CN109608396B - Composite probe composition for MicroRNA detection and application thereof - Google Patents

Abstract

The composite probe composition for MicroRNA detection comprises a fluorescent component and a phenylphosphine component, wherein the fluorescent component is a compound of a general formula I, and the phenylphosphine component is a compound of a general formula II. The fluorescent probe molecule is connected with a specific sequence DNA molecule to form a dye-DNA composite probe, and after the dye-DNA composite probe is combined with target MicroRNA through base complementary pairing hybridization, the reaction sites of the two molecules undergo Staudinger reaction, so that the fluorescence intensity of a fluorescence part quenched by an azide group is greatly improved. Therefore, the method can be used for detecting the target MicroRNA in biological samples such as living cells, tissues, blood and the like in a physiological environment under mild conditions with high selectivity and high sensitivity. In addition, the composition has the advantages of good solubility, high sensitivity, no influence of common oxides and reducing substances in the body, and the like.

Description

Composite probe composition for MicroRNA detection and application thereof

Technical Field

The invention belongs to the field of fine chemical engineering, and relates to a specific composite probe composition kit for detecting MicroRNA in living cells and other biological samples and application thereof.

Background

MicroRNAs are a type of single-stranded small-molecule RNA with the length of 18-24 nt. Research shows that MicroRNAs have close relationship with various tumorigenesis, and the MicroRNAs can play an important regulation and control role in the tumorigenesis and development process like oncogenes or cancer suppressor genes. Among many cancers, pancreatic cancer is a highly malignant tumor with low diagnosis rate, early metastasis, and insensitivity to radiotherapy and chemotherapy. Therefore, if early diagnosis of pancreatic cancer can be realized by the MicroRNAs, the MicroRNAs have important significance for treating and curing the pancreatic cancer.

It has been demonstrated that the expression profile of serum MicroRNAs is considered to be a very valuable diagnostic biomarker, since the presence of MicroRNAs in the circulation is well established. It is estimated that MicroRNAs can target more than 30% of the human genome, so the function and normal expression of MicroRNAs during physiological processes are important.

Research in the biological field related to the MicroRNAs has made great progress, so that the MicroRNAs are becoming a novel biological target molecule tool for researching intracellular regulation and providing a new target for designing and detecting for scholars. The abnormal expression of MicroRNAs is closely related to the occurrence of various cancers and serves as a novel biomarker, and MicroRNAs have great potential in the aspect of treating cancers, for example, the abnormal expression of tumor MicroRNAs is monitored to mark a novel method for cancer chemotherapy. In addition, abnormal expression of MicroRNAs is also detected in many other diseased tissues or cells. In the process of disease occurrence, a brand-new disease treatment method can be researched by regulating and controlling the expression or function of abnormal MicroRNAs. Exogenous molecules with a regulatory effect can sensitively disturb endogenous MicroRNAs, so that the exogenous molecules can be used as a powerful tool for regulating the MicroRNAs. Analyzing the expression of MicroRNAs in a tissue or cell sample can provide important information for researching the biological functions of the molecules, so that the detection of the MicroRNAs has important significance for molecular diagnosis of diseases and the development of novel biological medicine preparations.

Disclosure of Invention

The invention aims to provide a tool molecule which can be used for specific identification and detection of target MicroRNA so as to realize high-sensitivity detection of the target MicroRNA in a biological sample.

In the technical scheme of the invention, firstly, a composite probe composition for MicroRNA detection is provided, which comprises a fluorescent component and a phenylphosphine component,

wherein the fluorescent component is a compound of a general formula I, and the phenylphosphine component is a compound of a general formula II:

in formula I:

said L_IIs- (CH)₂)_m-, said m is an integer of 0 to 8;

f is selected from formula F₁-F₄The compound of (1):

formula F₁-F₄In, R₃-R₁₂Each independently selected from H, halogen, phenyl, substituted phenyl, C_1-8Substituted or unsubstituted alkyl of (a); the substituted phenyl is formed by halogen and C_1-6Alkyl or C_1-6Alkoxy is optionally substituted; the substituted alkyl is selected from halogen or C_1-6The alkoxy group is optionally substituted,

in formula II:

said R₁And R₂Each independently selected from phenyl or substituted phenyl; the substituted phenyl is represented by C_1-8Alkyl, halogen, C_1-6Alkoxy is optionally substituted;

said L_IIIs phenyl or- (CH)₂)_n-, said n is an integer of 1 to 8.

The composite probe composition forms a composite detection molecule of a dye molecule and DNA after connecting carboxyl carried by the composite probe composition with a specific sequence DNA molecule modified by amino, and after the composite probe composition is combined with target MicroRNA through base complementary pairing hybridization, the reaction sites of the two molecules generate Staudinger reaction, so that the fluorescence intensity of a naphthalimide part quenched by an azide group is greatly improved, and the composite probe composition can realize high-selectivity and high-sensitivity detection on the target MicroRNA in biological samples such as living cells, tissues and the like in a physiological environment under mild conditions. Meanwhile, the probe has the advantages of good solubility, no influence of common oxides and reducing substances in a receptor, two-photon excitation (the excitation wavelength is 785nm) and the like, and can reach 1.329 multiplied by 10^{- 15}Lower detection limit of M.

Based on the above, the invention provides a method for detecting MicroRNA, which uses the composite probe composition of the invention, and comprises the following steps:

firstly, based on a MicroRNA sequence to be detected, a complementary single-stranded DNA sequence is obtained according to the Watson-Crick base complementary pairing principle; the 5' -end part sequence of the single-stranded DNA sequence is DNA_IThe 3' terminal part sequence is DNA_II，

② Single-stranded DNA_IConnecting with a compound of a general formula I to obtain a detection molecule I; single-stranded DNA_IIConnecting with a compound of a general formula II to obtain a detection molecule II;

thirdly, adding the detection molecule i and the detection molecule ii into a system to be detected according to the molar ratio of 1:1, and judging whether the system contains the MicroRNA to be detected according to the change of the system fluorescence.

Further, the invention provides a detection reagent for specific target MicroRNA, which contains an effective dose of the fluorescent probe with the structure of formula I, II. The detection reagent is used for marking and detecting cancer cells with abnormal expression of a specific MicroRNA. And is suitable for single-photon and double-photon confocal fluorescence imaging detection means. The reagent can realize effective depth discrimination and fluorescence imaging of tumor tissues in normal tissues and tumor tissues, and can be used for early detection of specific cancer cells in laboratory or clinical biological samples.

Drawings

The invention is shown in figure 16, which is as follows:

FIG. 1 is a graph showing the change of the fluorescent probe according to the present invention with concentration and the corresponding linear relationship. Wherein, FIG. 1A is the fluorescence spectrum of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 under the condition of MicroRNA with different concentrations, which are involved in the test in the embodiment, and FIG. 1B is the linear relation graph of the target MicroRNA addition and the fluorescence intensity value.

FIG. 2 is a graph of the variation of the control probe with concentration and the corresponding linear relationship. Wherein, FIG. 2C is the fluorescence spectrum of the fluorescent probes I-LMX-SEQ ID NO.2 and DB under the condition of MicroRNA with different concentrations, which are involved in the test in the embodiment, and FIG. 2D is the linear relationship between the target MicroRNA addition and the fluorescence intensity value.

FIG. 3 is a graph showing the comparison of the fluorescence intensity of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 to be tested in this example with that of the comparative probes I-LMX-SEQ ID NO.2 and DB.

FIG. 4 is a graph showing the comparison of the specific recognition fluorescence intensities of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 referred to in the test in this example. The targets were MicroRNA (50nM) and non-matching RNA templates, mis-a and mis-b, which differ by only one base (100 nM each).

FIG. 5 is a graph showing the comparison of the selective fluorescence intensities of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 referred to in the test in this example with time. The test substances are glutathione, homocysteine, cysteine, creatine, hydantoin, phenylboronic acid, glucose, bovine serum albumin, human serum albumin, and H₂S (100 eq each 5 mM), bars increasing with time 0.5h, 1h, 1.5h, 2h, 3h, 4h, 5 h.

FIG. 6 is a graph showing the comparison of the anti-interference fluorescence intensity of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3, which are tested in this example.

FIG. 7 is a single-photon confocal microscopy image of fluorescence intensity changes of fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 in NIH-3T3 cells with time in the present example.

FIG. 8 is a single-photon confocal microscopy image of the fluorescence intensity contrast of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 (first row), I-LMX-SEQ ID NO.5 and II-LMX-SEQ ID NO.6 (second row), I-LMX-SEQ ID NO.8 and II-LMX-SEQ ID NO.9 (third row) in different cells in the present example, with reference to the test.

FIG. 9 is a graph showing the fluorescence intensity of the RNA extracts of different cells with respect to the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 tested in this example.

FIG. 10 is a bar graph showing comparison of fluorescence intensity values obtained by quantifying the fluorescence intensity of the cells in FIG. 8 using FV10-ASW3.0viewer software.

FIG. 11 is a comparison of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 involved in the test of this example with the fluorescent probes I-LMX-SEQ ID NO.2 involved in the test of this example when added alone in an intracellular single-photon confocal microscopy.

FIG. 12 is a two-photon confocal microscopy image of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 participating in the test in three different cells in this example.

FIG. 13 is a two-photon confocal fluorescence depth microscopy image of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 used in the mice normal tissue (a-g) and the mice tumor tissue (h-n) in this example with reference to the test.

FIG. 14 is a two-photon confocal fluorescence depth map of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 participating in the test in the normal tissues of mice in this example.

FIG. 15 is a two-photon confocal fluorescence depth map of the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 used in mouse tumor tissues in this example.

FIG. 16 is a graph showing the fluorescence intensity of the fluorescent probes I-LMX-SEQ ID NO.5 and II-LMX-SEQ ID NO.6 and I-LMX-SEQ ID NO.8 and II-LMX-SEQ ID NO.9 involved in the test in this example in actual blood sample detection.

Detailed Description

The invention firstly provides a composite probe composition for MicroRNA detection, which comprises a fluorescent component and a phenylphosphine component, wherein the fluorescent component is a compound of a general formula I, and the phenylphosphine component is a compound of a general formula II:

in formula I:

said L_IIs- (CH)₂)_m-, said m is an integer of 0 to 8, m is preferably an integer of 0 to 6, more preferably an integer of 0 to 3; most preferably m-2;

f is selected from formula F₁-F₄In which in turn F₁The group (2) is more suitable:

formula F₁-F₄In, R₃-R₁₂Each independently selected from H, halogen, phenyl, substituted phenyl, C_1-8Substituted or unsubstituted alkyl of (a); the substituted phenyl is formed by halogen and C_1-6Alkyl or C_1-6Alkoxy is optionally substituted; the substituted alkyl is selected from halogen or C_1-6Alkoxy is optionally substituted; in a preferred technical scheme, R is₃-R₁₂Each independently selected from hydrogen and C_1-6Alkyl and phenyl; more preferably, R is₃-R₁₂Each independently selected from hydrogen and C_1-3Alkyl groups of (a); most preferably, said R₃-R₁₂Are all hydrogen;

in formula II:

said R₁And R₂Each independently selected from phenyl or substituted phenyl; the substituted phenyl is represented by C_1-8Alkyl, halogen, C_1-6Alkoxy is optionally substituted. Preferably, R is₁And R₂Each independently selected from phenyl or methylphenyl. Most preferably, R is₁And R₂Are all phenyl groups.

Said L_IIIs phenyl or- (CH)₂)_n-n is an integer from 1 to 8, preferably n is an integer from 1 to 6, more preferably n is an integer from 1 to 3, and most preferably n ═ 2.

In a more specific technical scheme, the composite probe composition comprises compounds of formula I-LMX and formula II-LMX:

the invention further provides a preparation method of the composite probe composition, which comprises the following steps:

A. general formula I

(1) When F ═ F₁Of compounds of the formula IThe preparation method comprises the following steps:

firstly, reacting a compound 1 with sodium azide according to a molar ratio of 2:3 to prepare a (intermediate) compound 2; the solvent for the reaction is N, N-dimethylformamide; the temperature of the reaction system is room temperature; the reaction time is 5-10 h;

② the compound 2 and the compound 3 are mixed according to the mol ratio of 1: 1.3-1: 1.5 in the presence of carbonyl activating reagent and then react to obtain F ═ F₁A dye molecule of formula I; the carbonyl activating reagent is 4-dimethylamino pyridine; the reaction solvent is ethanol; the temperature of the reaction system is the reflux temperature of the ethanol; the reaction time is 4-8 h;

(2) when F ═ F₂A process for the preparation of a compound of formula I, comprising the steps of:

firstly, reacting a compound 4 with a compound 5 according to a molar ratio of 1: 1-0.8: 1 to prepare a compound 6; the solvent of the reaction is ethyl acetate; the temperature of the reaction system is 78 ℃; the reaction time is 0.5-1.5 h;

reacting the compound 6 with the compound 7 according to a molar ratio of 1: 1.5-1: 2 to prepare a prepared compound 8; the solvent of the reaction is ethanol; the temperature of the reaction system is 80 ℃; the reaction time is 2-4 h;

reacting the compound 8 with sodium azide according to a molar ratio of 1: 1.2-1: 1.5 to obtain F ═ F₂A dye molecule of formula I; the solvent of the reaction is DMSO; cuprous iodide as a catalyst; the temperature of the reaction system is room temperature; the reaction time is 3-8 h;

(3) when F ═ F₃A process for the preparation of a compound of formula I, comprising the steps of:

compound 9 and compound 10 (R)₇/R₈) Reacting according to a molar ratio of 1: 0.5-0.8 to prepare an (intermediate) compound 11; the solvent of the reaction is trifluoroacetic acid; the temperature of the reaction system is room temperature; the reaction time is 1-3 h;

reacting the compound 11 with N-chlorosuccinimide according to a molar ratio of 1: 1.1-1: 1.3 to prepare an intermediate compound 12; the solvent of the reaction is tetrahydrofuran; the temperature of the reaction system is-78 ℃; the reaction time is 0.5-2 h;

③ reacting the compound 12 with p-chlorobenzoquinone according to the molar ratio of 1: 0.8-1: 1.2 to prepare an (intermediate) compound 13; the solvent of the reaction is dichloromethane; the temperature of the reaction system is room temperature; the reaction time is 0.5-1.5 h;

fourthly, reacting the compound 13 with boron trifluoride diethyl etherate in the presence of triethylamine serving as an alkali according to a molar ratio of 1: 15-1: 20 to prepare a (intermediate) compound 14; the solvent of the reaction is toluene; the temperature of the reaction system is 110 ℃; the reaction time is 2-4 h;

reacting 14 with sodium azide according to a molar ratio of 1: 2-1: 5 to obtain F ═ F₃A dye molecule of formula I; the solvent of the reaction is acetonitrile; the temperature of the reaction system is room temperature; the reaction time is 3-5 h.

(3) When F ═ F₄A process for the preparation of a compound of formula I, comprising the steps of:

compound 15 and compound 16 (R)₁₀/R₁₁) Reacting in the presence of acid perchloric acid at a molar ratio of 1:1.5 to 1:2.0 to prepare an (intermediate) compound 17; the solvent of the reaction is water; the temperature of the reaction system is 80 ℃; the reaction time is 0.5-1.5 h;

reacting compound 17 with sodium nitrite in the presence of hydrochloric acid according to a molar ratio of 1: 1.5-1: 2, and then adding sodium azide according to a molar ratio of 1: 3-1: 5 to prepare F ═ F₄A dye molecule of formula I; the solvent of the reaction is water; the temperature of the reaction system is 0 ℃; the reaction time was 48 h.

B. A process for the preparation of a compound of formula II, which process comprises the steps of:

reacting the compound 18 with the compound 19 according to a molar ratio of 1: 1.5-1: 2, and then adding dilute hydrochloric acid into a reaction system for acidification to obtain a molecular structure shown in a formula II; the reaction solvent is dimethyl sulfoxide; the temperature of the reaction system is room temperature; the reaction time is 12-18 h.

Wherein the molecule of formula I is linked to the amino-modified specific sequence DNA molecule via its carboxyl group and purified by HPLC.

Based on the composite probe composition of the invention, the invention further provides a detection method of MicroRNA, which comprises the following steps:

firstly, based on a MicroRNA sequence to be detected, a complementary single-stranded DNA sequence is obtained according to the Watson-Crick base complementary pairing principle; the 5' -end part sequence of the single-stranded DNA sequence is DNA_IThe 3' terminal part sequence is DNA_II；

In a specific embodiment, for example, based on the sequence of MicroRNA to be detected, a complementary single-stranded DNA sequence with the base number of x is obtained according to Watson-Crick base complementary pairing principle, wherein x is 18-An integer of 24; when x is an odd number, 1 to (x-1)/2 bases from the 5 'end to the 3' end of the resulting complementary single-stranded DNA sequence constitute a single-stranded DNA_I(x +3)/2 to x bases constituting a single-stranded DNA_II(ii) a When x is an even number, 1 to x/2-1 bases from the 5 'end to the 3' end of the resulting complementary sequence constitute a single-stranded DNA_IX/2+1 to n bases constituting a single-stranded DNA_II；

② Single-stranded DNA having 5' -terminus modified with amino group_IConnecting with a compound of a general formula I to obtain a detection molecule I; single-stranded DNA having 3' -end modified with amino group_IIConnecting with a compound of a general formula II to obtain a detection molecule II;

thirdly, adding the detection molecule i and the detection molecule ii into a system to be detected according to the molar ratio of 1:1, and judging whether the system contains the MicroRNA to be detected according to the change of the system fluorescence; the concentrations of both in vitro assays were 1. mu.M each, and in cell experiments 2. mu.g/. mu.L each. The detection molecule i and the detection molecule ii are carried out by DNA_IAnd DNA_IIThe DNA is matched with the target MicroRNA through a base complementary pairing principle_IThe 5' end of (A) is linked to a molecule of the general formula I_IIThe molecular distance of the general formula II connected with the 3' end is shortened, the Staudinger reaction can be carried out between the azide group and the diphenylphosphine group, the fluorescence intensity of the fluorescent dye part quenched by the azide group can be greatly improved in the process, and the target MicroRNA in biological samples such as living cells, tissues and blood can be detected in a physiological environment under mild conditions in a high-selectivity and high-sensitivity manner.

Further, the present specification provides, by way of example, 3 sets of target MicroRNAs to be detected, and corresponding DNAs in the method of the present invention_I、DNA_IIThe base sequence is designed to explain the present invention, but it should not be construed as limiting the present invention, particularly to the detection of MicroRNA. As can be seen from the above description, the present invention has no strict limitation on the target MicroRNA to be detectedAnd (4) determining. These groups are exemplified by MicroRNA and the corresponding DNA_IAnd DNA_IIRespectively as follows:

firstly, the MicroRNA to be detected has a base sequence of SEQ ID NO.1, and the DNA_IAnd DNA_IIRespectively has base sequences of SEQ ID NO.2 and SEQ ID NO. 3;

SEQ ID NO.1:5’-GAA AGA CAG UAG AUU GUA UAG-3’(Let-7a)

SEQ ID NO.2:5’-ACT GTC TTT C-3’

SEQ ID NO.3:5’-CTA TAC AAT C-3’

② the MicroRNA to be detected has a base sequence of SEQ ID NO.4, the DNA_IAnd DNA_IIHaving base sequences of SEQ ID NO.5 and SEQ ID NO.6, respectively;

SEQ ID NO.4:5’-UGG CUC AGU UCA GCA GGA ACA G-3’(Mir-24)

SEQ ID NO.5:5’-AAC TGA GCC A-3’

SEQ ID NO.6:5’-TCG TCC TTG TC-3’

③ the MicroRNA to be detected has the base sequence of SEQ ID NO.7, and the DNA_IAnd DNA_IIHas base sequences of SEQ ID NO.8 and SEQ ID NO.9, respectively.

SEQ ID NO.7:5’-UGU AAA CAU CCU ACA CUC UCA GC-3’(Mir-30c)

SEQ ID NO.8:5’-GGA TGT TTA CA-3’

SEQ ID NO.9:5’-GCT GAG AGT GT-3’

The fluorescent probe composition of the present invention is most specifically represented by the following formula I-LMX-DNA_I、II-LMX- DNA_IIThe structure of (1):

in order to compare the test effect, a group of comparison probes is additionally designed according to the structure, and the structure is as follows:

the invention provides a preparation method of the dye molecule with the structure of formula I-LMX, which comprises the following steps:

(1) reacting the compound 20 with sodium azide according to a molar ratio of 2:3 to prepare a (intermediate) compound 21;

(2) mixing a compound 21 and a compound 22 in a molar ratio of 1: 1.3-1: 1.5 in the presence of a carbonyl activating reagent, and reacting to obtain a dye molecule I-LMX;

in a specific embodiment, the fluorescent probe of the present invention can be prepared by the following method:

(1) dissolving 2.77g (10mmol) of compound 20 in 50ml of N, N-dimethylformamide as a reaction solvent; subsequently, 0.957g (15mmol) of sodium azide was slowly and carefully added to 2ml of water to completely dissolve it. And slowly adding an aqueous solution of sodium azide into the reaction system, and stirring at room temperature for 5 hours. And then adding ice water into the reaction system to generate a large amount of solid, and performing suction filtration and washing to obtain the compound 21.

(2) 10ml of ethanol as a reaction solvent, and 239mg (1mmol) of compound 21 and 134.06 mg (1.3mmol) of compound 22, in that order, followed by 12.2mg (0.1mmol) of 4-Dimethylaminopyridine (DMAP), were added. Heating and refluxing for 4h, cooling after the reaction is finished, and performing column chromatography separation to obtain a yellow I-LMX compound.

1H NMR(400MHz,DMSO-d₆),δ:12.02(s,1H),8.47(d,J＝7.2Hz,1H),8.45(d,J＝ 7.3Hz,1H),8.39(d,J＝8.4Hz,1H),7.85(t,J＝7.9Hz,1H),7.73(d,J＝8.0Hz,1H),4.07 (t,J＝6.9Hz,2H),2.31(t,J＝7.3Hz,2H),1.92–1.85(m,2H).

The structure of 13C NMR (126MHz, DMSO), delta: 173.91,163.32,162.87,142.73,131.51,131.43, 128.34,128.24,127.23,123.50,122.21,118.24,115.89,31.30,22.97.I-LMX was characterized by nuclear magnetism.

The subsequent step of linking the carboxyl group of compounds 22, 3- (diphenylphosphine) propionic acid and 4-pentynoic acid to amino-modified DNA molecule of specific sequence was carried out by Takara Bio Inc. to obtain composite probe composition structure:

SEQ ID NO.1: let-7a (5'-GAA AGA CAG UAG AUU GUA UAG-3') has 21 bases,

I-LMX-SEQ ID NO.2：

in this case, I-LMX-DNA_IDNA of middle origin_I5'-ACT GTC TTT C-3';

II-LMX-SEQ ID NO.3：

in this case II-LMX-DNA_IIDNA of middle origin_IIIs 5'-CTA TAC AAT C-3'

DB：

In this case II-LMX-DNA_IIDNA of middle origin_IIIs 5 '-CTA TAC AAT C-3'

SEQ ID NO. 4: mir-24(5 '-UGG CUC AGU UCA GCA GGA ACA G-3') has 22 bases,

I-LMX-SEQ ID NO.5：

in this case, I-LMX-DNA_IDNA of middle origin_I5'-AAC TGA GCC A-3';

II-LMX-SEQ ID NO.6：

in this case, I-LMX-DNA_IIDNA of middle origin_II5'-TCG TCC TTG TC-3';

SEQ ID NO. 7: mir-30c (5'-UGU AAA CAU CCU ACA CUC UCA GC-3') has 23 bases,

I-LMX-SEQ ID NO.8：

in this case, I-LMX-DNA_IDNA of middle origin_I5'-GGA TGT TTA CA-3';

II-LMX-SEQ ID NO.9：

in this case, I-LMX-DNA_IIDNA of middle origin_II5'-GCT GAG AGT GT-3';

the following examples are presented to enable one of ordinary skill in the art to more fully understand the teachings of the present invention and are not intended to limit the invention in any way. Unless otherwise specified, the fluorescent probes described in the following examples are DNA of the formula I-LMX-DNA synthesized by the above method and structurally characterized_I、II-LMX-DNA_IIAnd a fluorescent probe for DB.

Example 1

The intensity of the response of the fluorescent probe to different concentrations of target MicroRNA (SEQ ID NO.1) in PBS (10mM pH 7.4) buffer was examined.

Preparing the probe molecules I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 into mother liquor. Then, sampling was performed with a microsyringe to ensure that the total test system was 100. mu.L, and test fluorescent probe molecules (each 1. mu.M) and target MicroRNA concentrations were added thereto at from 10fM, 50fM, 100fM, 1pM, 100pM, 10nM, and 50 nM. The fluorescence spectrum was scanned by a microplate reader to obtain a fluorescence spectrum as shown in FIG. 1A. The fluorescence intensity value at the position of the maximum emission peak is taken as the ordinate, the target object to be measured is taken as the abscissa, and the linear relationship between the addition amount and the fluorescence intensity value is drawn (fig. 1B). It was found that the concentration and the absorption intensity showed a strong linear relationship (R) with increasing concentration²0.99659) indicating that the fluorescent probes I-LMX-SEQ ID No.2 and II-LMX-SEQ ID No.3 referred to in the test in this example were buffered in PBS (10mM pH 7.4)Has better response to target MicroRNA (SEQ ID NO.1) in the solution.

Example 2

The intensity of the response of the comparative probes I-LMX-SEQ ID NO.2 and DB to different concentrations of the target MicroRNA (SEQ ID NO.1) in PBS (10mM pH 7.4) buffer solution was examined.

Preparing the probe molecules I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 into mother liquor to prepare the mother liquor. Then, sampling was performed with a microsyringe to ensure that the total test system was 100. mu.L, and probe molecules (1. mu.M each) and SEQ ID NO.1 were added thereto at concentrations of from 10nM, 20nM, 30nM, 60nM, 100 nM. The fluorescence spectrum was scanned by a microplate reader to obtain a fluorescence spectrum as shown in FIG. 2C. The fluorescence intensity value at the position of the maximum emission peak is taken as the ordinate, the target object to be measured is taken as the abscissa, and the linear relationship between the addition amount and the fluorescence intensity value is drawn (fig. 2D). It was found that with increasing concentration, the concentration and the absorption intensity showed a linear relationship (R)²0.95742). A bar graph comparing the fluorescence intensity values as shown in FIG. 3 is plotted, indicating that the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 referred to in this example were more responsive in PBS (10mM pH 7.4) buffer than the comparative probes to the target MicroRNA (SEQ ID NO. 1).

Example 3

The selectivity test of the fluorescent probe of the invention is examined.

Preparing the probe molecules I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 into mother liquor. Then, sampling was performed with a microsyringe so as to ensure that the total test system was 100. mu.L, probe molecules (1. mu.M each) were added thereto, followed by addition of the target MicroRNA (SEQ ID NO.1) at a concentration of 50nM and two non-matching RNA templates mis-a and mis-b (100 nM each) differing in only one base, respectively, and the fluorescence spectra thereof were scanned with a microplate reader, and a bar graph comparing the fluorescence intensity values as shown in FIG. 4 was plotted. The increase in fluorescence intensity was found to be exhibited only in the presence of the target MicroRNA (SEQ ID NO.1), indicating that the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 referred to in this example have good selectivity in a simulated physiological environment.

Example 4

The interference resistance of the fluorescent probe of the present invention was examined.

Preparing the probe molecules I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 into mother liquor. Then sampling with a microsyringe to ensure that the total test system is 100 μ L, adding probe molecules (each 1 μ M), and respectively adding small molecules of glutathione, homocysteine, cysteine, creatine, hydantoin, phenylboronic acid, glucose, bovine serum albumin, human serum albumin, and H, which are common in cells₂S (5 mM 100eq each), increasing with time 0.5h (black), 1h (red), 1.5h (blue), 2h (pink), 3h (green), 4h (orange), 5h (violet), the fluorescence spectrum of which was scanned with a microplate reader, and a bar graph of fluorescence intensity values versus time as shown in FIG. 5 was plotted. The fluorescence intensity enhancement is only shown when the target MicroRNA (SEQ ID NO.1) exists, and no obvious response is found to other small molecules, which indicates that the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 participating in the test in the embodiment have good anti-interference performance under the simulated physiological environment.

Example 5

The stability of the fluorescent probe of the present invention was examined.

Preparing the probe molecules I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 into mother liquor. Then, sampling was performed with a microsyringe to ensure that the total test system was 100. mu.L, probe molecules (each 1. mu.M) were added thereto, and oxides (TCEP), reductants (hydrogen peroxide) and sulfur-containing compounds (cysteine) (each 5mM, 100eq) which are common in cells were added, respectively, and at the same time, the target MicroRNA (SEQ ID NO.1, 50nM) was added, and the fluorescence spectrum thereof was scanned with a microplate reader to draw a bar graph showing a comparison of fluorescence intensity values as shown in FIG. 6. The added active substances are found not to influence the response intensity of the probe of the invention to the target MicroRNA (SEQ ID NO.1), which shows that the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 participating in the test in the embodiment have better stability in the simulated physiological environment.

Example 6

The fluorescent probe can realize the function of distinguishing normal cells from cancer cells by detecting target MicroRNA (SEQ ID NO. 1).

Selecting normal cell mouse embryo fibroblast NIH-3T3, respectively adding 2 mug/ml probe molecule I-LMX-SEQ ID NO.2 and 2 mug/ml probe molecule II-LMX-SEQ ID NO.3, and performing fluorescence confocal imaging once every half hour. As shown in fig. 7, the fluorescence intensity reached a maximum in all four cells after 4 hours.

Example 7

In order to better illustrate that the fluorescent probe can realize the function of distinguishing normal cells from cancer cells, the fluorescent probe is used for the confocal fluorescent imaging contrast experiment of cancer cells (human pancreatic cancer PANC-1, SW1990, Bxpc-3, human breast cancer MCF-7) and normal cells (mouse embryo fibroblast NIH-3T3, human normal pancreatic cell HPDE 6-C7). Each group of cells is incubated for 4 hours for imaging after adding the fluorescent probe (each 2 mug/ml), the first group is added with I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3, the second group is added with I-LMX-SEQ ID NO.5 and II-LMX-SEQ ID NO.6, and the third group is added with I-LMX-SEQ ID NO.8 and II-LMX-SEQ ID NO. 9. It was found that SEQ ID NO.1 was down-regulated in pancreatic cancer cells and breast cancer cells, while SEQ ID NO.4 was up-regulated in pancreatic cancer cells and breast cancer cells, and SEQ ID NO.7 was up-regulated in pancreatic cancer cells and down-regulated in breast cancer cells. As shown in FIG. 8, it is further demonstrated that the three groups of fluorescence tested in this example have the function of distinguishing normal cells from cancer cells.

Example 8

To demonstrate that the fluorescent signal generated by the fluorescent probe of the present invention in the cell is indeed a response signal to the target MicroRNA (SEQ ID NO.1), we tested all RNA extracts of four cells. Total RNA from cancer cells (human pancreatic cancer PANC-1, SW1990, human breast cancer MCF-7) and normal cells (mouse embryonic fibroblast NIH-3T3) was extracted using TRIzol reagent, chloroform, isopropanol, 75% ethanol and sterilized ultrapure water. Using NanoDrop^TMThe total amount of extracted RNA was calibrated by UV spectrophotometer to maintain the ratio of A260 nm/A280nm between 1.8 and 2.0, indicating that the extracted RNA was purer and less residual protein and compounds were present. Subsequently, the concentration of the four cellular RNA extracts was adjusted to 200 ng/. mu.l, and I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 (each)1. mu.M), using NanoDrop^TMThe 3300 spectrofluorometer scans its fluorescence spectrum, and takes the fluorescence intensity value at the position of its maximum emission peak as the ordinate to draw a bar graph comparing the fluorescence intensity values as shown in fig. 9. Subsequently, we quantified the fluorescence intensity of the cells in FIG. 8 using FV10-ASW3.0viewer software to make a bar graph of fluorescence intensity value comparison (FIG. 10). The result shows that the fluorescent probe has better anti-interference performance, and the obtained signal is the fluorescent signal generated on the target MicroRNA.

Example 9

In order to intuitively show that the fluorescent probe tool can better avoid false positive signals, normal cell mouse embryonic fibroblast NIH-3T3(A), human pancreatic cancer cell PANC-1(B), SW1990 (C) and human breast cancer MCF-7(D) are still selected, and then I-LMX-SEQ ID NO.2 is independently added into four cells to be compared with single-photon confocal imaging experiments in which I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 are completely added into four cells. After the cells in each group were incubated for 4 hours with fluorescent probes (2. mu.g/ml each), it was found that the fluorescence intensity of only adding I-LMX-SEQ ID NO.2 was significantly lower than that of the cells with complete addition of I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3, both in cancer cells and in normal cells (FIG. 11). This result indicates that the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 involved in the test in this example can better avoid false positive signals, and the obtained signals are the fluorescent signals generated for the target MicroRNA (SEQ ID NO. 1).

Example 10

In order to expand the application of the fluorescent probe and make up for the defects caused by short-wavelength excitation. The two-photon performance of the probe was explored. We used the fluorescent probe of the present invention in the confocal fluorescence imaging contrast experiment of cancer cells (human pancreatic cancer PANC-1, SW1990, human breast cancer MCF-7) and normal cells (mouse embryo fibroblast NIH-3T 3). Each group of cells was imaged by adding I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 (each 2. mu.g/ml) for 4 hours of incubation, and the expression of SEQ ID NO.1 was found to be down-regulated in pancreatic cancer cells and breast cancer cells by using a two-photon excitation wavelength of 785nm as shown in FIG. 12. The results show that the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 involved in the test in the embodiment have good two-photon performance, and can be used for laser confocal imaging of deep tissue living bodies.

Example 11

To verify the applicability of the probe of the present invention to other biological samples, we explored the imaging performance of the probe on tissue samples.

A breast cancer model mouse is deburred to obtain a tumor tissue and a normal lung tissue, the two tissues are cut into tissue sections with the thickness of 200 mu m by adopting a freezing microtome, probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 (each 2 mu g/ml) are added, tissue depth imaging is carried out after 30min of incubation, and a confocal fluorescence depth imaging graph of the fluorescent probe used for the mouse tumor tissue and the mouse normal tissue is obtained by adopting a two-photon excitation wavelength of 785nm and taking one probe per 10 mu m, as shown in figure 13. FIG. 14 and FIG. 15 show that the fluorescent probes I-LMX-SEQ ID NO.2 and II-LMX-SEQ ID NO.3 involved in the test in this embodiment have a good tissue penetration depth, can reach 100 μm in 30min, and can be applied to the detection of target MicroRNA (SEQ ID NO.1) in a tissue biological sample, thereby realizing the effect of distinguishing normal tissues from tumor tissues.

Example 12

To verify the applicability of the probe of the present invention to other biological samples, we explored the ability of the probe to detect on actual blood samples.

Blood samples of 6 normal persons (N1-N6), 21 pancreatitis patients (I1-I21) and 3 pancreatic cancer patients (T1-T3) are collected, probes I-LMX-SEQ ID NO.5& II-LMX-SEQ ID NO.6 (each 1 μ M) and I-LMX-SEQ ID NO.8& II-LMX-SEQ ID NO.9 (each 1 μ M) corresponding to two MicroRNAs of SEQ ID NO.4 and SEQ ID NO.7 are respectively added, incubation is carried out at 37 ℃, an enzyme reader is used for detecting once every 30min, the fluorescence intensity can reach the maximum at 1h, and the fluorescence intensity in the cancer patient sample has 5-7 times of signal enhancement relative to that in the normal person sample (FIG. 16). The two groups of fluorescent probes involved in the test in the embodiment can be applied to the detection of the target MicroRNA in the blood sample, so that the effect of distinguishing the normal sample from the cancer sample is realized.

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Claims (7)

1. A composite probe composition for MicroRNA detection comprises a fluorescent component and a phenylphosphine component,

wherein the fluorescent component is a compound of a general formula I, and the phenylphosphine component is a compound of a general formula II:

in formula I:

said L_IIs- (CH)₂)_m-, said m is an integer of 0 to 8;

said F is of formula F₁Wherein R is₃And R₄Each independently selected from hydrogen and C_1-6Alkyl and phenyl;

in formula II:

said R₁And R₂Each independently selected from phenyl or methylphenyl;

said L_IIIs phenyl or- (CH)₂)_n-, said n is an integer of 1 to 8.

2. The composite probe composition according to claim 1, wherein m is an integer of 0 to 6.

3. The composite probe composition according to claim 1, wherein n is an integer of 1 to 6.

4. The composite probe composition according to claim 1, wherein: a compound comprising formula I-LMX and formula II-LMX:

5. a method for detecting MicroRNA, which is characterized by using the composite probe composition of claim 1, and comprises the following steps:

firstly, based on a MicroRNA sequence to be detected, a complementary single-stranded DNA sequence is obtained according to the Watson-Crick base complementary pairing principle; the 5' -end part sequence of the single-stranded DNA sequence is DNA_IThe 3' terminal part sequence is DNA_II，

② Single-stranded DNA having 5' -terminus modified with amino group_IConnecting with a compound of a general formula I to obtain a detection molecule I; single-stranded DNA having 3' -end modified with amino group_IIConnecting with a compound of a general formula II to obtain a detection molecule II;

thirdly, adding the detection molecule i and the detection molecule ii into a system to be detected according to the molar ratio of 1:1, and judging whether the system contains the MicroRNA to be detected according to the change of the system fluorescence.

6. The method for detecting MicroRNA of claim 5, comprising the steps of:

firstly, based on a MicroRNA sequence to be detected, obtaining a complementary single-stranded DNA sequence x with the base number of x as an integer of 18-24 according to the Watson-Crick base complementary pairing principle; when x is an odd number, 1 to (x-1)/2 bases from the 5 'end to the 3' end of the resulting complementary single-stranded DNA sequence constitute a single-stranded DNA_I(x +3)/2 to x bases constituting a single-stranded DNA_II(ii) a When x is an even number, 1 to x/2-1 bases from the 5 'end to the 3' end of the resulting complementary sequence constitute a single-stranded DNA_IX/2+1 to n bases constituting a single-stranded DNA_II；

② Single-stranded DNA having 5' -terminus modified with amino group_IConnecting with a compound of a general formula I to obtain a detection molecule I; single-stranded DNA having 3' -end modified with amino group_IIConnecting with a compound of a general formula II to obtain a detection molecule II;

thirdly, adding the detection molecule i and the detection molecule ii into a system to be detected according to the molar ratio of 1:1, and judging whether the system contains the MicroRNA to be detected according to the change of the system fluorescence; the concentrations of both in vitro assays were 1. mu.M each, and in cell experiments 2. mu.g/mL each.

7. The method for detecting MicroRNA of claim 5, wherein the MicroRNA and the corresponding DNA are present in the sample_I、DNA_IIThe base sequences are respectively as follows:

firstly, the MicroRNA to be detected has a base sequence of SEQ ID NO.1, and the DNA_IAnd DNA_IIRespectively has base sequences of SEQ ID NO.2 and SEQ ID NO. 3;

② the MicroRNA to be detected has a base sequence of SEQ ID NO.4DNA of (a)_IAnd DNA_IIHaving base sequences of SEQ ID NO.5 and SEQ ID NO.6, respectively;

③ the MicroRNA to be detected has the base sequence of SEQ ID NO.7, and the DNA_IAnd DNA_IIHas base sequences of SEQ ID NO.8 and SEQ ID NO.9, respectively.

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