KR20140108913A - Method for quantitative analysis of interactions between Lin 28 protein and pre-let-7 miRNA and method for screening interaction inhibitors using the same - Google Patents

Method for quantitative analysis of interactions between Lin 28 protein and pre-let-7 miRNA and method for screening interaction inhibitors using the same Download PDF

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KR20140108913A
KR20140108913A KR1020130022795A KR20130022795A KR20140108913A KR 20140108913 A KR20140108913 A KR 20140108913A KR 1020130022795 A KR1020130022795 A KR 1020130022795A KR 20130022795 A KR20130022795 A KR 20130022795A KR 20140108913 A KR20140108913 A KR 20140108913A
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김광록
김채원
최상운
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Abstract

The present invention relates to a method for quantitatively analyzing the binding between Lin28 protein and pre-let-7 miRNA using a fluorescence probe, and a method for screening inhibitors inhibiting the binding between Lin28 protein and pre-let-7 miRNA using the same method. More specifically, the present invention relates to a method for the quantitative analysis of the binding between Lin28 protein and pre-let-7 miRNA comprising: a) a step for preparing the fluorescent probe which is labeled with a quencher at the 5` end and a fluorescent material at the 3` end of pre-let 7 miRNA; (b) a step for making Lin28 ptorein react with the fluorescent probe prepared at the step (a); and (c) a step for measuring fluorescence value of the reactant. Additionally, the analyzing method can be useful for mass screening the inhibitors inhibiting the binding.

Description

Method for quantitative analysis of binding of Lin28 protein to pre-let-7 miRNA and screening method for inhibitors that inhibit binding using the method screening interaction inhibitors using the same}

The present invention relates to a method for quantitatively analyzing the binding between a Lin28 protein and a pre-let7 miRNA using a fluorescent probe, and a screening method of an inhibitor that inhibits binding thereof.

Lin28 protein is an RNA-binding protein that regulates development, pluripotency, tumorgenesis and metabolism in the cell.

Within the cell, the Lin28 protein is mainly expressed in the cytoplasm and can be found in the processing of RNA such as mRNAs, polysomes, processing bodies and stress granules. In addition, according to recent reports, it is speculated that the Lin28 protein can reciprocate between the nucleus and the cytoplasm. It is also known that Lin28 is highly expressed in embryonic stem cells and is highly expressed in tissues that have progressed to cancer rather than normal tissues and that the progression of cancer is accelerated by the expression of Lin28.

Recently, Lin 28 has transformed cells into cancerous states, which are abundant in advanced human cancers such as liver cancer, ovarian cancer, chronic myeloid cancer, germ cell tumors, Wilm's tumor (infant kidney cancer) . Therefore, Lin 28 is overexpressed in about 15% of all cancers, and it is expected that various resistance will be a new target of high cancer treatment.

In addition, Lin 28 protein is known to abundantly exist in embryonic stem cells as well as cancer cells, thereby inhibiting the differentiation of stem cells into specific cells. Lin 28 is an important protein that acts on both stem cells and cancer cells. Screening for substances that interfere with their binding and expression may be useful in the development of drugs for the treatment or differentiation of cancer or stem cells.

Lin28 is a regulator of strong let-7 microRNAs (miRNAs). In embryonic stem cells, where Lin28 protein is highly expressed, almost no let-7 miRNA is expressed, The expression of let-7 miRNA is increased when the expression of Lin28 is decreased. These changes in expression of Lin28 and let-7 miRNA are identical in most cells and tissues as well as embryonic stem cells. Figure 1 shows the maturation process of Let-7 miRNA.

Although let-7 miRNA is a very short strand of RNA of ~ 22 nucleotides, it binds with the RNA-induced silencing complex (RISC) to bind to the 3'-UTR of the target mRNA, By inhibiting the gene related to the cell cycle, oncogene, etc., it reduces the proliferation. The let-7 miRNA is produced by primary RNA-7 miRNA (pri-let-7) by RNA synthesis enzyme (polymerase II) in the nucleus and Drosha / DGCR8 is inserted into precursor let-7 miRNA (pre-let-7).

When pre-let-7 is transferred to cytoplasm via exportin, Dicer recognizes and produces a mature form of let-7. The RISC then binds to a single let-7 miRNA and targets and targets mRNA with an identical sequence of 7-8 nt.

Lin28 protein controls the maturation of pre-let-7 miRNA, but let-7 miRNA also regulates the expression of Lin28, allowing feedback control.

Lin28 regulates mRNA stability as well as let-7 miRNA. Lin28, along with RNA helicase (RHA), binds to the 5'-UTR of mRNA and promotes translation. Target genes include genes related to cell cycle such as IGF-2 and cyclin A, cyclin B, and Cdk4, which are related to muscle cell differentiation. The expression of Lin28 is influenced by the differentiation of stem cells and the oncogene expression is increased in cancer cells to accelerate the progression of cancer and the insulin-PI3K-mTOR pathway Regulate the expression of genes related to glucose metabolism by regulating the expression of various genes within the cell.

The regulation of expression of Lin28 and let-7 miRNAs is very important in stem cells, cancer cells and cells. An accurate analysis of these linkages can predict the expression level of Let-7, It can be very important to regulate proliferation.

Previously, binding of Lin28 protein to let-7 miRNA was confirmed by binding of Lin28 protein using radioactivity-labeled pre-let-7 with EMSA (Electrophoric Mobility Shift Assay). In addition, RNA-protein complex (RNP) complex immunoprecipitation is used. After Lin28 protein is precipitated by antibody antigen reaction, RNA is isolated and subjected to northern blotting or RT-PCR, And that the Lin28 protein and the let-7 miRNA bind to each other.

However, since the above method has a complicated and costly disadvantage, a method for simply analyzing the binding of Lin28 protein to pre-let-7 miRNA is required. Accurate analysis of these associations can not only help to observe various life phenomena such as cancer development, diagnosis and stem cell differentiation, but also can be utilized as a high efficiency screening method in the development of drugs such as anticancer drugs. . ≪ / RTI >

S. R Viswanathan et al., "Lin28 promotes transformation and is associated with advanced human malignancies" Published online: 31 May 2009, doi: 10.1038 / ng.392 S. R. Viswanathan et al., "Selective blockade of microRNA processing by LIN28 ", Science, 320, p97-100, 2008. S. R Viswanathan et al., "Lin28 promotes transformation and is associated with advanced human malignancies ", Nature Genetics, vol 10.1038 / ng.392

The present invention aims at providing a simple and accurate quantitative analysis method of binding to Lin28 protein and pre-let7 miRNA, and a method of screening inhibitors that inhibit the binding of Lin28 protein and Let7 miRNA using the above assay method.

In order to achieve the above object, the present invention provides a method for producing a fluorescent probe, comprising the steps of: a) preparing a fluorescent probe having a quencher substance attached to the 5'-end of pre-let-7 miRNA and a fluorescent substance attached to the 3'- b) reacting the Lin28 protein with the fluorescent probe of step a); c) measuring the fluorescence value of the reactant; Lt; RTI ID = 0.0 > miRNA < / RTI >

Preferably, the light minerals in step a) are dimethylaminoazobenzenesulfonic acid (DABSYL), the fluorescent substance is fluorescein amide (FAM), fluorescenecarboxylic acid (FCA), fluorescein isothiocyanate (FITC) Yl, 2 ', 7'-dichlorofluorescein-5-yl, 2'-fluorocycloheptadecane, ', 7'-dichlorofluorescein-6-yl is preferable. The pre-let-7 miRNA has the nucleotide sequence of SEQ ID NO: 1 or 2, and the Lin28 protein preferably has the amino acid sequence of SEQ ID NO: 3. In step b), the Lin28 protein and the pre-let-7 miRNA are preferably reacted at a molar ratio of 2: 1 to 5: 1, and the reaction temperature is preferably 20 to 40 ° C.

In addition, the present invention provides a method for detecting a protein comprising the steps of: a) adding an inhibitor candidate substance to a reaction solution of a fluorescent probe having a quencher substance attached to the 5'-end of pre-let-7 miRNA and a fluorescent substance attached to the 3'- ; b) measuring the fluorescence of the reaction solution, and observing changes in fluorescence according to whether the inhibitor candidate is added; And c) determining the candidate substance as an inhibitor when the fluorescence of the reactant of the Lin28 protein and the pre-let-7 miRNA is decreased by the addition of the inhibitor candidate substance. lt; RTI ID = 0.0 > miRNA < / RTI >

Preferably, the light minerals in step a) are dimethylaminoazobenzenesulfonic acid (DABSYL), the fluorescent substance is fluorescein amide (FAM), fluorescenecarboxylic acid (FCA), fluorescein isothiocyanate (FITC) Yl, 2 ', 7'-dichlorofluorescein-5-yl, 2'-fluorocycloheptadecane, ', 7'-dichlorofluorescein-6-yl is preferable. The pre-let-7 miRNA has the nucleotide sequence of SEQ ID NO: 1 or 2, and the Lin28 protein preferably has the amino acid sequence of SEQ ID NO: 3. In step a), the Lin28 protein and the pre-let-7 miRNA are preferably reacted at a molar ratio of 2: 1 to 5: 1, and the temperature is preferably 20 to 40 ° C.

In addition, the present invention provides a pharmaceutical composition for treating cancer comprising an inhibitor of binding of Lin28 protein and pre-let-7 miRNA as an active ingredient. The inhibitor inhibits the binding of Lin28 protein and pre-let-7 miRNA to promote maturation of pre-let-7 miRNA. Preferably, the cancer is selected from liver cancer, ovarian cancer, benign bone marrow cancer, germ cell tumor, Wilm's tumor, infant kidney cancer) and lung cancer.

The present invention also provides a composition for inducing differentiation of stem cells comprising an inhibitor of binding of Lin28 protein and pre-let-7 miRNA as an active ingredient. The inhibitor may inhibit the binding of Lin28 protein to pre-let-7 miRNA and promote maturation of pre-let-7 miRNA.

The present invention will be described in more detail below.

A) preparing a fluorescent probe having a quencher substance attached to the 5'-end of pre-let-7 miRNA and a fluorescent substance attached to the 3'-end thereof; b) reacting the Lin28 protein with the fluorescent probe of step a); c) measuring the fluorescence value of the reactant; Lt; RTI ID = 0.0 > miRNA < / RTI >

Lin28 is critical for the maturation process of let-7 miRNA and binds to precursor let-7 miRNA to inhibit its expression. The Lin28 protein binds to the stem loop of pre-let-7 and recruits the TUT4 protein. TUT4 degrades the maturation of the let-7 miRNA by degrading it by uridylation at the end of pre-let7 . TUT4 was found to be highly dependent on Lin28 protein (Newman et al, RNA. 2008 August; 148 (8) p 1539-1549. "Lin-28 interaction with the let-7 precursor loop mediates regulated microRNA processing"; V. Narry Kim, Molecular Cell, 32 (2), p276-284, 24 October 2008, "Lin28 Mediates the Terminal Uridylation of let-7 Precursor MicroRNA"). When Lin28 binds to the pre-let-7, recognizing and combining a specific sequence called "GGAG" will cause the structure of the cleavage site to be modified so that the Dicer plays a role Maturation can be suppressed by making it inoperable.

Lin28 uses a CCHC domain containing a pair of a cold shock domain (CSD) and a retroviral type cys-cys-his-cys (CCHC) zinc finger, and binds to target RNA to regulate expression.

Thus, a fluorescent probe was prepared by attaching a small mineral substance to the 5'-end of pre-let-7 miRNA and attaching a fluorescent substance to the 3'-end. When the Lin28 protein is not bound, as shown in Fig. 2, the ends of both ends are bound to each other, so that the minerals are prevented from fluorescence by suppressing fluorescence. However, when the Lin28 protein recognizes and binds to the GGAG region of the pre-let-7 miRNA, the miRNA is expanded and the minerals are not activated, resulting in fluorescence. Therefore, by measuring the fluorescence value, the binding of pre-let-7 miRNA and Lin28 protein can be quantitatively analyzed. If the fluorescence value is low, the binding is low or not. If the fluorescence value is large, the pre-let-7 miRNA and Lin28 complex are produced in large amounts.

The fluorescent probe of the present invention is labeled with a fluorescent dye and specifically binds to pre-let-7 miRNA to measure the fluorescence value. Fluorescent probes have regions that are capable of binding to the Lin28 protein and are usually capable of controlling their length depending on the target protein and may be shorter or longer. Probes can include any base sequence as long as they do not affect the binding capacity of Lin28 and pre-let-7 miRNA. The fabrication or selection of probes can be easily done by those skilled in the art and can be modified using known conditions.

Preferably, the pre-let-7 miRNA has the nucleotide sequence of SEQ ID NO: 1 or 2, and Lin28 has the amino acid sequence of SEQ ID NO: 3.

The small mineral of step a) is Dabsyl (dimethylaminoazobenzenesulfonic acid), and the fluorescent material is fluorescein amidite (FAM), fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC) 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichloroheptadecane, -'-dichlorofluorescein-6-yl. ≪ / RTI >

The reaction of step b) is preferably carried out by mixing the Lin28 protein and the pre-let-7 miRNA in a suitable buffer solution at a molar ratio of 2: 1 to 5: 1, most preferably 4: 1 . When the Lin28 protein is added to the pre-let-7 miRNA at less than 2 times, sufficient binding reaction does not occur. If the protein is added more than 5 times, the amount of Lin28 protein in the reaction solution increases. The reaction temperature is preferably in the range of 20 to 40 캜. When it is out of the above range, the combination of Lin28 protein and pre-let-7 miRNA is not sufficiently formed and it is difficult to obtain accurate fluorescence value.

A probe is a material that selects only a specific substance from among various substances in a sample. For diagnosis of a disease, a nucleic acid, a protein, a peptide, or an antibody that specifically binds to a specific disease marker may be used.

The present invention can use a pre-let-7 miRNA whose nucleotide sequence is regulated as a fluorescent probe. For example, miRNA labeled with fluorescein can be used.

Specific examples of the fluorescent dye include, but are not limited to, rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, erythrosine and its derivatives , Eosin and its derivatives, and 4-acetamido-4'-isothiocyanatostilbene-2,2 'disulfonic acid. The fluorescent material that can be used in the present invention is more specifically described as follows.

(ROX), 6-carboxyhodamine (R6G), lysaminrodamine B sulfonyl chloride, rhodamine, rhodamine B, rhodamine 123, N, N, N ' -tetramethyl-6-carboxyhodamine < RTI ID = 0.0 > (R) < / RTI & (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosol acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, Alexa-547 and Alexa- Can be heard;

(FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy-4 ' (JOE), fluorescein, fluorescein isothiocyanate, QFITC (XRITC), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-picoerythrin and o-phthalaldehyde;

(AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanosine, 4'-6-diaminodino- 2-phenylindole (DAPI), 5 ', 5 " -dibromopirogol Bromopyrogallol Red, 7-diethylamino- 3- (4 ' -isothiocyanatophenyl) -4 methylcoumarin Diethylenetriaminepentaacetate, 4- (4'-diisothiocyanato dihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene- Dimethylamino] naphthalene-1-sulfonyl chloride, 4- (4'-dimethylaminophenylazo) benzoic acid (DABCYL) and 4-dimethylaminophenyl azophenyl- Isothiocyanate (DABITC) and the like;

Acridine and its derivatives include acridine, acridine isothiocyanate, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino- Phenyl] naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N- (4-aniline

Naphthyl) maleimide, anthranylamide and Brilliant Yellow, and the like;

Pyrene and derivatives thereof include pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron Brilliant Red 3B-A) and the like;

Erythrosine and its derivatives such as erythrosine B, erythrosine isothiocyanate, and ethidium;

Eosin and its derivatives include eosin and eosin isothiocyanate, and the like;

4-acetamido-4'-isothiocyanatostilbene-2,2 'disulfonic acid.

In particular, fluorescent dyes can utilize materials having various emission wavelengths of Dylight. In the present invention, the fluorescent substance bound to one end of the nucleic acid probe may be any fluorescent substance that can be used for bio (bio) imaging.

After the binding reaction of the Lin28 protein and the pre-let-7 miRNA using the fluorescent probe of the above step c), the fluorescence value of the reactant can be measured and the presence or absence of the binding can be quantitatively analyzed. The measurement of the fluorescence value is not particularly limited, and fluorescence microscopy, fluorescence spectrometer or well plate-based fluorescence detector (well plate reader) can be used. When the light of a specific wavelength is irradiated, it absorbs the light and emits light of a longer wavelength having a lower energy, so that the wavelength of the fluorescent microscope can be changed according to the fluorescent dye.

In addition, the present invention provides a method for screening an inhibitor candidate compound, which comprises the steps of: a) adding a quencher substance to the 5'-end of a pre-let-7 miRNA and a fluorescent probe having a fluorescent substance attached at its 3'- Adding and reacting; b) measuring the fluorescence of the reaction solution, and observing changes in fluorescence according to whether the inhibitor candidate is added; And c) determining the candidate substance as an inhibitor when the fluorescence of the reactant of the Lin28 protein and the pre-let-7 miRNA is decreased by the addition of the inhibitor candidate substance. lt; RTI ID = 0.0 > miRNA. < / RTI >

Preferably, the light minerals in step a) are dimethylaminoazobenzenesulfonic acid (DABSYL), the fluorescent substance is fluorescein amide (FAM), fluorescenecarboxylic acid (FCA), fluorescein isothiocyanate (FITC) Yl, 2 ', 7'-dichlorofluorescein-5-yl, 2'-fluorocycloheptadecane, ', 7'-dichlorofluorescein-6-yl is preferable. The pre-let-7 miRNA has the nucleotide sequence of SEQ ID NO: 1 or 2, and the Lin28 protein preferably has the amino acid sequence of SEQ ID NO: 3. In step a), the Lin28 protein and the pre-let-7 miRNA are preferably reacted at a molar ratio of 2: 1 to 5: 1, and the reaction temperature is preferably 20 to 40 ° C.

The candidate inhibitor of step (b) may be an antibody, peptide, oligonucleotide or synthetic compound which competitively adheres to pre-let-7 with a fluorescent substance and can inhibit binding to the Lin28 protein .

In addition, the present invention provides a pharmaceutical composition for treating cancer comprising an inhibitor of binding of Lin 28 protein and pre-let-7 miRNA as an active ingredient. The inhibitor inhibits the binding of Lin 28 protein and pre-let-7 miRNA to promote maturation of pre-let-7 miRNA. Preferably, the cancer is selected from liver cancer, ovarian cancer, benign bone marrow cancer, (Wilm's tumor, infant kidney cancer), lung cancer.

Increased expression of Lin28 in the cells inhibits Let-7-inhibiting cells from becoming mature and leads to more stem-like cells. In addition, normal cells are more likely to be cancer cells, and Lin28 is considered to be a factor causing tumor. Therefore, an inhibitor that inhibits the binding of Lin28 protein to pre-let-7 miRNA is an anticancer agent that inhibits cancer by inducing maturation of pre-let-7 miRNA and controlling the expression of Lin28.

The inhibitor may be administered as a single active pharmaceutical formulation, but may also be used in combination with one or more inhibitors of the present invention or other agents. When administered as a combination, the therapeutic agent may be formulated as a separate composition administered at the same time or continuously at different times, or may be provided as a single composition. Administration of such inhibitors may be combined with additional therapies known to those of skill in the art of chemotherapeutic treatment such as radiation therapy or the administration of cytostatic or cytotoxic agents.

The present invention also provides a composition for inducing differentiation of stem cells comprising an inhibitor of binding of Lin28 protein and pre-let-7 miRNA as an active ingredient. The inhibitor may inhibit the binding of Lin28 protein to pre-let-7 miRNA and promote maturation of pre-let-7 miRNA. In particular, it can be used to regulate and control the differentiation and maturation of human stem cells. Stem cells include, but are not limited to, umbilical cord, blood, placenta and stem cells isolated from other sources.

The present invention provides a new method for quantitatively analyzing the binding of Lin28 protein and pre-let-7 miRNA, which is an important mechanism in cancer cells or stem cells, to rapidly and accurately analyze the binding of these and to inhibit the binding of anticancer agents or differentiation inducing agents It can be useful for screening large quantities of various inhibiting drugs.

Figure 1 shows the maturation process of let-7 miRNA.
Figure 2 shows the principle of measuring the binding of pre-let-7 and Lin28 proteins labeled with Dabcyl and FAM.
FIGS. 3 (a) to 3 (d) show pre-let-7a, g miRNA (cold) stored in ice and pre-let-7a and g miRNA And the absorbance value in the linearly expanded state (heat) was measured and analyzed.
FIG. 4 is a graph showing the absorbance values measured according to the reaction temperature when the concentration of pre-let-7a and pre-let-7g miRNA was fixed and the concentration of Lin28 protein was increased 2-fold or 4-fold.
FIG. 5 is a graph showing absorbance values measured by fixing the concentrations of pre-let-7a and pre-let-7g miRNA and increasing the concentration of Lin28 protein by 10 times.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are illustrative only and are not intended to be limiting.

Example 1: Fluorescence probe and recombinant Lin28 protein

Dabcyl and FAM were attached to the 5 'and 3' ends of the precursors let-7a and let-7g, respectively, and fluorescence probes were synthesized with the sequences shown in Table 1 below. Recombinant Lin28 protein (Peprotech Co. Cat. No. 110-06) was prepared.

The pre-let-7a, pre-let-7g and recombinant Lin28 proteins have the sequences shown in Table 1, respectively.

order FAM-pre-let-7a
(SEQ ID NO: 1) Dabcyl-GUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUAA-FAM FAM-pre let-7g
(SEQ ID NO: 2) Dabcyl-CAGUUUGAGGGUCUAUGAUACCACCCGGUACAGGAGAUAACUA-FAM recombinant Lin28 a TAT
(SEQ ID NO: 3) GPSVSNQQFA GGCAKAAEEA PEEAPEDAAR AADEPQLLHG AGICKWFNVR MGFGFLSMTA RAGVALDPPV DVFVHQSKLH MEGFRSLKEG EAVEFTFKKS AKGLESIRVT GPGGVFCIGS ERRPKGKSMQ KRRSKGDRCY NCGGLDHHAK ECKLPPQPKK CHFCQSISHM VASCPLKAQQ GP

Example 2: Measurement of maximum fluorescence value of fluorescent probe

(Dabcyl, FAM) adapters and Pam-labeled precursors let-7a and let-7g labeled with 10, 20, and 50 μg in a 96 well plate (Greiner bio-one, Cat. No. 655900.) 7a and g (heat), which were stored in ice (4 ℃) and heated at 95 ℃ for 10 min, were added to the cells in the concentrations of 40, 60, 80 and 100 nM.

The fluorescence value was measured with a plate reader (Molecular Device, SpectraMax Me5, manufactured by Molecular Device, Inc.) at an excitation wavelength of 490 nm and an emission wavelength of 515 nm in 100 mu l of buffer solution (20 mM Tris-HCL, pH 7.5; KCl 60 mM; MgCl2 10 mM; DTT 1 mM) ).

The measurement results are shown graphically in Fig. As shown in (a) and (c), the fluorescence value increased with increasing concentrations of pre-let-7a and pre-let-7b in the heat (heat) As the miRNA is linearized, the maximum value of the fluorescent material, FAM, can be obtained with the minerals and the fluorescent material being farthest away from each other. (B) and (d) analyzed by the ratio (heat / cold) are increased by 2 to 2.5 times.

Example 3: Dependent on temperature and concentration Analysis of binding force between fluorescent probe and Lin28 protein

(Dabcyl, FAM) adapter and Pam-labeled precusor let-7a, let-7g 5nM and Lin28 protein in a 96 well plate (Greiner bio-one, Cat. No. 655900.) (25 ° C, RT) and 4 ° C and 37 ° C for 1 hour, respectively, at a low rate.

The fluorescence value was measured with a plate reader (Molecular Device, SpectraMax Me5, manufactured by Molecular Device, Inc.) at an excitation wavelength of 490 nm and an emission wavelength of 515 nm in 100 mu l of buffer solution (20 mM Tris-HCL, pH 7.5; KCl 60 mM; MgCl2 10 mM; DTT 1 mM) ).

In Example 3 Except that the Lin28a protein was changed as shown in Table 2 below, and the fluorescence values of the respective proteins were measured. Tables 2 and 3 below show the ratios of fluorescence values measured by repeating the above procedure three times for each of pre-let-7a and pre-let-7g.

FAM-pre-let-7a recombinant Lin 28 a TAT 5 nM 0 10 nM 20 nM 온도 25 ℃ One 1.2 1.5 37 ℃ One 1.25 1.6 4 ℃ One 1.14 1.29

FAM-pre-let-7g recombinant Lin 28 a TAT 5 nM 0 10 nM 20 nM 온도 25 ℃ One 1.37 2.52 37 ℃ One 1.36 1.92 4 ℃ One 1.16 1.42

As a result of comparing the reaction efficiency with the above temperature condition, it was confirmed that the reaction was effected at 25 ° C for 1 hour. Therefore, it was confirmed that the combination of Lin28 protein and pre-let-7 miRNA is most well formed at 25 ° C. The results are shown in Fig.

Example 4: Analysis of binding force between fluorescent probe and Lin28 protein

(Dabcyl, FAM) adapter and Pam-labeled precusor let-7a, let-7g 5nM and Lin28 protein in a 96 well plate (Greiner bio-one, Cat. No. 655900.) The concentration was 50 nM, which is 10 times the molar concentration of pre-let-7, and reacted at low speed for 1 hour at room temperature.

The fluorescence value was measured with a plate reader (Molecular Device, SpectraMax Me5, manufactured by Molecular Device, Inc.) at an excitation wavelength of 490 nm and an emission wavelength of 515 nm in 100 mu l of buffer solution (20 mM Tris-HCL, pH 7.5; KCl 60 mM; MgCl2 10 mM; DTT 1 mM) ).

As a result of comparing the reaction efficiency with the Lin28 protein concentration, it was confirmed that even when the Lin28 protein was added 10 times more than the pre-let-7 miRNA, the effect of Lin28 and pre-let- The results are shown in Fig. As can be seen from the graph, when the ratio of Lin28 protein and pre-let-7 miRNA was 4: 1 at 25 ° C, it was found that the complex formed well.

From the above results, it was confirmed that the binding pattern of Lin28 protein and pre-7 miRNA can be easily quantitatively analyzed by fluorescent probe.

The foregoing description is merely illustrative of the technical idea of the present invention and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, and thus the scope of the technical idea of the present invention is not limited to this embodiment. The scope of protection of the present invention should be construed in accordance with the following claims and all technical ideas within the technical scope of the claims should be construed as falling within the scope of the present invention.

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY <120> Method for quantitative analysis of interactions between Lin 28          protein and pre-let-7 miRNA and method for screening interaction          inhibitors using the same <130> P12111321499 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> pre-let-7a <400> 1 guauaguuuu agggucacac ccaccacugg gagauaacua uaa 43 <210> 2 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> pre-let-7g <400> 2 caguuugagg gucuaugaua ccacccggua caggagauaa cua 43 <210> 3 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of recombinant Lin28 protein <400> 3 Gly Pro Ser Val Ser Asn Gln Gln Phe Ala Gly Gly Cys Ala Lys Ala   1 5 10 15 Ala Glu Glu Ala Pro Glu Glu Ala Pro Glu Asp Ala Ala Arg Ala Ala              20 25 30 Asp Glu Pro Gln Leu Leu His Gly Ala Gly Ile Cys Lys Trp Phe Asn          35 40 45 Val Arg Met Gly Phe Gly Phe Leu Ser Met Thr Ala Arg Ala Gly Val      50 55 60 Ala Leu Asp Pro Pro Val Asp Val Phe Val His Gln Ser Lys Leu His  65 70 75 80 Met Glu Gly Phe Arg Ser Leu Lys Glu Gly Glu Ala Val Glu Phe Thr                  85 90 95 Phe Lys Lys Ser Ala Lys Gly Leu Glu Ser Ile Arg Val Thr Gly Pro             100 105 110 Gly Gly Val Phe Cys Ile Gly Ser Glu Arg Arg Pro Lys Gly Lys Ser         115 120 125 Met Gln Lys Arg Arg Ser Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly     130 135 140 Leu Asp His His Ala Lys Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys 145 150 155 160 Cys His Phe Cys Gln Ser Ser Ser His Met Val Ala Ser Cys Pro Leu                 165 170 175 Lys Ala Gln Gln Gly Pro             180

Claims (20)

a) preparing a fluorescent probe to which a quencher substance is attached to the 5'-end of the pre-let-7 miRNA and a fluorescent substance is attached to the 3'-end thereof; b) reacting the Lin28 protein with the fluorescent probe of step a); c) measuring the fluorescence value of the reactant; Lt; RTI ID = 0.0 &gt; miRNA &lt; / RTI &gt;
The method according to claim 1, wherein the small mineral of step a) is dimethylaminoazobenzenesulfonic acid (Dabsyl).
The method of claim 1, wherein the fluorescent material of step a) is selected from the group consisting of fluorescein amidite (FAM), fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH) 2'7'-dichlorofluorescein-5-yl, 2 ', 7'-dichlorofluore-3'-fluorocyclin 6-yl. &Lt; / RTI &gt;
The method according to claim 1, wherein the pre-let-7 miRNA of step a) has the nucleotide sequence of SEQ ID NO: 1.
The method according to claim 1, wherein the pre-let-7 miRNA of step a) has the nucleotide sequence of SEQ ID NO: 2.
2. The method according to claim 1, wherein the Lin28 protein of step a) has the amino acid sequence of SEQ ID NO: 3.
The method according to claim 1, wherein the Lin28 protein and the pre-let-7 miRNA are added at a molar ratio of 2: 1 to 5: 1 in the step a).
The method according to claim 1, wherein the reaction of Lin28 protein and pre-let-7 miRNA in step b) is carried out at 20 to 40 ° C
a) adding a candidate inhibitor to a reaction solution of a fluorescent probe having a quencher substance attached to the 5'-end of the pre-let-7 miRNA and a fluorescent substance attached to the 3'-end thereof and a Lin28 protein, ; b) measuring the fluorescence of the reaction solution, and observing changes in fluorescence according to whether the inhibitor candidate is added; And c) determining the candidate substance as an inhibitor when the fluorescence of the reactant of the Lin28 protein and the pre-let-7 miRNA is decreased by the addition of the inhibitor candidate substance. lt; RTI ID = 0.0 &gt; miRNA. &lt; / RTI &gt;
The method according to claim 9, wherein the small mineral of step a) is dimethylaminoazobenzenesulfonic acid (DABSYL).
10. The method of claim 9, wherein the fluorescent material of step a) is selected from the group consisting of fluorescein amidite (FAM), fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH) 2'7'-dichlorofluorescein-5-yl, 2 ', 7'-dichlorofluore-3'-fluorocyclin 6-yl. &Lt; / RTI &gt;
10. The method according to claim 9, wherein the pre-let-7 miRNA of step a) has the nucleotide sequence of SEQ ID NO: 1.
10. The method of claim 9, wherein the pre-let-7 miRNA of step a) has the nucleotide sequence of SEQ ID NO: 2.
[10] The method of claim 9, wherein the Lin28 protein and the pre-let-7 miRNA are added in a molar ratio of 2 : 1 to 5: 1 in step a).
The method according to claim 9, wherein the reaction of step a) is carried out at a temperature of 20 to 40 ° C.
A pharmaceutical composition for treating cancer comprising an inhibitor of binding of Lin 28 protein and pre-let-7 miRNA as an active ingredient.
18. The pharmaceutical composition of claim 16, wherein said inhibitor inhibits the binding of Lin28 protein to pre-let-7 miRNA to promote maturation of pre-let-7 miRNA.
18. The pharmaceutical composition according to claim 16, wherein the cancer is liver cancer, ovarian cancer, benign bone marrow cancer, germ cell tumor, Wilm's tumor, lung cancer.
A composition for inducing stem cell differentiation comprising an inhibitor of binding of Lin 28 protein and pre-let-7 miRNA as an effective ingredient.
20. The composition of claim 19, wherein said inhibitor inhibits binding of Lin28 protein to pre-let-7 miRNA to promote maturation of pre-let-7 miRNA.
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* Cited by examiner, † Cited by third party
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KR20180024705A (en) 2016-08-31 2018-03-08 전주대학교 산학협력단 Detecting method for controlling miRNA ID and its application of biomarker for breast cancer
KR20180031980A (en) 2016-09-21 2018-03-29 전주대학교 산학협력단 micro-RNA ID analysis method through data mining of micro RNA
KR20180035383A (en) 2016-09-29 2018-04-06 전주대학교 산학협력단 Detecting method for controlling miRNA and its application of biomarker for colon cancer
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KR20180024705A (en) 2016-08-31 2018-03-08 전주대학교 산학협력단 Detecting method for controlling miRNA ID and its application of biomarker for breast cancer
KR20180031980A (en) 2016-09-21 2018-03-29 전주대학교 산학협력단 micro-RNA ID analysis method through data mining of micro RNA
KR20180035383A (en) 2016-09-29 2018-04-06 전주대학교 산학협력단 Detecting method for controlling miRNA and its application of biomarker for colon cancer
KR20190001799A (en) 2017-06-28 2019-01-07 전주대학교 산학협력단 Method for analysis of Alzheimer biomarker microRNA ID using correction of expression gene
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