first layer, the molar ratio of Y and Yb is (4-5): (2-3). In the second layer, the molar ratio of Gd, Yb and Nd is (4-5): (1-2): (5-6).

[0014] Furthermore, the mass ratio of the core particles, the first layer, the second layer and the third layer is (11-12):(5-6):(6-7):(5-6).

[0015] Furthermore, the upconversion nanoparticles have an apparent morphology of nanoparticles with an average particle size of 25-50 nm.

[0016] Furthermore, the core particle includes NaGdF4:Yb / Tm; the first layer includes NaYF4:Yb; the second layer includes NaGdF4:Yb / Nd; and the third layer includes NaGdF4.

[0017] It is understandable that, based on the selection of the absorption wavelength and emission wavelength of the upconversion nanoparticles, those skilled in the art can select the number of layers, the type of rare earth element, and the doping amount to achieve different absorption wavelengths and emission wavelengths of the upconversion nanoparticles.

[0018] In the above method, the hydrophilic layer also has good biocompatibility and can be an organic acid layer, a hydrophilic copolymer layer, etc. Further, the hydrophilic layer can be obtained by oxidizing the surface of the upconversion nanoparticles with a Lemieux-von Rudloff reagent.

[0019] In the above method, the hydrophilic upconversion nanocomposite material can be prepared by a preparation method comprising the following steps: S1) dispersing a precursor compound containing a rare earth element, a precursor compound containing a sodium element, and a fluoride in an organic solvent containing a fatty acid to obtain a mixture, and heating the mixture to prepare the upconversion nanoparticles; S2) forming a hydrophilic layer on the surface of the upconversion nanoparticles to obtain the hydrophilic upconversion nanocomposite material.

[0020] Furthermore, the precursor compound containing rare earth elements can be specifically a soluble salt of rare earth elements, such as acetate, halide, nitrate, etc. Further, the precursor compound containing rare earth elements can be, for example, Gd(CH3CO2)3, Yb(CH3CO2)3, Tm(CH3CO2)3, Cl3Y, Gd(CH3CO2)3, Nd(CH3CO2)3, etc.

[0021] Furthermore, the compound containing sodium element may be, for example, NaOH; and the fluoride may be, for example, NH4F.

[0022] Furthermore, the organic solvent containing fatty acid may be, for example, an organic solvent including oleic acid and octadecene, wherein the volume ratio of oleic acid to octadecene is 1:1.

[0023] Furthermore, during the preparation process, the required rare earth element-containing precursor compound is dispersed in an organic solvent containing fatty acids according to a certain mass ratio, and heated to 140-160° C. for 0.5-1.5 hours to dissolve the soluble salt of the rare earth element and form a lanthanide oleate complex. After the mixed system becomes a clear solution, it is cooled to 30-50° C.; a compound containing sodium element and fluoride are added to the cooled mixed solution, and stirring is maintained at 50-60° C. for 0.5-1 hour, and the temperature is increased to 290-305° C. for reaction for 1-2 hours.

[0024] Furthermore, the mass ratio of the required rare earth element-containing precursor compounds is Gd(CH3CO2)3:Yb(CH3CO2)3:Tm(CH3CO2)3:Yb(CH3CO2)3:Cl3Y:Gd(CH3CO2)3:Yb(CH3CO2)3:Nd(CH3CO2)3:Gd(CH3CO2)3 is (67-134):(69-138):(1-2):(27-52):(38-72):(65-123):(14-26):(63-118):(63-108).

[0025] Furthermore, the above reactions are all carried out in an inert atmosphere; further, the inert atmosphere can be an argon atmosphere.

[0026] Furthermore, the above steps also include post-processing steps such as washing, centrifugation, and re-dispersion to remove impurities and non-reactants and obtain uniformly dispersed nanoparticles.

[0027] Furthermore, the hydrophilic layer formed on the surface of the upconversion nanoparticles may be an organic acid layer, and the organic acid layer may be obtained by oxidizing the surface of the upconversion nanoparticles with a Lemieux-von Rudloff reagent. Furthermore, the method comprises: dispersing the prepared upconversion nanoparticles in a mixed solvent including cyclohexane, tert-butyl alcohol and a 5% K2CO3 aqueous solution, then adding a Lemieux-von Rudloff reagent for oxidation reaction, and after the reaction is completed, preparing the hydrophilic upconversion nanocomposite material through post-treatment steps such as washing and centrifugation.

[0028] In the method as described above, the photosensitizer can be at least one of porphyrin and its derivatives, and chlorophyll derivatives. Further, the photosensitizer is a chlorophyll derivative, for example, it can be at least one of tetra-m-hydroxyphenyl chlorin (mTHPC), p-bromophenyl-hydrazone-methyl pyroxyporphyrin-a (BPMppa), chlorophyll-a (Chl-a), pheophorbide a (Pheoa), pyropheophorbide-a (Ppa), pyropheophorbide a hexyl ether (HPPH), dihydrochlorin e6 (Ce6), and mono-asparagine dihydrochlorin-e6 (NPe6). Further, the photosensitizer is pheophorbide A, and its chemical structure is shown below: .

[0029] Furthermore, the pheophorbide A has strong absorption spectra at 600-700nm and 400-500nm, and accordingly, the upconversion nanoparticles can emit visible light at 600-700nm and 400-500nm. Furthermore, the upconversion nanoparticles can emit visible light at 450nm and 650nm.

[0030] In the method as described above, the probe is selected from at least one of a singlet oxygen detection probe, a labeling probe, and an enrichment probe.

[0031] In a specific embodiment, the probe is a singlet oxygen detection probe. Since the hydrophilic upconversion nanocomposite material emits visible light with the same wavelength as the photosensitizer absorption after absorbing near-infrared light, it generates singlet oxygen through FRET with the photosensitizer. The singlet oxygen detection probe is used to perform fluorescence intensity detection or fluorescence imaging analysis on a microplate reader to determine the generation of singlet oxygen.

[0032] Furthermore, the singlet oxygen detection probe may be DCFH-DA.

[0033] Furthermore, when the biological sample to be tested is an in vitro cell, the above-mentioned hydrophilic upconversion nanocomposite material, photosensitizer, and singlet oxygen detection probe can be co-incubated with the cells to be tested. After incubation for a certain period of time, the cells are irradiated with 808nm near-infrared light to promote in situ cross-linking of RNA and RNA protein in the cells and catalyze the generation of singlet oxygen; then, the fluorescence of the singlet oxygen probe is detected for analysis.

[0034] Furthermore, the final concentration of the hydrophilic upconversion nanocomposite material in the cell culture medium can be 100-750 μg / mL, specifically 350 μg / mL; the incubation time with the cells is 1-2 h, specifically 1 h. The concentration of the photosensitizer can be 1-4 μM, specifically 2.5 μM, and the incubation time with the cells is 3-4 h, specifically 3 h. The concentration of the singlet oxygen detection probe can be 10-20 μM, specifically 10 μM, and the incubation time with the cells is 20-30 min, specifically 20 min.

[0035] Furthermore, the power of the near-infrared light is 5 W, and the irradiation time is 0.5-3 min, specifically 1 min.

[0036] Furthermore, when the biological sample to be tested is a tissue and / or organ of an organism, the hydrophilic upconversion nanocomposite material and the photosensitizer can be intravenously injected into the organism, an 808 nm laser is directly illuminated to the tested part of the organism to observe the luminescence of the hydrophilic upconversion nanocomposite material, the organism is anesthetized and the tested tissue and / or organ of the organism is obtained, and the singlet oxygen detection probe is incubated with the tissue and / or organ; then, the tissue and / or organ is irradiated with near-infrared light to promote in situ cross-linking of RNA and protein in the tissue and / or organ and the generation of singlet oxygen; finally, the tissue and / or organ is lysed and analyzed.

[0037] Further, based on 8-week-old mice, the injection amount of the hydrophilic upconversion nanocomposite material can be 0.5-0.8 mg, specifically 0.8 mg. The concentration of the photosensitizer can be 0.1-0.4 mM, specifically 0.15 mM. The concentration of the compound described in Formula 1 can be 10-20 mM, specifically 18 mM.

[0038] Furthermore, the power of the near-infrared light is 5 W, and the irradiation time is 1-4 min, specifically 2 min.

[0039] In another specific embodiment, the compound shown in Formula 1 and streptavidin labeled with a fluorescent group can be used as a labeled probe to achieve labeling of RNA-RNA binding protein complexes and / or RNA binding proteins in biological samples. The structure of the compound shown in Formula 1 is as follows: .

[0040] Specifically, when the hydrophilic upconversion nanocomposite material absorbs near-infrared light and reacts with a photosensitizer to generate singlet oxygen through FRET, the oxoquinoline group in the compound shown in Formula 1 can react with RNA under singlet oxygen oxidation, thereby binding to the RNA-RNA binding protein complex, an in situ cross-linked product of RNA and RNA binding protein; at the same time, streptavidin has a strong affinity for the biotin group in the compound shown in Formula 1, and by labeling streptavidin with a fluorescent group, the RNA-RNA binding protein complex in the biological sample to be tested and the RNA binding protein cross-linked with RNA can be located and the content detected.

[0041] Furthermore, the fluorescent group-labeled streptavidin may be FITC-labeled streptavidin.

[0042] Furthermore, when the biological sample to be tested is an in vitro cell, the above-mentioned hydrophilic upconversion nanocomposite material, the compound shown in Formula 1 and the photosensitizer can be co-incubated with the cells, and the cells can be irradiated with 808nm near-infrared light to promote the formation of RNA-RNA binding protein complexes in the cells, and the compound shown in Formula 1 can be bound to the RNA-RNA binding protein complexes; and specific labeling of all RNA-RNA binding protein complexes in the cells can be achieved by using streptavidin labeled with a fluorescent group.

[0043] Further, the final concentration of the hydrophilic upconversion nanocomposite material in the cell culture medium can be 100-750 μg / mL, and further can be 350 μg / mL; the incubation time with the cells is 1-2 h, and specifically can be 1 h. The concentration of the photosensitizer can be 1-4 μM, and specifically can be 2.5 μM, and the incubation time with the cells is 3-4 h, and specifically can be 3 h. The concentration of the compound represented by Formula 1 can be 100-750 μM, and further can be 300 μM; the incubation time with the cells is 1-3 h, and specifically can be 2 h.

[0044] Furthermore, the power of the 808 nm near-infrared light is 5 W, and the irradiation time is 0.5-3 min, specifically 1 min.

[0045] Furthermore, a laser scanning confocal microscope can be used to label the RNA-RNA binding protein complex or the RNA binding protein.

[0046] In another specific embodiment, the compound shown in Formula 1 and streptavidin-modified magnetic beads can be used as probes to achieve enrichment and analysis of RNA-RNA binding protein complexes and RNA binding proteins in the biological sample to be tested.

[0047] Furthermore, when the organism to be detected is an in vitro cell, the method is the same as the method corresponding to the aforementioned labeling probe.

[0048] Furthermore, when the biological sample to be tested is an organism, such as a mouse, the hydrophilic upconversion nanocomposite material, the photosensitizer and the compound described in Formula 1 can be intravenously injected into the organism; then, the organism is anesthetized, and the organs and / or tissues of the organism are irradiated with near-infrared light; then, the organs and / or tissues irradiated with near-infrared light are excised and lysed, and the RNA-RNA binding protein complex is enriched using streptavidin-modified magnetic beads.

[0049] Furthermore, the RNA-RNA binding protein complex enriched by the magnetic beads can be hydrolyzed with ribonuclease (RNase) to obtain the RNA binding protein bound to the RNA.

[0050] Further, based on 8-week-old mice, the injection amount of the hydrophilic upconversion nanocomposite material can be 0.5-0.8 mg, and further can be 0.8 mg. The concentration of the photosensitizer can be 0.1-0.4 mM, and further can be 0.15 mM. The concentration of the compound shown in Formula 1 can be 6-36 mM, and further can be 18 mM. It can be understood that when the age or weight of the mouse changes, the injection amount of the above material can be appropriately adjusted, and the adjusted injection amount also belongs to the protection scope of the present invention.

[0051] Furthermore, the above material can be injected into the mouse through the tail vein of the mouse, and the injection can be followed by waiting for 3-6 hours, and further for 3 hours.

[0052] In a second aspect, the present invention provides a product for in situ labeling and / or enrichment and / or analysis of RNA-RNA binding protein complexes and / or RNA binding proteins in biological samples, comprising the hydrophilic upconversion nanocomposite material, photosensitizer and probe in any of the above methods.

[0053] The product as described above may specifically be a kit.

[0054] For the product as described above, the selection of the hydrophilic upconversion nanocomposite material, photosensitizer and probe are all as described above.

[0055] In a third aspect, the present invention provides a hydrophilic upconversion nanocomposite material, comprising upconversion nanoparticles and a hydrophilic layer coated on the surface of the upconversion nanoparticles; the upconversion nanoparticles have a heterogeneous core-multi-shell structure, can absorb near-infrared light and emit ultraviolet light with a wavelength of 254 nm and visible light with a wavelength of 390 nm-780 nm.

[0056] In a fourth aspect, the present invention provides a method for preparing the above-mentioned hydrophilic upconversion nanocomposite material, comprising: S1) dispersing a precursor compound containing a rare earth element, a precursor compound containing a sodium element, and a fluoride in an organic solvent containing a fatty acid to obtain a mixture, and heating the mixture to prepare the upconversion nanoparticles; S2) forming a hydrophilic layer on the surface of the upconversion nanoparticles to obtain the hydrophilic upconversion nanocomposite material.

[0057] In a fifth aspect, the present invention provides a compound, the structure of which is shown in Formula 1: .

[0058] In a sixth aspect, the present invention provides a method for preparing the compound represented by the above formula 1, comprising: The compound of formula 1 is synthesized by amidation reaction using biotin-PEG2-NH3 and 3-(2-oxopyrrolidine) propionic acid as substrates. The reaction process involved is shown in formula 2: .

[0059] According to the preparation method described above, the molar ratio of 3-(2-oxoquinoline) propionic acid to biotin-PEG2-NH3 can be 1:1-2:1; further, the molar ratio of 3-(2-oxoquinoline) propionic acid to biotin-PEG2-NH3 is 1.5:1.

[0060] In the preparation method as described above, the amidation reaction is carried out in an organic solvent containing an acid binding agent, the organic solvent may be dimethylformamide (DMF), the acid binding agent may be N,N-diisopropylethylamine (DIPEA), and the volume ratio of the organic solvent to the acid binding agent is (1.5-2.5):1, and may further be 2:1.

[0061] In the preparation method as described above, the amidation reaction system further comprises N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) for activating the carboxyl group to promote the amidation reaction of the carboxyl group and the amine group. Further, the molar ratio of 3-(2-oxopyrrolidine)propionic acid, NHS and EDC can be 1:1:1-1:5:5, and can further be 5:5:6.

[0062] In the preparation method as described above, the temperature of the amidation reaction can be 21-25° C., and can further be 25° C.; the reaction time can be 4-24 hours, and can further be 12 hours.

[0063] The present invention provides a method for in-situ labeling, enrichment and analysis of RNA-RNA binding protein complexes and RNA binding proteins in biological samples. Specifically, when the hydrophilic upconversion nanocomposite material used in the present invention is excited by near-infrared light, there is ultraviolet light emission of 254nm and visible light emission with the same absorption wavelength as that of a photosensitizer, wherein the ultraviolet light of 254nm coincides with the base absorption peak of ribonucleic acid, so that the hydrophilic upconversion nanocomposite material and the base of ribonucleic acid undergo a FRET process, thereby promoting amino acid cross-linking of the excited base of ribonucleic acid and the ribonucleic acid. An RNA-RNA binding protein complex, a cross-linked product of RNA and protein, is formed in situ in the biological sample; the emitted visible light with the same absorption wavelength as the photosensitizer is absorbed by the photosensitizer, and the photosensitizer acts as an energy acceptor to catalyze the production of singlet oxygen. The oxoquinoline group in the compound shown in Formula 1 has the ability to react with ribonucleic acid under singlet oxygen oxidation, thereby binding to the RNA-RNA binding protein complex; finally, the RNA-RNA binding protein complex and RNA binding protein in the biological sample are specifically labeled, enriched and analyzed by fluorescent group-modified streptavidin or streptavidin magnetic beads.

[0064] The method provided by the present invention can realize the in situ cross-linking of RNA and RNA-binding proteins in an organism, and can be used in combination with the compound shown in Formula 1 to achieve the labeling and enrichment of all types of RNA (coding RNA and non-coding RNA) binding proteins, with a broad spectrum; at the same time, the compound shown in Formula 1 can only achieve the labeling of RNA bases, avoiding the contamination of protein-protein cross-linking and DNA-protein cross-linking, and has high specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 A flow chart for preparing a hydrophilic upconversion nanocomposite material provided by one embodiment of the present invention; Figure 2 A high-angle annular dark field transmission electron microscope image and an element mapping image of a single nanoparticle of a hydrophilic upconversion nanocomposite material provided by an embodiment of the present invention; wherein a is a high-angle annular dark field transmission electron microscope image, and b is an element mapping image of a single nanoparticle; Figure 3 The emission spectrum of the hydrophilic upconversion nanocomposite material provided in one embodiment of the present invention under 808nm near-infrared light excitation; wherein a is the emission spectrum within the range of 210-300nm, and b is the emission spectrum within the range of 325-690nm; Figure 4 A hydrogen nuclear magnetic resonance spectrum of the compound represented by Formula 1 provided in one embodiment of the present invention; Figure 5 The carbon nuclear magnetic resonance spectrum of the compound represented by formula 1 provided in one embodiment of the present invention; Figure 6 Fluorescence intensity detection and fluorescence confocal microscopy imaging of singlet oxygen generated at the cellular level; the left side is the fluorescence intensity detection result, and the right side is the fluorescence confocal microscopy imaging; Figure 7 Fluorescence intensity detection and fluorescence confocal microscopy imaging of singlet oxygen generated at the tissue level; the left side is the fluorescence intensity detection result, and the right side is the fluorescence confocal microscopy imaging; Figure 8 Sodium dodecyl sulfate-PAGE gel electrophoresis characterization of the enriched products obtained by the method provided by the present invention and the control method; Fig. 9 Fluorescence confocal microscopic imaging of RNA-protein in situ cross-linked products in cells labeled with the compound of Formula 1; Fig.10 Schematic diagram of the process for enriching RNA-protein in situ cross-linking products in mouse kidney; Fig.11 For electrophoretic characterization of the enriched products in mouse kidney; Fig.12 The scatter plot and correlation analysis of protein identification in mouse kidney; the left side is the protein scatter plot, and the right side is the correlation analysis result; Fig.13 Gene annotation for identification of RNA-protein in situ cross-linking products in mouse kidney. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0067] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0068] The reagents used in the following examples were prepared using RNase-free solutions or RNase-free solvents (such as DEPC-treated water) as much as possible, and the materials used were RNase-free or DEPC-treated as much as possible.

[0069] Example 1 Synthesis and characterization of hydrophilic upconversion nanocomposite materials like Figure 1 As shown, the synthesis of the hydrophilic upconversion nanocomposite material includes the following steps: Step 1, Synthesis of NaGdF4:Yb / Tm: A certain amount of Gd(CH3CO2)3, Yb(CH3CO2)3 and Tm(CH3CO2)3 are dissolved in a mixed solvent of oleic acid and octadecene in a volume ratio of 1:1 to obtain a first mixed solution of Gd / Yb / Tm; the first mixed solution of Gd / Yb / Tm is heated to 150°C and maintained at this temperature for 1 hour, and then cooled to 50°C for standby use. 0.05g of ammonium fluoride (NH4F) and 0.04g of sodium hydroxide (NaOH) are dissolved in 4.4ml of methanol, and mixed evenly to obtain a second mixed solution of NH4F / NaOH. The second mixed solution of NH4F / NaOH is added to the first mixed solution of Gd / Yb / Tm cooled to 50°C, and the mixed solution is heated to 80°C-100°C, maintained at this temperature for 15-25 minutes to remove methanol, and then the resulting solution is heated to 300°C and reacted at this temperature for 1.5 hours. After the reaction is completed, the reaction system is cooled to room temperature. After adding ethanol, the obtained nanoparticles NaGdF4:Yb / Tm were collected by centrifugation at 6000-7500 rpm for 5-10 minutes, and then washed three times with ethanol and cyclohexane in sequence, and finally dispersed in cyclohexane for use.

[0070] Step 2: Synthesis of NaGdF4:Yb / Tm@NaYF4:Yb A certain amount of Cl3Y and Yb(CH3CO2)3 is dissolved in a mixed solvent of oleic acid and octadecene in a volume ratio of 1:1 to obtain a third mixed solution of Y / Yb; the third mixed solution of Y / Yb is heated to 150°C and maintained at this temperature for 1 hour, and then cooled to 50°C for use. 4 ml of the cyclohexane dispersion containing nanoparticles NaGdF4:Yb / Tm prepared in step 1 and the second mixed solution of NH4F / NaOH are added to the cooled solution, stirred at 50°C for 30 minutes, and the mixed solution is heated to 85°C-100°C, maintained at this temperature for 15-25 minutes to remove methanol and cyclohexane, then heated to 300°C and reacted at this temperature for 1.5 hours, and then cooled to room temperature. After adding ethanol to the reaction system, the obtained nanoparticles NaGdF4:Yb / Tm@NaYF4:Yb were collected by centrifugation at 6000-7500 rpm for 5-10 minutes, and then washed three times with ethanol and cyclohexane in sequence, and finally dispersed in cyclohexane for use.

[0071] Step 3. Synthesis of NaGdF4:Yb / Tm@NaYF4:Yb@NaGdF4:Yb / Nd A certain amount of Gd(CH3CO2)3, Yb(CH3CO2)3 and Nd(CH3CO2)3 are dissolved in a mixed solvent of oleic acid and octadecene in a volume ratio of 1:1 to obtain a fourth mixed solution of Gd / Yb / Nd; the fourth mixed solution of Gd / Yb / Nd is heated to 150°C and maintained at this temperature for 1 hour, and then cooled to 50°C for use. 4 ml of the cyclohexane dispersion containing nanoparticles NaGdF4:Yb / Tm@NaYF4:Yb prepared in step 2 and the second mixed solution of NH4F / NaOH are added to the cooled solution, stirred at 50°C for 30 minutes, and the mixed solution is heated to 85°C-100°C, maintained at this temperature for 15-25 minutes to remove methanol and cyclohexane, then heated to 300°C and reacted at this temperature for 1.5 hours, and then cooled to room temperature. After adding ethanol to the reaction system, the obtained nanoparticles NaGdF4:Yb / Tm@NaYF4:Yb@NaGdF4:Yb / Nd were collected by centrifugation at 6000-7500 rpm for 5-10 minutes, and then washed three times with ethanol and cyclohexane in sequence, and finally dispersed in cyclohexane for use.

[0072] Step 4. Synthesis of NaGdF4:Yb / Tm@NaYF4:Yb@NaGdF4:Yb / Nd@NaGdF4 A certain mass of Gd(CH3CO2)3 is dissolved in a mixed solvent of oleic acid and octadecene in a volume ratio of 1:1 to obtain a fifth mixed solution; the fifth mixed solution is heated to 150°C and maintained at this temperature for 1 hour, and then cooled to 50°C for use. 4 ml of the cyclohexane dispersion containing nanoparticles NaGdF4:Yb / Tm@NaYF4:Yb@NaGdF4:Yb / Nd prepared in step 3 and the second mixed solution of NH4F / NaOH are added to the cooled solution, stirred at 50°C for 30 minutes, and the mixed solution is heated to 85°C-100°C, maintained at this temperature for 15-25 minutes to remove methanol and cyclohexane, then heated to 300°C and reacted at this temperature for 1.5 hours, and then cooled to room temperature. After adding ethanol to the reaction system, the system was centrifuged at 6000-7500 rpm for 5-10 minutes to collect the obtained nanoparticles NaGdF4:Yb / Tm@NaYF4:Yb@NaGdF4:Yb / Nd@NaGdF4, i.e., upconversion nanoparticles.

[0073] The mass ratio of the reaction materials in the above steps 1-4 is: Gd(CH3CO2)3:Yb(CH3CO2)3:Tm(CH3CO2)3:Yb(CH3CO2)3:Cl3Y:Gd(CH3CO2)3:Yb(CH3CO2)3:Nd(CH3CO2)3:Gd(CH3CO2)3=67:69:1:28:39:67:14:64:67; the reactions are all carried out in an argon atmosphere.

[0074] Step 5. Synthesis of hydrophilic upconversion nanocomposites 0.1 g of the upconversion nanoparticles prepared in step 4, 100 mL of cyclohexane, 70 mL of tert-butanol, and 10 mL of 5% K2CO3 aqueous solution were mixed, and the mixture was stirred at room temperature for 30 minutes; then 20 mL of Lemieux-von Rudloff reagent (5.7 mM potassium permanganate and 0.105 M sodium periodate aqueous solution) was added dropwise at a rate of 2 s / drop; the mixture was stirred at 40°C for 48 hours, the product was separated by centrifugation, and the nanoparticles were washed with deionized water, acetone and ethanol in turn, the washed product was dispersed in 50 ml of hydrochloric acid solution with a pH of 4-5, stirred for 40 minutes, and the oxidation product of the nanoparticles was obtained after centrifugation, which was washed twice with deionized water and dried under vacuum for use.

[0075] The hydrophilic upconversion nanocomposite prepared in step 5 was analyzed by high-angle annular dark field transmission electron microscopy. The analysis results are as follows: Figure 2 As shown, according to Figure 2 It can be seen that the hydrophilic upconversion nanocomposite material with heterogeneous core-multi-shell structure was successfully synthesized in this example.

[0076] The hydrophilic upconversion nanocomposite prepared in step 5 was subjected to fluorescence emission detection under 808 nm excitation. The detection results are as follows Figure 3 As shown, it can be seen that the nanocomposite material prepared in this embodiment has at least emissions at wavelengths of 254 nm, 450 nm and 650 nm at 808 nm.

[0077] Example 2 Synthesis of the compound represented by formula 1 The compound shown in Formula 1 is synthesized according to the reaction route shown in Formula 2, specifically comprising the following steps: 1.5 mM 3-(2-oxopyrrolidine) propionic acid, 1.5 mM N-hydroxysuccinimide (NHS), 1.8 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1 mM biotin-PEG2-NH3 were reacted in 10 mL dimethylformamide (DMF) for 4 hours, and then 5 mL N,N-diisopropylethylamine (DIPEA) was added and reacted at 25°C for 12 hours. After the reaction was completed, the reaction product was concentrated under reduced pressure and further washed and purified with ethyl acetate to obtain the compound shown in Formula 1.

[0078]

[0079] The prepared compound of formula 1 was subjected to nuclear magnetic resonance analysis, and the obtained H spectrum and C spectrum were as follows: Figure 4-5 As shown. The nuclear magnetic signal is: 1 HNMR (600 MHz, DMSO- d 6) δ 7.93(dd, J =87.1,5.9 Hz,1 H),6.41(s,0H),6.39(d, J =32.1 Hz,1H),6.33(dd, J =3.2,1.9Hz,0H),6.14(t, J =6.0Hz,1H),6.09–6.01(m,2H),4.16–4.09(m,1H),3.50(d, J =5.0Hz,2H),3.39(ddd, J =8.2,5.4,2.3 Hz,10 H),3.24–3.14(m,2H),3.05(q, J =6.5 Hz,3H),2.99(t, J =7.1Hz,4 H),2.07(t, J=7.4 Hz, 1H), 1.99 (s, 1 H), 1.82-1.68 (m, 3 H), 1.63-1.32 (m, 2 H).

[0080] 13 CNMR (151 MHz, DMSO- d 6) δ173.22,163.17,158.85,155.22,141.71,110.79,105.45,70.00,69.63,69.58,61.5 1,60.22,59.68,55.89,54.97,42.49,40.55,35.57,34.59,33.87,28.66,28.51,25.76.

[0081] The above results indicate that the compound represented by Formula 1 was successfully synthesized.

[0082] Example 3 Hydrophilic upconversion nanocomposites catalyze singlet oxygen generation via FRET-driven photosensitizers 3.1 Cellular level: HeLa cells of appropriate density were inoculated in 6-well plates and 20 mm glass-bottomed culture dishes, and cultured in a 37°C incubator. When the cells grew to a density of 70%-80%, the cells were washed with PBS. The washed cells were incubated with 2.5 μM photosensitizer pheophorbide A for 3 h at 37°C; then incubated with 350 μg / mL of the hydrophilic upconversion nanocomposite prepared in Example 1 for 1 h; then incubated with 10 μM / L singlet oxygen detection probe DCFH-DA for 20 min; after the incubation, the cells were washed with PBS 3 times and irradiated with 5W 808 nm laser for 1 min.

[0083] The cells seeded in the 6-well plate were digested with trypsin and added to a black 96-well plate. The fluorescence intensity was then detected in an ELISA reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The results are shown in Figure 2. Figure 6 As shown in the left figure, the hydrophilic upconversion nanocomposite can catalyze singlet oxygen generation in cells via FRET-driven photosensitizer.

[0084] The cells seeded in a 20 mm glass-bottomed culture dish were fixed with 4% paraformaldehyde (diluted with PBS) at room temperature for 15 min, and then washed three times with PBS, followed by the addition of 10 μL Hoechst for further labeling at room temperature for 10 min to locate the cell nucleus; finally, washed three times with PBS and imaged using a laser scanning confocal microscope. Figure 6As shown in the middle right, obvious fluorescence intensity can be observed, indicating the generation of singlet oxygen in the cell.

[0085] 3.2 Organizational level 0.8 mg of upconversion nanocomposite, 0.15 mM photosensitizer and 18 mM compound described in formula 1 were injected into C57BL / 6J mice (8 weeks old) or BALB / c-nu mice (5 weeks old) through the tail vein, and waited for 3 hours; 808 808 nm laser was directly illuminated to the test part of BALB / c-nu mice to observe the luminescence of hydrophilic upconversion nanocomposite; C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (dissolved in saline); blood was removed from the kidneys by portal vein liver perfusion, and the required parts were cut off; 30 μM singlet oxygen probe was used for incubation for 30 min. 5W 808 nm laser was irradiated for 2 min; the tissue was ground and lysed with steel beads in a tissue grinder, and centrifuged at 1000 g for 5 min. 100-200 μL of lysate was taken to a black 96-well plate and excited at 488 nm in an ELISA reader, and the emission at 525 nm was detected. The results are shown in Figure 7 As shown on the left, the hydrophilic upconversion nanocomposite is able to catalyze singlet oxygen generation in tissues via FRET-driven photosensitizers.

[0086] In addition, some tissues were fixed with 4% paraformaldehyde at 4°C for 24 h; then immersed in 15% sucrose solution until the tissue sank to the bottom, and then changed to 30% sucrose solution until the tissue sank to the bottom. OCT was used for embedding, and 9 μm sections were cut using a freezing microtome. The OCT was removed by soaking in PBS for 10 min, and 20 μL of anti-fluorescence quenching sealing solution (containing DAPI) was added, and the coverslip was used for sealing, and imaging was performed using a laser scanning confocal microscope. The results are shown in Figure 7 As shown in the middle right, there is an obvious difference in fluorescence intensity between the experimental group and the blank tissue control group, indicating the generation of singlet oxygen in the tissues and organs.

[0087] Example 4 Comparison of the ability of hydrophilic upconversion nanocomposites and 254 nm to fix RNA-RNA binding protein complexes in vivo Eight-week-old male C57BL / 6 mice were divided into two groups. One group of mice was injected with 0.8 mg of hydrophilic upconversion nanocomposite materials through the tail vein. After waiting for 3 hours, the kidneys of the mice in the other group were irradiated with 808 nm laser for 2 min. The kidneys of the mice in the other group were irradiated with 254 nm light for 2 min. After irradiation, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (dissolved in saline); the blood in the kidneys was removed by portal vein liver perfusion, and the irradiated parts were cut off and quickly frozen in liquid nitrogen and stored at -80°C for later use. At the same time, biotin psoralen probes were introduced in vitro to label RNA in tissue lysate to enrich RBPs, and the enriched products were characterized by sodium dodecyl sulfate-PAGE gel electrophoresis. The silver staining results are shown in Figure 2. Figure 8 As shown, taking the whole lysate of the tissue before enrichment as a reference (lysate), under the premise that there is no difference in the lysate, there are obvious protein bands after the hydrophilic upconversion nanocomposite material is irradiated with 808 nm near-infrared light, but there are no protein bands after 254 nm light treatment, which fully demonstrates the superior ability of the hydrophilic upconversion nanocomposite material to cooperate with 808 nm laser in cross-linking RNA and RNA-binding proteins in living animals.

[0088] Example 5: Use of a hydrophilic upconversion nanocomposite material in combination with the compound of formula 1 to label RNA-RNA binding protein complexes in cells HeLa cells of appropriate density were inoculated in glass bottom culture dishes with a diameter of 20 mm, cultured in a 37°C incubator, and washed with PBS when the density reached 70%-80%. The cells were incubated with 2.5 μM photosensitizer for 3 h at 37°C; then incubated with 300 μM compound shown in Formula 1 for 2 h; then incubated with 350 μg / mL hydrophilic upconversion nanocomposite for 1 h; after the incubation, the cells were irradiated with 5W 808 nm laser for 1 min, washed with PBS 3 times, fixed with 4% paraformaldehyde (diluted with PBS) at room temperature for 15 min, permeabilized with 0.5% Triton X-100 (diluted with PBS) at room temperature for 15 min, then washed with PBS 3 times, incubated with 5 μg / mL streptavidin-FITC at 25°C for 25 min, washed with PBS 3 times, and then added with 10 μL Hoechst to continue labeling at room temperature for 10 min to locate the cell nucleus; finally washed with PBS 3 times and imaged with laser scanning confocal microscopy. The imaging results are shown in Fig. 9 As shown, there is obvious green fluorescence in the cells, which indicates that the compound described in Formula 1 can successfully enter the cells and simultaneously achieve labeling of the RNA-RNA binding protein complex in the cell nucleus and cytoplasm.

[0089] Example 6: Combination of hydrophilic upconversion nanocomposite materials and the compound of formula 1 to achieve enrichment and analysis of RNA-RNA binding protein complexes in vivo according to Fig.10 The schematic diagram of the process shown in the figure uses the hydrophilic upconversion nanocomposite material and the compound shown in Formula 1 to enrich and identify the in situ cross-linked products of RNA and RNA-binding proteins in mouse kidneys. The specific steps are as follows: Step 1: Mouse tail vein injection and organ extraction: 0.8 mg of hydrophilic upconversion nanocomposite, 0.15 mM photosensitizer and 18 mM compound of formula 1 were injected into the tail vein of 8-week-old C57BL / 6 male mice, and the mice were irradiated with 808 nm laser for 2 min. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (dissolved in saline). The blood in the kidneys was removed by portal vein hepatic perfusion, and the irradiated parts were cut out and quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0090] Step 2: Enrichment of RNA-protein cross-linked products in mouse kidneys using streptavidin-modified magnetic beads: The mouse kidney was transferred to a centrifuge tube with grinding balls, and 500 μL of lysis buffer [including Tris-HCl (20mM, pH 7.5), 500 mM lithium chloride, 1 mM EDTA, 5 mM dithiothreitol, 0.5% lithium dodecyl sulfate, RNase inhibitors and EDTA-free protease inhibitors] was added. The kidney was ground using a tissue grinder at 4°C for 1 min, and the supernatant was collected and transferred to a 1.5mL RNase-free centrifuge tube. 80 μL of pre-cleaned streptavidin-modified magnetic beads were added (the pre-cleaning steps were as follows: first use solution A (0.1 M sodium hydroxide, 0.05 M sodium chloride, DEPC-treated water) to wash the magnetic beads twice, then use solution B (0.1 M sodium chloride, DEPC-treated water) to wash the magnetic beads 3 times, and finally resuspend the magnetic beads in 80 μL Tris-HCl), and enrich at 4°C for 90 min. After enrichment, the magnetic beads were collected and washed twice with 200 μL of PBS solution containing 0.2% sodium dodecyl sulfate, twice with 200 μL of PBS solution containing 6 M urea, twice with 200 μL of PBS, and twice with 200 μL of 50 mM sodium bicarbonate aqueous solution to remove nonspecific adsorption and obtain pure RNA-protein in situ cross-linked products; finally, 20 μL of 0.01 μg / μL RNase A solution was added and incubated at 37°C for 1 h to elute and release RBPs.

[0091] At the same time, an RNaes cleaning group was set up. The mouse kidney lysate was enzymatically hydrolyzed with high concentration of RNase at 37°C for 1 hour and then enriched with magnetic beads. Subsequently, the same method as the experimental group was used to process and collect the processed products.

[0092] Step 3: Characterization of RNA-protein in situ cross-linked products by sodium dodecyl sulfate-PAGE gel electrophoresis: Take 20 μL of the treated products of the experimental group and the RNase cleaning group and load them onto 10% sodium dodecyl sulfate-PAGE gel. Keep the voltage at 80 V for 20 min. After the voltage is increased to 120 V, electrophoresis is performed until the indicator bromophenol blue reaches the bottom of the gel and then the electrophoresis is stopped. The gel was transferred to a culture dish and silver-stained using a silver staining kit according to the instructions. The specific steps are as follows: 1. Washing: Wash the gel twice with ultrapure water, 5 min each time; 2. Fixation: Fix the gel twice with 25 mL of fixative (ultrapure water: ethanol: glacial acetic acid = 6:3:1), 15 min each time; 3. Elution: Wash the gel twice with elution solution (10% ethanol), 5 min / time; 4. Washing: Wash the gel twice with ultrapure water, 5 min / time; 5. Sensitization: Place the gel washed in the previous step in silver staining sensitizer working solution (50 μL SilverStain Sensitizer mixed with 25 mL ultrapure water), incubate at room temperature for 1 min, then wash the gel 3 times with ultrapure water, 20 s / time; 6. Silver staining: Discard the ultrapure water, place the gel in silver staining working solution (25 mL Silver Stain added with 50 μL SilverStain Enhancer and mix well) and incubate for 30 min; 7. Washing: Wash the gel quickly twice with ultrapure water, and each wash should be accurately controlled to 20s; 8. Development: Immediately immerse the washed gel in the developer (25 mL Silver Stain Developer added to 30 μL Silver Stain Enhancer and mixed well), and incubate at room temperature until the protein bands are clearly displayed; 9. Stopping: After washing the developer on the gel with the stop solution (5% glacial acetic acid), immerse the gel in the new stop solution for 10 min.

[0093] Silver staining results Fig.11 As shown in the gel image, it is obvious that there are protein bands in the experimental group, while the protein bands in the RNase group disappear, which shows that the enriched protein is dependent on RNA enrichment and is directly related to RNA.

[0094] Example 7 Proteomic Identification of RBPs Enriched by Combining the Hydrophilic Upconversion Nanocomposite with the Compound of Formula 1 The magnetic beads enriched with the in situ cross-linked products of RNA and protein in Example 6 were divided into an experimental group and a control group. The magnetic beads in the experimental group were washed twice with 200 μL of PBS solution containing 0.2% sodium dodecyl sulfate, washed twice with 200 μL of PBS solution containing 6 M urea, washed twice with 200 μL of PBS, and washed twice with 200 μL of 50 mM sodium bicarbonate aqueous solution to remove non-specific adsorption and obtain pure RNA-protein in situ cross-linked products; 20 μL of 0.01 μg / μL RNase A solution was added to the RNA-protein in situ cross-linked products, and the RNA was enzymatically hydrolyzed by incubation at 37°C for 1 h to collect RBPs.

[0095] The magnetic beads in the control group were first incubated with 0.1 μg / μL RNase A solution at 37°C for 1 h to enzymatically hydrolyze RNA, and the supernatant was removed by magnetic separation. The beads were then washed twice with 200 μL PBS solution containing 0.2% sodium dodecyl sulfate, twice with 200 μL PBS solution containing 6 M urea, twice with 200 μL PBS, and twice with 200 μL 50 mM sodium bicarbonate aqueous solution. Finally, 20 μL 0.01 μg / μL RNase A solution was added to resuspend the magnetic beads.

[0096] The treated proteins of the experimental group and the control group were processed as follows: Step 1: Trypsin digestion and dimethylation labeling quantification. The specific steps are as follows: add 80 μL of sodium bicarbonate solution, add dithiothreitol with a final concentration of 10 mM and reduce at 56°C for 45 min. After cooling to room temperature, add iodoacetamide with a final concentration of 20 mM and react in the dark for 45 min; add 0.02 μg of trypsin and digest at 37°C for 12 h, then add the same amount of trypsin and continue digestion at 37°C for 4 h. After the digestion, the supernatant containing the enzymatic peptides was recovered by magnetic separation and desalted using StageTip C18. The steps were as follows: a C18 desalting column was made using a three-layer C18 membrane. The C18 desalting column was activated and balanced with acetonitrile, 50% acetonitrile containing 0.1% trifluoroacetic acid, and 0.1% trifluoroacetic acid in sequence; then the mixed sample was added to the desalting column to retain the peptides in the column, and the salt on the desalting column was removed with 0.1% trifluoroacetic acid. Finally, the peptides were eluted with 50% acetonitrile containing 0.1% trifluoroacetic acid into a new centrifuge tube, and concentrated and dried using a vacuum rotary dryer. The peptides after heat drying were dimethylated. First, the peptides were redissolved in 200 μL 100 mM TEAB buffer solution, and 8 μL 4% formaldehyde solution and 8 μL 0.6 M sodium cyanoborohydride were added to the experimental group, vortexed and mixed, and reacted at 21°C for 1 h. After the reaction was completed, 32 μL of 1% ammonia solution was added to terminate the reaction, and then 16 μL of formic acid was added to acidify the solution. Finally, the light and heavy isotope labeled experimental groups and control groups were mixed, and then desalted using StageTip C18, and the eluted peptides were concentrated and dried using a vacuum rotary dryer.

[0097] Step 2, mass spectrometry identification: The heat-dried peptides were redissolved in 0.1% formic acid solution and centrifuged at 14,000 g for 10 min. The supernatant was loaded onto a 2 cm self-assembled pre-column (100 μm inner diameter, filled with 3 μm inner diameter C18 filler). A homemade reversed-phase analytical column with an inner diameter of 150 μm and a length of 15 cm (filled with Ultimate XB-C18 filler with a particle size of 1.9 μm) was used for separation. Easy nLC 1000 nanoliter liquid chromatography system was used, with a liquid phase gradient of 78 min (mobile phase A: 0.1% formic acid-water solution, mobile phase B: acetonitrile solution containing 0.1% formic acid, flow rate of 600 nL / min), elution gradient: 0-8 min, 5%-8% B; 8-58 min, 8%-22% B; 58-70 min, 22%-32% B; 70-71 min, 32%-90% B; 71-78 min, 90% B. The eluted peptides were sequentially analyzed by the Orbitrap Fusion™Tribrid™ mass spectrometer through the electrospray ion source. The spray voltage was 2.0 kV, the data acquisition mode was data-dependent mode, the scanning method was positive ion scanning mode, the ion transfer tube temperature was set to 320 °C, the primary full scan detection range was 300-1400 m / z, the scanning resolution was 120000, the maximum injection time was 100 ms, the automatic gain control was set to 5e5, and the parent ion was fragmented into secondary fragments using high-energy collision-induced dissociation mode with a collision energy of 32%. The linear ion trap fast mode was used for secondary data acquisition, the AGC was set to 5000, the maximum injection time was set to 35 ms, and the dynamic exclusion time was set to 18 s.

[0098] Step 3. Mass spectrometry data retrieval and analysis: The raw files obtained by mass spectrometry were further analyzed using MaxQuant software and searched using the UniProt Mouse (Release on 202103, 17082 entry) database. Trypsin was selected as the enzymatic mode, the maximum allowed missed cleavage sites were set to 2, the minimum number of amino acids per peptide was 6, cysteine ​​carbamidomethylation was set as a fixed modification, and methionine oxidation and N-terminal acetylation were set as variable modifications. For dimethylation labeling, check DimethLys0 and DimethNter0 for the light label, and check DimethLys4 and DimethNter4 for the heavy label. The maximum mass tolerances for the parent ion and secondary fragment ion were 20 ppm and 0.5 Da, respectively, and the false positive rate of the protein level and the spectrum was no more than 1%. The missing values ​​were filled according to the minimum value filling principle, and the enrichment multiple of each protein (the ratio of the signal intensity of the experimental group to the signal intensity of the control group) was calculated by Persus software, and the degree of enrichment significance was calculated by T-test, which was expressed by P-Value. The smaller the P-Value, the more significant the difference between the experimental group signal and the control group signal. The obtained data were screened to obtain RBPs according to the following principles: 1. The protein was identified at least twice in three experiments; 2. The protein contained at least 2 specific peptides; 3. The enrichment multiple of the experimental group / control group was not less than 2 times and P-Value < 0.01; those that met the above three conditions were considered to be high-confidence RBPs.

[0099] The results are as follows Fig.12 As shown in the figure, the left side is a scatter plot of proteins identified in mouse kidney, and a total of 1709 highly reliable RBPs were identified. The enrichment multiples of RBPs in the three groups of biological replicates were further compared pairwise, and it was found that there was a good correlation between each two groups. The results are shown in Fig.12 As shown in the right side of the figure, the correlation analysis of RBPs identified in mouse kidney, the calculated Pearson correlation coefficient is above 0.90, indicating that the RBPs identified by mass spectrometry in the three groups of biological replicates have similar enrichment multiples, proving that the enrichment method provided by the present invention has good reproducibility.

[0100] Gene annotation analysis was performed on the identified RBPs, and the results were as follows Fig.13 As shown, from the analysis results, it was found that the most enriched items were all related to RNA. For example, the most enriched items in biological processes were mostly related to "translation", and the most enriched items in molecular functions were all "RNA binding". This shows that the enriched RBPs have high credibility, and also proves that the method provided by the present invention has the ability to enrich and identify RBPs in living animals.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for in situ labeling and / or enrichment and / or analysis of RNA-RNA binding protein complexes and / or RNA binding proteins in a biological sample, characterized in that: include: contacting a hydrophilic upconversion nanocomposite material and a photosensitizer with a biological sample to be tested, and after the hydrophilic upconversion nanocomposite material and the photosensitizer enter a target position of the biological sample to be tested, irradiating the target position of the biological sample to be tested with near-infrared light, so as to promote in situ cross-linking between RNA and RNA-binding protein in the target position of the biological sample to be tested, thereby forming an RNA-RNA binding protein complex; Using the probe to label and / or enrich and / or analyze the RNA-RNA binding protein complex and / or RNA binding protein in the biological sample to be tested; The hydrophilic upconversion nanocomposite material comprises upconversion nanoparticles and a hydrophilic layer coated on the surface of the upconversion nanoparticles; the upconversion nanoparticles have a heterogeneous core-multi-shell structure, can absorb near-infrared light and emit at least ultraviolet light with a wavelength of 254 nm and visible light with a wavelength of 390 nm-780 nm, which is the same as the absorption spectrum of the photosensitizer.

2. The method according to claim 1, characterized in that: The biological sample to be tested is an organism or at least one of cells, tissues and organs derived from an organism.

3. The method according to claim 1 or 2, characterized in that: The up-conversion nanoparticles are fluorides formed by rare earth elements and sodium elements; The rare earth element is selected from at least one of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, and Y.

4. The method according to any one of claims 1 to 3, characterized in that: The upconversion nanoparticles include a core particle and a first layer, a second layer and a third layer sequentially coated on the surface of the core particle; wherein the core particle is doped with rare earth elements Gd, Yb and Tm, the first layer is doped with rare earth elements Y and Yb, the second layer is doped with rare earth elements Gd, Yb and Nd, and the third layer is doped with rare earth element Gd.

5. The method according to any one of claims 1 to 4, characterized in that: The hydrophilic layer is biocompatible.

6. The method according to any one of claims 1 to 5, characterized in that: The probe is selected from at least one of a singlet oxygen detection probe, a labeling probe, and an enrichment probe; the labeling probe comprises a compound as shown in Formula 1 and streptavidin labeled with a fluorescent group; the enrichment probe comprises a compound as shown in Formula 1 and magnetic beads modified with streptavidin; 。 7. A product for in situ labeling and / or enrichment and / or analysis of RNA-RNA binding protein complexes and / or RNA binding proteins in a biological sample, characterized in that: The invention comprises the hydrophilic upconversion nanocomposite material, a photosensitizer and a probe in the method according to any one of claims 1 to 6.

8. A hydrophilic upconversion nanocomposite material, characterized in that: It comprises upconversion nanoparticles and a hydrophilic layer coated on the surface of the upconversion nanoparticles; the upconversion nanoparticles have a heterogeneous core-multi-shell structure, can absorb near-infrared light and emit ultraviolet light with 254 nm and visible light with a wavelength of 390 nm to 780 nm.

9. A compound, characterized in that The structure of the compound is shown in Formula 1: 。 10. The method for preparing the compound according to claim 9, characterized in that: include: The compound of formula 1 is synthesized by amidation reaction using biotin-PEG2-NH3 and 3-(2-oxopyrrolidine) propionic acid as substrates. The reaction process involved is shown in formula 2: 。

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