KR20170123776A - Aptamers that specifically bind to MLL1 and the use thereof - Google Patents

Abstract

The present invention relates to: aptamers specifically binding to mixed lineage leukemia 1 (MLL1); a pharmaceutical and diagnostic composition for preventing or treating MLL1-mediated diseases comprising the same; a composition for imaging an MLL1 mediated disease site; and a method for providing information for diagnosis of MLL1-mediated diseases using the aptamers. The aptamers of the present invention specifically bind to MLL1, and thus can effectively inhibit the activity of MLL1 protein. Accordingly, the composition comprising the aptamers can be usefully used for preventing, treating or diagnosing the MLL1 mediated diseases including leukemia.

Description

Aptamers that specifically bind MLL1 and the use thereof < RTI ID = 0.0 >

The present invention relates to a platemer specifically binding to MLL1 (Mixed Lineage Leukemia 1), a pharmaceutical composition for preventing or treating MLL1 mediated diseases, a diagnostic composition, a composition for imaging MLL1 mediated disease site, Lt; RTI ID = 0.0 > MLL1 < / RTI > mediated disease.

MLL1 (Myeloid / lymphoid, or Mixed lineage leukemia 1) is a transcription co-activator required to maintain Hox gene expression in hematopoiesis and developmental stages. MLL1 binds to several types of proteins to form multiple protein complexes, and each complex modulates the H3K4 methylation activity against a unique target. In particular, the SET domain of the MLL1 protein catalyzes mono-, di- or trimethylation at the H3K4 site, and each of these methylations has a unique and specific role.

On the other hand, the methylation is closely related to chromatin transcription activity and various types of cancer, while functional or non-functional variants of MLL1 maintain enzyme activity under stringent control. For example, MLL1 is known to be one of the most important proteins involved in various types of acute lymphocytic or myeloid leukemia. The fusion of MLL with one of the 60 partner genes results in the formation of a chimeric tumor gene that up-regulates the HOX gene, ultimately leading to the blockage of blood cell differentiation that leads to acute leukemia (Eguchi et al., Int J Hematol, 2003, 78 (5): 390-401). Leukemia patients with MLL translocation have a very poor prognosis (35% 5-year survival) and new treatment strategies are needed to treat these leukemia (Slany, Hematol OncoI, 2005, 23 (1): 1-9) . Recently, it has been reported that MLL1 is required for hormone-dependent expression of the estrogen receptor alpha target gene and growth of MCF-7 cells. In addition, the present inventors' previous studies (Jeong et al., Nucleic Acids Res, 2014, 42: 2245-2256; Jeong et al., Nat Struct Mol Biol, 2011, 18: 1358-1365; Won Jeong et al. Some important facts about MLL1 in Mol Endocrinol, 2012, 26: 955-966 include: 1) the fact that MLL1 regulates cancer cell-specific gene expression and 2) the growth of breast cancer cell lines upon reduction of intracellular MLL1 levels using siRNA Has been confirmed that MLL1 can be usefully used as a cancer treatment target molecule. Further, as a result of the above-described studies, it was confirmed that the decrease in the intracellular MLL1 level using siRNA also showed that not only a breast cancer cell line but also a cell line (for example, It has been reported that the growth of various cancer cells such as colon cancer, stomach cancer and cervical cancer is inhibited.

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology was developed by Ellington, AD, and is a test tube that concentrates ssRNA / ssDNA molecules (named asatamera) bound to specific ligands with high selectivity and sensitivity ( In vitro ) screening method. The selected aptamer is considered to be a good substitute for the antibody since SELEX is cheap, easy to chemically deform without loss of function, easy to fix compared to antibody, and has various advantages (Nieuwlandt D, Curr Issues Mol Biol, 2000. 2: 9-16).

Accordingly, the present inventors have made intensive efforts to develop an amphoter capable of inhibiting MLL1 activity for the treatment of MLL1-mediated diseases, and as a result, selected aptamer specifically binding to MLL1 was selected, and the above- The present invention has been completed. Thus, the compositions comprising the platemer of the present invention can be usefully used for the treatment, diagnosis, and imaging of MLLl mediated disease sites of MLL1 mediated diseases.

It is an object of the present invention to provide a platamer that specifically binds to MLL1 (Mixed Lineage Leukemia 1).

Another object of the present invention is a pharmaceutical composition for preventing or treating an MLL1 mediated disease comprising the above-mentioned platemer.

Another object of the present invention is a composition for the diagnosis of MLL1 mediated diseases comprising the above-mentioned platemer.

Another object of the present invention relates to a composition for imaging an MLL1 mediated disease site comprising the above squamotome.

Another object of the present invention is to provide a method for detecting a biological sample, comprising the steps of: (a) And (b) measuring the degree of binding of the platamer in the biological sample, and determining that the disease is MLL1 mediated disease when the degree of binding is higher than the degree of binding in the normal sample. And a method for providing the same.

In the present invention, a platamer specifically binding to 10 kinds of MLL1 was developed. The above-mentioned aptamer specifically binds to MLL1 protein and inhibits its activity, so that it can be usefully used for prevention or treatment of MLL1 mediated diseases.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

To achieve the above object, one embodiment of the present invention is an umbilical cord speculum that specifically binds to MLL1 (Mixed Lineage Leukemia 1).

In the present invention, the term "MLL1 (Mixed Lineage Leukemia 1)" is a protein encoded by the KMT2A gene, and is also called histone-lysine N-methyltransferase 2A. MLL1 is a protein belonging to the histone-modified protein group, which contains a transcriptional activation domain 9aaTAD, which broadly regulates gene transcription with histone methyltransferases.

MLL1 is a transcriptional coactivator that plays a key role in regulating gene expression in early development and hematopoiesis. In particular, the SET domain of MLL1 has histone H3 lysine 4 (H3K4) methyltransferase activity and mediates chromatin modification in relation to post-sexual transcriptional activity. Chromosomal translocation of MLL1 is known to cause acute lymphocytic and acute myelogenous leukemia.

The term "aptamer " in the present invention refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer of the present invention may be in the form of single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA), but may be in the form of single-stranded DNA , But is not limited thereto. The aptamer can specifically bind to a selected target. A typical aptamer is 10-15 kDa (30-45 nucleotides) in size and can bind to its target with a sub-nanomolar affinity. The aptamer can specifically bind to a target selected through binding interactions (e.g., hydrogen bonding, electrostatic complementation, hydrophobic contact, steric exclusion) or specificity in an antibody-antigen complex.

Specifically, the aptamer may have a base sequence of any one of base sequence shown in SEQ ID NO: 10 to SEQ ID NO: 12 and a base sequence complementary thereto, or may have a base sequence of 70% or more, specifically 80% More specifically 90% or more, more specifically 95% or more, and most particularly 99% or more homology to the MLL1, and it is within the scope of the present invention that the plasmid having substantially the binding activity to MLL1 .

Also, an aptamer having a base sequence in which a part of the sequences in the above-mentioned homology is deleted, modified, substituted or added substantially has the same or a corresponding biological activity as that of the base sequence of SEQ ID NOS: 10 to 12 or a complementary base sequence It is obvious that the present invention is also included in the scope of the present invention.

In the above, the term "homology" means the degree to which a given amino acid sequence or base sequence is consistent and can be expressed as a percentage. In the present invention, a homologous sequence having the same or similar activity as a given amino acid sequence or base sequence is represented as "% homology ". For example, standard software for calculating parameters such as score, identity and similarity, specifically BLAST 2.0, or by sequential hybridization experiments under defined stringent conditions, And the appropriate hybridization conditions to be defined are within the skill of the art and can be determined by methods well known to those skilled in the art (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). The term " stringent conditions " as used herein refers to conditions that allow specific hybridization between polynucleotides. For example, these conditions are specifically described in the literature (e.g., J. Sambrook et al., Same as above).

In the present invention, a platamer that specifically binds to MLL1 was developed using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process. The above-mentioned "SELEX" was developed by Ellington, AD, etc. and is an in vitro screening method for concentrating ssRNA / ssDNA molecules (aptamer) having high selectivity and sensitivity to bind to a specific ligand through a circulation process . The aptamer is considered to be a good substitute for the antibody because it is low in price of SELEX, easy to be chemically modified without loss of function, and easy to fix compared with antibody immobilization and has various advantages over antibody.

The method applied to SELEX in the present invention is a method of obtaining double stranded DNA (dsDNA) by i) binding an RNA library to an MLL1 protein, ii) elimination of a non-binding RNA sequence, and iii) RT-PCR of the bound RNA- , iv) preparing an RNA library by reverse transcription using a coupled DNA library, and v) purifying the RNA library using a polyacrylamide gel electrophoresis (PAGE) method. The purified RNA was used again in the next SELEX cycle.

In the present invention, the first RNA library used for SELEX circulation was named S0 library, and named as S1, S2, S3 as the SELEX circulation turn-up was increased. The S0 library is an RNA tymator library containing a central randomized region sequence of 25 nucleotides, which contains a flanking region including the T7 promoter and primer region and XhoI and HindIII restriction enzyme sites I have. In one specific example of the present invention, the S0 library was subjected to 16 positive rounds of selection using the Sterpine (SELEX) to produce the S16 library (FIG. 1).

One disadvantage of the SELEX technology, however, is that it obtains the RNA with the highest affinity only at the target site through several purification and amplification steps, and thus the range and relative affinity of the RNA-sequence preference of the RNA binding protein Can not be fully specified. Thus, we further analyzed the selected platamers using HT-SELEX, a relatively inexpensive, high-throughput sequencing method. This method is a more quantitative and comprehensive method than the SELEX process. This method is performed with a small number of selection cycles (1 to 3 times in most cases) and analyzes millions of RNA oligonucleotides. This method can help to establish more quantitatively the RNA binding protein sequence-binding preferences.

In a specific embodiment of the present invention, S1, S3, and S16 library sequences were analyzed using HT-SELEX, and ten kinds of platamers (APT1 to APT10, SEQ ID NO: 10 to 19) were selected (Table 1 and Fig. 2).

In another embodiment of the present invention, the structure of the selected squamometer is predicted using an RNA structure prediction program (FIGS. 3 and 4), and the squamogram sequence has an interaction propensity of 37% and 85 It has been confirmed that it has a discriminative power and an interaction strength of about 100% (FIG. 5).

In addition, in another specific embodiment of the present invention, it was confirmed that the FAM (Fluorescein Amidite) modified ssRNA aptamer showed strong fluorescence intensity against MLL1 (Fig. 7), and the binding dissociation constant was measured (Fig. 8) (FIG. 9) and an electrophoretic mobility shift assay (EMSA) (FIG. 10), confirming that the aptamers selected in the present invention specifically bind to MLL1.

Further, it was confirmed that the activity of MLL1 protein was inhibited to a level similar to that of known MLL1 inhibitor GS-13041 by performing a chemiluminescence test using the above-mentioned selected platemer (FIG. 9).

Taken together, these results indicate that the aptamer of the present invention specifically binds to MLL1 and has an effect of inhibiting protein activity.

On the other hand, when the plummeter is used, stability and physical properties can be changed through chemical modification of the plummeter residue. Such a method is commonly performed in the art, and it is obvious that the same is also included in the scope of the present invention. Specifically, the aptamer may be a modified nucleotide analogue of at least one or more nucleotides in SEQ ID NO: 10 to SEQ ID NO: 19 (or a complementary sequence thereof), wherein the modified nucleotide analogue is at the 2'position of the ribosome of the nucleotide May be substituted with a hydrogen atom, a fluorine atom, an -O-alkyl group, an -O-acyl group or an amino group. Also, the aptamer may further comprise at least one selected from the group consisting of PEG (polyethylene glycol), inverted deoxythymidine (idT), LNA (Locked Nucleic Acid), 2'-methoxy nucleoside, A modified form of the known tyrosinase residue, a modified form of the known tyrosine residue, a modified form of the known tyrosine residue, a modified form of the known tyrosine residue, Can be appropriately selected and applied to the present invention. For example, Acta Naturae, 2011, 3 (4): 12-29; And http://www.genelink.com/Literature/ps/Aptamer_PG_Ver3.2.pdf may all fall within the scope of the present invention.

Another embodiment of the present invention is a pharmaceutical composition for the prevention or treatment of MLL1 mediated diseases comprising said platemer.

The aptamer specifically binding to MLL1 of the present invention shows an inhibitory activity against MLL1 protein and can be effectively used for the prevention or treatment of MLL1 mediated diseases.

The term " MLL1-mediated disease " in the present invention refers to a disease caused by activation of MLL1 protein, specifically, an increase in expression upon increase of transcription activity of a gene encoding MLL1, or post-translational modification of MLL1 protein ) Or all diseases caused by increased activity of MLL1 protein by gene mutation or the like.

The MLL1 mediated disease may be, for example, leukemia, and may be, but is not limited to, acute lymphocytic, acute myelogenous, chronic lymphocytic or chronic myelogenous leukemia. In addition, the MLL1 mediated diseases may include all kinds of cancers that can be mediated by MLL1, such as breast cancer, colon cancer, stomach cancer, and cervical cancer. By reducing the activity of the MLL1 protein using the platemer of the present invention, MLLl mediated diseases can be prevented or treated.

As used herein, the term " prevention " means any action that inhibits or delays the onset of MLL1 mediated disease by administration of the composition, and " treatment " means that administration of the composition alleviates symptoms due to MLL1 mediated disease It means all acts that benefitfully change.

The effective amount of the platemer of the present invention in the pharmaceutical composition is not particularly limited and may be in the range of 0.00001% by weight to 99.99% by weight based on the total weight of the pharmaceutical composition, or may be included in the total amount.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, in addition to comprising an < RTI ID = 0.0 > platamer. ≪ / RTI > The pharmaceutical composition of the present invention can be formulated and used together with the carrier.

In the present invention, the type of the carrier is not particularly limited and any carrier conventionally used in the art can be used. In the present invention, the carrier comprises a natural carrier and a non-natural carrier. Examples of the carrier include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, . These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition may be supplemented with other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, and may be used as fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, binders Or a lubricant or the like may be additionally used.

The pharmaceutical composition may be formulated into various formulations suitable for oral administration or parenteral administration.

Non-limiting examples of such oral dosage forms include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs .

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; Excipients such as dicalcium phosphate and the like; Disintegrating agents such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be used, and sweeteners, fragrances, syrups and the like may also be used. .

Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be further used.

Non-limiting examples of the parenteral preparation include injectable solutions, suppositories, respiratory inhalation powders, aerosol formulations for spray, mouth spray, mouthwash, toothpaste, ointment, powder for application, oil, have.

In order to formulate the pharmaceutical composition for parenteral administration, a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, a freeze-dried preparation, an external agent and the like may be used. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, Vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used.

More specifically, when the pharmaceutical composition is formulated into an injection, the composition is mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which is formulated for unit administration of an ampoule or a vial can do. In addition, when the pharmaceutical composition is formulated into an aerosol formulation, a propellant or the like may be blended with the additive such that the water-dispersed concentrate or the wet powder is dispersed.

When the pharmaceutical composition is formulated into ointment, cream, or the like, it is possible to use an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, Can be used as a carrier.

The pharmaceutical composition may be formulated into parenteral dosage forms, including, but not limited to, gels, patches, sprays, ointments, alerts, lotions, liniments, pastas, And a plasma agent.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term " pharmaceutically effective amount " of the present invention means a therapeutic or prophylactic treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the amount of the effective amount of the composition of the present invention, and the effective dose level is determined depending on the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, The duration of the treatment, factors including the drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. Considering these factors, it is important to administer an amount that can achieve the maximum effect in the least amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined, for example, by administering the pharmaceutical composition of the present invention to a mammal, including a human, at 1 to 20 mg / kg, more particularly 1 to 10 mg / kg, And the frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into several doses.

Another aspect of the present invention provides a method of preventing or treating MLL1 mediated diseases comprising administering to a subject a pharmaceutical composition comprising an abtamer that specifically binds to MLL1.

The prevention or treatment of the platemer, pharmaceutical composition and MLL1 mediated diseases is as described above.

As described above, the aptamer specifically binding to MLL1 provided by the present invention can be used as an active ingredient of a pharmaceutical composition for preventing or treating MLL1 mediated diseases, so that the composition can prevent or treat MLL1 mediated diseases Can be used.

The term " individual " as used herein includes, without limitation, mammals, including rats, livestock, humans, and the like, which have been or are likely to develop MLL1 mediated diseases.

The preventive or therapeutic method of the present invention specifically includes a step of administering to a subject suffering from or at risk of developing MLL1 mediated disease a composition comprising a platemma specifically binding to MLL1 in a pharmaceutically effective amount . The appropriate daily use amount of the composition comprising the platemma that specifically binds to MLL1 can be suitably determined by those skilled in the art within the scope of sound medical judgment.

The specific pharmaceutically effective amount of the composition comprising the platemper specifically binding to MLL1 to a particular animal will vary depending on factors such as the type and degree of reaction to be achieved, the age, weight, general health status, sex or diet of the individual , The administration time of the composition containing the platamer that specifically binds to MLL1 as an active ingredient, the administration route and the fraction of the composition, the treatment period, etc., and the drug or other composition used simultaneously or at the same time And the like, and similar factors well known in the medical arts.

The administration route and method of administering the composition comprising the platemma specifically binding to the MLL1 in the above-mentioned preventive or therapeutic method of the present invention are not particularly limited and may be selected depending on the pressure specifically binding to the MLL1 Any route of administration and mode of administration may be used as long as the composition containing the tramer is reachable. Specifically, the composition comprising the platemper specifically binding to MLL1 can be administered via various routes of oral or parenteral administration. Non-limiting examples of the administration route include oral, rectal, topical, intravenous, intraperitoneal Intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, or inhalation, and the like.

Another embodiment of the present invention is a diagnostic composition for an MLL1 mediated disease comprising an abatumer that specifically binds to MLL1.

The platemaker and MLL1 mediated diseases are as described above.

Since MLL1 can be overexpressed in patients with MLL1 mediated diseases such as leukemia, breast cancer, colorectal cancer, stomach cancer and cervical cancer, the aptamer of the present invention can be used as a diagnostic composition for the MLL1 mediated diseases.

Another aspect of the present invention relates to a method for preparing a biological sample, comprising the steps of: (a) reacting an isolated biosample with a platemper specifically binding to the MLL1; And (b) measuring the degree of binding of the platamer in the biological sample, and determining that the disease is MLL1 mediated disease when the degree of binding is higher than the degree of binding in the normal sample. .

The platemaker and MLL1 mediated diseases are as described above.

The biological sample may be, but is not limited to, a mammalian organism other than a human, a cell, tissue, blood, body fluids, saliva, etc. isolated from mammals including humans.

The step of measuring the degree of binding of the platemer in the biological sample may be performed using a DNA platamer binding assay technique commonly used in the related art. For example, fluorescence or a radioactive substance may be labeled at the end of the platemaker Binding to biotin to measure fluorescence or radiation intensity, or imaging and observing the fluorescence or radiation intensity. However, the present invention is not limited thereto.

The detectable label may be a fluorescent substance or a radioactive substance label (or a substance capable of reacting with a fluorescent substance or a radioactive substance), for example, a coloring enzyme (e.g., peroxidase, alkaline phosphatase), a radioactive isotope ^{: 124 I, 125 I, 111} In, 99m Tc, 32 P, 35 S), chromotherapy pores (chromophore), FITC, RITC, fluorescent protein (GFP (Green fluorescent protein); EGFP (Enhanced Green fluorescent protein), RFP (Red Fluorescent Protein), DsRed ( Discosoma sp. Red fluorescent protein), CFP (Cyan Fluorescent Protein), CGFP (Cyan Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), Cy3, Cy5 and Cy7.5 But is not limited thereto.

As described above, the presence or overexpression of MLL1 in a sample using the platemer of the present invention is significantly superior to detection using a conventional antibody or peptide.

Another embodiment of the present invention is a composition for imaging an MLL1 mediated disease site comprising an abtamer that specifically binds to MLL1.

The imaging and diagnosis of MLL1 mediated diseases may include, but is not limited to, the primary purpose of MLLl mediated disease, as well as progress, progress, and response to treatment. The aptamer may be provided as a link (for example, covalently bonded or bridged) to a detectable label to facilitate identification, detection and quantification of binding.

Specifically, the detectable label for MLL1 mediated disease site imaging can be a radioisotope, a fluoropore, a quantum dot, or a magnetic particle, such as super paramagnetic particles or ultrasuper paramagnetic particles, and the like.

Another embodiment of the present invention is a nanoparticle contrast agent comprising a composition for imaging the MLL1 mediated disease site.

The aptamers of the present invention may be provided in the form of nanoparticle contrast agents for imaging of MLLl mediated disease sites by binding to nanoparticles. Here, the aptamer of the present invention specifically binds to MLL1 to serve as a target ligand for targeting MLL1-mediated disease site, and thus it is possible to rapidly and accurately diagnose MLL1-mediated disease site by an active targeting method have.

In the present invention, the term "contrast agent" refers to an agent used for visualizing blood vessels and tissues by artificially forming a difference in contrast degree for diagnostic purposes. Contrast agents can determine the presence and degree of disease or damage by increasing the visibility and contrast of the surface under study.

In order to be used as a contrast agent, biocompatibility and biodegradability must be excellent in the living body, the stability in the living body is excellent, and the biodistribution in the blood is high, so that it is necessary to continuously accumulate the cancer tissue for a sufficient time. The nanoparticle contrast agent combined with an aptamer of the present invention is highly safe for use as a contrast agent because it has a very high cancer cell scale efficiency, is not toxic to living organisms, and does not show abnormal findings.

Nuclear imaging including magnetic resonance imaging (MRI), X-ray imaging techniques, and positron emission tomography (PET) may be used to implement the contrast agent of the present invention, no.

Magnetic resonance imaging (MRI) is an imaging technique that acquires the anatomical, physiological, and biochemical information of the body using images of spin relaxation of hydrogen atoms in a magnetic field. When the present invention is applied to a nanoparticle contrast agent for MRI, paramagnetic nanoparticles or superpowder nanoparticles widely used in the art can be used. For example, in the case of paramagnetic particles, transition metal ions such as Gd, Fe, and Mn may be used, and superparamagnetic iron oxide superparamagnetic iron oxide nanoparticles may be used.

The aptamer of the present invention specifically binds to MLL1, and thus can effectively inhibit the activity of MLL1 protein. Therefore, the composition comprising the platemaker can be usefully used for the prophylaxis and treatment of MLLl mediated diseases including leukemia.

Figures 1a and 1b measure the specificity of the S16 library for MLL1. (a) Equivalent amounts (10 ng) of RNA libraries S0 and S16 were applied to 1 쨉 g of MLL1 protein, and then the Ct value for the bound oligonucleotide was measured using qRT-PCR, and the Ct value was substituted for the standard curve to calculate the amount . Binding affinities were expressed as a ratio of bound forms (MLL-binding RNA / total RNA x 100). Each value is expressed as mean ± SEM. * P <0.001 is the value for the initial library control. (b) Various amounts of RNA (S0, S3 and S16) were applied to 1 쨉 g of MLL1 protein and 1 / Ct values confirmed by qRT-PCR for the log of RNA amount (ng).
Figure 2 shows an MLL1 binding force tamer that occupies a high specific gravity in the library.
3 shows the KineFold structure of the plummeter.
Figure 4 shows the secondary structure of the selected platamater. The expected secondary structure of the selected RNA-platamer using the Mfold official web server was shown.
FIG. 5 is a graph for estimating the binding tendency of the plumbers to the MLL1 using the web server catRAPID graphic and the catRAPID intensity. Discriminative power (DP) is expressed in the range of 0% (unpredictable) to 100% (predictable). A DP value of more than 50% means that there is almost no interaction, and a DP value of more than 75% indicates a high-confidence prediction.
FIGS. 6A and 6B illustrate motifs of 50 extruders by Multiple EM Motif Elicitation (MEME) and Gapped Local Alignment of Motifs (GLAMS). The top five abdomens with the highest marginal score were shown.
Fig. 7 shows the MLL1 specificity of the platemaker using a fluorescence microscope. The affinity and specificity of the three most enriched FAM-labeled ssRNA extracellular (APT1, APT2 and APT3 for A, B and C, respectively) and control (D) were tested for their MLL1. E and F represent FAM-APT1 combined with agarose beads and TNFa, respectively.
FIG. 8 shows Kd values determined by FLAA (Fluorescence Linked Aptamer Assay).
Figure 9 compares the binding affinity of the library and selected platamer for the MLL1 protein by qRT-PCR analysis.
FIG. 10 shows the results of performing electrophoretic mobility shift assay (EMSA) using unmodified APT1 and MLL1.
Figure 11 shows the pH effect for APT1 binding.
Figure 12 shows the temperature resistance in the APT1-MLL1 complex binding.
Figure 13 shows the 3D structure of the platamer-MLL1 complex. (A) is a 3D structure of APT1, (B) is a docking model between MLL1 and APT1, and (C) is a result of observing three-dimensional interaction of MLL1 SET domain and APT1 nucleotide at two points.
Figures 14A and 14B show chemiluminescent assays of the MLL1 complex. (a) the effect of APT1, APT2 and APT3 on MLL1 activity. NEG: negative control (without enzyme), DMSO: DMSO-treated, S0: SELEX first library. *** p <0.001 is the value for the DMSO group. (b) the effect of APT1 on MLL1 activity. NEG: negative control (without enzyme), DMSO: DMSO-treated, S0: SELEX original library, GS-13041: MLL1 inhibitor. *** p <0.001 is the value for the DMSO group.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  1: Library manufacture

The first ssDNA library with XhoI and HindIII restriction sites, along with a flanking region comprising the T7 promoter and primer region, contains a central randomized region sequence of 25 nucleotides.

Specifically, the initial ssDNA library sequence is 5'-CGACTCACTATA GGCTCGAGG-N (25) -GGAAGCTTACGGTACCTAGC-3 '. The forward primer 5'-GCTAATACGACTCACTATAGGCTCGAGG-3 '(SEQ ID NO: 1) and the reverse primer 5'-GCTAGGTACCGTAAGCTTCC-3' (SEQ ID NO: 2) were used for transcription, screening, reverse transcription and PCR amplification in the SELEX screening process. The RNA plasmid library was purified using polyacrylamide gel electrophoresis and dissolved in NTEN buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40).

Example  2: MLL1  Preparation and purification of proteins

The MLL1 (SET) expression plasmid was obtained from Yali Dou (University of Michigan). His-tagged MLL1 (SET) protein (aa 3762-3970) was expressed in E. coli BL21 (DE3) strain and purified by incubation with Ni-NTA agarose beads. The bound protein was washed with PBS (phosphate-buffered saline) buffer containing 25 mM imidazole and eluted twice with PBS containing 250 mM imidazole. The concentration of the separated proteins was confirmed by Bradford analysis.

Example  3: SELEX  Screening process

First, the RNA plamer library was exposed to agarose beads three times to remove the platamer non-specifically bound to the beads. Then 10 μg of RNA library was mixed with 1 μg of MLL1 immobilized Ni-NTA beads suspended in NTEN buffer solution (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) Lt; / RTI &gt; for 1 hour. Unbound RNA was washed three times with NTEN buffer solution (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) to remove RNA, and the RNA-protein complex was directly reverse- To generate a cDNA library to be used for the next PCR reaction. The obtained DNA library was used for the next step of RNA production through an in vitro transcription (IVT) process.

After the first 5 rounds of SELEX, the amount of RNA library was reduced to half (5 [mu] g). After every third selection, the selection pressure was increased to capture the most strongly bound species, and the amount of the target was reduced by half. We performed 16 selections.

Example  4: qRT - PCR  (quantitative reverse transcription polymerase chain reaction)

The initial library (S0) and the library (S16) after 16 rounds of selection were used as a template for cDNA synthesis using an IVT kit (Invitrogen) according to the manufacturer's instructions. A standard curve was generated based on the results obtained by performing the qRT-PCR with the pre-binding RNA library (SO) at various RNA contents (0 ng, 1 ng, 10 ng and 100 ng). A partial sample (1/100) of the cDNA reaction mixture was analyzed by qRT-PCR in Stratagene Mx3005P (Agillent biotechnology) according to the manufacturer's instructions.

Example  5: High throughput  High-throughput sequencing technology

The S1, S3, and S16 libraries were purified by agarose gel, and then 10 ng of each purified library was ligated to the custom adapter sequence, the forward 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3` (SEQ ID NO: 3) (SEQ ID NO: 4) - (index) - GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3` (SEQ ID NO: 5). The reverse adapter sequence is linked to a specific index sequence in which the nucleotide sequence of SEQ ID NO: 4 and SEQ ID NO: 5 is able to distinguish the S1, S3 or S16 library. After sequencing, the library can be distinguished through an index sequence. To check each library, illumina Various indicators of TruSeq® Targeted RNA Expression (R702, R704 and R707) were used for each library. PCR was performed on the DNA library sequences linked to the adapter and the fragments with sizes ranging from 150-200 bp were separated by gel extraction Pippin pre (Sage Science) using a 3% agarose gel cassette and software V. 6.00. The quality of the library was quantified as the concentration measured by Agilent bioanalyzer 2011 (Serial Number 72,901,419), and by using the SynergyH1 hybrid reader (biotek) Picogreen method (Quant-iT TM Picogreen ®). Sequencing was performed using MiSeq ^™ illumina ^® using 7 pM of each library according to the manufacturer's instructions.

Example  6: Sequencing analysis

The data obtained by the sequencing was used to identify the correct population of MLL1 specific platamers. First, sequences that do not include adapter sequences and flanking regions were removed. The adapter sequences and flanking regions were then filtered to find the correct sequence. After filtering, the data was analyzed using Perl Script software.

Example  7: Abtamer's  Computer analysis

7-1. Identify secondary structure

KineFold web server ( http://kinefold.curie.fr/cgi-bin/form.pl ) for predicting RNA folding, including pseudo knots and entangled helix, and Mfold official server http://mfold.rna.albany.edu ) to obtain the secondary structures of the five top plummers. In Mfold, the parameter adjustments for all plummers are: suboptimality number = 5, the upper bound on the number on computed folding = 4, maximum internal bulge / maximum interior bulg / loop size) = 30. The folding temperature and salinity were pre-fixed and unchanged.

7-2. QGRS  ( Quadruplex  forming G-Rich Sequences)

The nucleotide sequence of the selected platamer was analyzed to identify the putative QGRS. The existence of the quadruplex structure was used by the official web server http://bioinformatics.ramapo.edu/QGRS/analyze.php .

7-3. catRAPID  Graphics and catRAPID  burglar

The catRAPID graphics module ( http://service.tartaglialab.com/update_submission/ ) predicts the interaction propensity of protein-RNA pairs and, with the corresponding training set, the discriminative power ), And interaction scores. The carRAPID Strength Module ( http://service.tartaglialab.com/update_submission/ ) calculates the interaction strength of a protein-RNA pair in response to a reference set. The reference sequence has the same length as the protein-RNA pair of interest.

7-4. RNA Abtamer's  The common motif (consensus motif)

The common motifs of RNA ablation are GLAMS2 (Gapped Local Alignment of Motifs, http://meme.nbcr.net/meme/tools/glam2 ) and MEME (Multiple Em for Motif Elicitation, http://meme.nbcr.net / meme / tools / meme ) using a web server.

Example  8: Fluorescence microscopy

The Fluorescein Amidite (FAM) modified ssRNA extracellular sequence was synthesized in Bioni (daejeon, Korea). The platemaker and FAM labeled control RNA were heated in binding buffer for 10 min at 90 &lt; 0 &gt; C, rapidly cooled in 0 [deg.] C ice, then incubated at room temperature for 10 min. One microgram of protein was incubated with the FAM-labeled platemaker or the control for 30 minutes. After the binding reaction, the supernatant was removed, and the unbound extracellular domain was removed by washing the FAM-RNA and protein complexes three times with binding buffer solution. 100 [mu] l of binding buffer was added to each tube and the complex was placed on a microscope slide. I took a picture of Abdomen using a Nikon Eclipse Ti U microscope.

Example  9: Dissociation constant  ( Kd ) decision

The dissociation constant (Kd) was determined using qRT-PCT and fluorescence method.

9-1. qRT - PCR  Used Dissociation constant  ( Kd ) decision

The original libraries of varying concentrations and S16 were each bound to 1 g of MLL1 protein (with Ni-agarose beads) in binding buffer (NTEN) in separate tubes and incubated for 30 minutes. The protein-abtamer complex was washed three times with binding buffer solution and the complex was used as a target for cDNA synthesis. A 1/100 split sample was prepared from each tube and the amount of the affhammer bound to the protein was quantified by qRT-PCR in Stratagene Mx3005P (Agillent biotechnology) according to the manufacturer's instructions.

9-2. Fluorescence Dissociation constant  Measure

The FAM-labeled ssRNA aptamer was synthesized in Bioni; 3 '(SEQ ID NO: 6), APT2-5' FAM-CGAGGACGUAACAGAGGGAGGCGAGUGGG-3 '(SEQ ID NO: 7), APT3-5 FAM-CGAGGACGGAAGUCGAGGGGGACGUGAGGG- - 5 'FAM-AUCGAAGUUAAUUGAGUCAUUUCAGUCUGA-3' (SEQ ID NO: 9). The platemaker and control RNA were heated in binding buffer for 5 minutes at 90 &lt; 0 &gt; C, rapidly cooled in 0 [deg.] C ice, then incubated at room temperature for 10 minutes. One microgram of protein was incubated with the FAM-labeled platemaker or control for 30 minutes. After the binding reaction, the supernatant was removed and the FAM-RNA and protein complexes were washed three times with NTEN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40). The combined compressors were then eluted by heating to 95 [deg.] C for 10 minutes. The amount of combined platamer was measured by quantifying fluorescence using a Synergy H1 hybrid reader (Biotek) and the dissociation constant was calculated by non-linear regression method using prism graph pad software 6.0.

Example  10: Temperature and pH tolerance test

10-1. Temperature tolerance test

The FAM labeled ssRNA APT1 was tested for temperature resistance. FAM-APT1 was heated at 95 DEG C for 10 minutes, rapidly cooled on ice for 5 minutes, and then incubated at room temperature. 2000 nM of RNA-APT1 in binding buffer in a total volume of 25 [mu] l was incubated with 1 [mu] g of NiTA beads conjugated with MLL1 protein. After 30 minutes, all supernatants, including uncompressed platemer, were removed and the APT-MLL1 complex washed three times with 200 μl of binding buffer. Finally, 200 [mu] l of binding buffer was added to the complex and allowed to settle at room temperature. To measure the temperature at which bound APT in the MLL1-APT1 complex drops, the complexes were incubated at different temperatures, namely 35, 45, 55, 65 75 &lt; 0 &gt; C, 85 &lt; 0 &gt; C and 95 &lt; 0 &gt; C. The complex was incubated at a specific temperature for 10 minutes, 100 占 퐇 of the supernatant was taken for analysis, and then 100 占 퐇 of buffer solution was added to the mixture to maintain a total volume of 200 占 퐇. The fluorescence intensity was measured with the supernatant removed.

10-2. pH tolerance test

The pH values of the binding buffer were varied (2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5 and 12.5) using HCl and NaOH solutions, Binding reaction between 200 nM of APT1 and 1 [mu] g of MLTA binding NiTA beads was confirmed. After 30 minutes of conjugation, the supernatant was removed with unbound amphotermer and the complex was washed three times with binding buffer solution of the corresponding pH value. The combined compressors were then eluted by heating to 95 [deg.] C for 10 minutes. The combined platelets were quantitated using a Synergy H1 hybrid reader (Biotek).

Example 11: 2 components  (binary) composite EMSA  ( Electrophoretic  Mobility Shift Assay) Analysis

The binding of APT1 to the MLL1 protein target in solution was analyzed by EMSA. In all solutions, the aptamer was incubated for 5 min at 95 ° C for denaturation, immediately frozen on ice for 5 min and then incubated at room temperature. The total volume was 25 [mu] l, and each platemaker solution was incubated at room temperature for 30 minutes while increasing the concentration of MLL1 protein. The complex was loaded onto a 10% unmodified polyacrylamide gel (native PAGE gel) in 1X TBE buffer (Tris-HCl 89 mM, Borate 89 mM, EDTA 2 mM). The gel-like aptamer was stained with gel green (Biotium), which binds to the RNA-abatumer. The gel was visualized by Bio Rad gel imager Doc ^® EZ.

Example  12: MLL1 - APT1  3D Structure Prediction of Composites

The secondary structure of the APT RNA was dot-bracketed through the RNAfold web server ( http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi ) to predict only the minimum free energy. The above file format is needed to fold and draw the Pdb format of APT1 with the RNA composer web server (httpL // rnacomposer.ibch.poznan.pl /). The 3D structure of the MLL1 SET domain was obtained from Protein Data Bank (PDB code: 2W5Y) and APT1 was drawn with iFoldRNA Ver 2.0 ( http://troll.med.unc.edu/ifoldrna.v2/index.php ). APT1-MLL1 (SET domain) docking is ZDOCK server Ver 3.0.2 ( http://zdock.umassmed.edu) which evaluates each structure using energy-based scoring function, Statistical Potential, Shape Complementarily and Electrostatics / ). Structural homology was also performed with Swiss-PdbViewer 4.1.0.

Example  13: MLL1  Complex chemiluminescence assay

The activity of MLL1 in the presence of squamous cell was quantitated by chemiluminescence assay (BPS Bioscience) according to the manufacturer's instructions. Specifically, 250 ng of MLL1 was added to a reaction mixture solution (4x HMT assay buffer solution 1, 20 μM S (pH 3) containing 3 μM each of platamater (APT1, APT2 and APT3), S0 control library or positive control (S-adenosylmethionine and inhibition buffer solution), and H ₂ O was added thereto to adjust the final volume to 50 μl. The mixture was incubated at room temperature for 30 minutes. The mixed solution was put on a 96-well plate and incubated at room temperature for 1 hour. Each well was washed with 200 [mu] l of 1X TBST buffer (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20), dried with a paper towel and incubated with 10 [mu] l of blocking buffer for 10 minutes. 100 [mu] l of 800-fold primary antibody 2 (diluted in blocking buffer) was added to each well and incubated for 1 hour. The wells were then washed three times with blocking buffer, dried and then added with HRP (horseradish peroxidase) labeled secondary antibody 1 (1,000-fold dilution in blocking buffer) to each well. After incubation for 30 minutes with gentle agitation at room temperature, each well was washed with 200 μl of 1X TBST buffer (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20), dried with paper towel, and then 100 μl of blocking buffer &Lt; / RTI &gt; for 10 minutes. 100 [mu] l of an equal volume fraction (50 [mu] l of HRP chemiluminescent substrate A and 50 [mu] l of HRP chemiluminescent substrate B) was mixed on ice and added to each well. Chemiluminescence was measured immediately using a Bio-Tek fluorescent microplate reader.

Experimental Example  One: SELEX  fair

In order to isolate RNA tyramaras binding to MLL1, SELEX optimized for obtaining high affinity and high specificity platamerm was performed 16 times. Prior to the actual SELEX process, five negative SELEXs were performed using Ni-NTA-beads to remove non-specific binding. To concentrate the platelets for MLL1, SELEX was performed more rigorously by lowering the ratio of RNA to protein (1: &lt; RTI ID = 0.0 &gt; 1/2) &lt; / RTI &gt; at positive screening angles, increasing the incubation time, . PCR was performed for 16 cycles to reduce artifacts and to maintain the library from abnormal replication sequences. Before each binding step, the RNA library was heated to 90 ° C, rapidly cooled on ice and normalized at room temperature to reduce the occurrence of false secondary structures in the RNA library.

Experimental Example  2: To MLL1  The binding affinity of the RNA library

To confirm the concentration of MLL1-binding RNA from each SELEX cycle, the amount of oligonucleotide was measured using qRT-PCR technique. Standard curves were drawn with pre-bound pure SO RNA libraries (0 ng, 1 ng, 10 ng and 100 ng). Ten ng of each of the original RNA library (S0) or library (S16) after 16 rounds of selection were ligated into 1 μg of MLL1. The Ct values of the two complexes were compared with the standard RNA curve. As a result, 0.7 ng (7% of 10 ng) of S0 was bound to MLL1 and 6.8 ng (68% of 10 ng) of S16 was bound (Fig. 1A). S16 showed about a 10-fold higher affinity for MLL1 compared to S0, indicating that the SELEX process was performed several times to increase library selectivity.

Further, 1 / Ct values were determined at various concentrations of the S0, S3 and S16 RNA libraries, and it was found that the expected bond strength for MLL1 was S16> S3> S0, which means that the SELEX process was performed correctly 1 of B).

Experimental Example  3: of the RNA library High throughput  Through high throughput sequencing Abtamer  Selection

High throughput sequencing SELEX (high throughput sequencing SELEX, HT-SELEX) is more efficient, stable, and flexible compared to conventional SELEX in terms of multiple screening, cloning and optimization steps, . Therefore, NGS (Next Generation Sequencing) was performed to analyze the sequences of the S1, S3, and S16 libraries.

Approximately 1.5 × 10 ⁶ sequences were obtained from each screening cycle and data were analyzed using Perl Script. Sequencing data analysis of the library confirmed that the ratio of the two platameras (APT1 and APT2) was significantly increased in the S3 and S16 libraries (Table 1 and Figure 2). As the SELEX screening count increased, the two platelet counts increased significantly.

Abtamer order group ( % ) Concentration (times) 5'-GGCUCGAGG-N (25) -GGAAGCUUACGGUACCUAGC-3 ' S16 S3 S1 S3 / S1 APT1 5'-GGCUCGAGG-AACGUACAGAGGGUGGAGAGUGGGU-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 10) 30.49 12.70 3 x 10 ^-2 464 APT2 5'-GGCUCGAGG-ACGUAACAGAGGGAGGCGAGUGGGU-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 11) 25.95 12.24 3 x 10 ^-2 384 APT3 5'-GGCUCGAGG-ACCGAAGUCGAGGGGGACGUGAGGG-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 12) 0.78 0.43 1 x 10 ^-3 534 APT4 5'-GGCUCGAGG-ACCUAAGUGGGAAGGUGAGCGGGUG-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 13) 0.27 0.28 6 x 10 ^-3 303 APT5 5'-GGCUCGAGG-AACGUACAGAGGGCGGAGAGUGGGU-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 14) 0.25 0.18 6 × 10 ^-4 376 APT6 5'-GGCUCGAGG-AAAUGCAAGGAGGUCGGAGGUCGGA-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 15) 0.22 0.14 6.8 x 10 ^-5 164 APT7 5'-GGCUCGAGG-ACCGACGACGAGGGGGACGUGAGGG-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 16) 0.20 0.12 6.8 x 10 ^-5 197 APT8 5'-GGCUCGAGG-AUAAGCAUGGGGGUCGAGAGGGGAC-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 17) 0.20 0.11 6.8 x 10 ^-5 294 APT9 5'-GGCUCGAGG-AACGUACAGAGGGAGGCGAGUGGGU-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 18) 0.17 0.10 6.8 x 10 ^-5 273 APT10 5'-GGCUCGAGG-AACGUACAGAGGGUGGAGAGUGGGU-GGAAGCUUACGGUACCUAGC-3 ' (SEQ ID NO: 19) 0.17 0.10 2 x 10 ^-3 268

In addition, the concentration ratio (ratio of the number of copies between the S3 / S1 sorting times) was analyzed, and it was confirmed that APT3 was concentrated at a higher ratio than APT1 and APT2 (Table 1).

The data show that aptamers with high copy numbers do not necessarily exhibit high enrichment ratios as reported by other researchers. The S16 / S3 enrichment ratio showed no significant difference (2.4, 2 and 2 times, respectively) between APT1, APT2 and APT3. The above results indicate that the HT-SELEX technique is an effective way to minimize the cycle of SELEX.

Experimental Example  4: Abtamer's  Structure prediction

The present inventors have applied various bioinformatics tools to analyze the structure of the selected plumbers. First, the secondary structure of platamater is used as an RNA secondary structural prediction program kineFold ( http://kinefold.curie.fr/cgi-bin/form ), which encompasses prediction algorithms for pseudo knots and entangled helices .pl ). As a result of selecting the structure having the lowest free energy for analysis, it was confirmed that APT3 and APT5 had the lowest energy as compared with other structures (Fig. 3).

In addition, the structure of the umbrella was analyzed by Mfold official server ( http://mfold.rna.albany.edu ). The electrometer structure with the lowest energy was selected and it was confirmed that the energy and stability were almost similar patterns as in the KineFold web server (FIG. 4). The plethora structure drawn by Mfold consists of one central multibranch (APT1, APT2, and APT3) surrounded by two hairpin loops and one terminal open branch, loop). APT3 was found to have the lowest free energy as compared with the rest. From the above results, the present inventors concluded that the stability of the platemaker structure is not inevitably correlated with the binding affinity of the platamer.

On the other hand, since the aptamer obtained through sequencing has a large number of guanine bases (Gs) at the 5 'terminus, it is thought that the Gs forms a G-quadruplex structure. We analyzed the top five tabameters for the estimated QGRS (Quadruplex forming G-Rich Sequences) using the official web server ( http://bioinformatics.ramapo.edu/QGRS/analyze.php ). As a result, it was confirmed that there is no aptamer having substantially the quadruplex form.

Experimental Example  5: MLL1 - Abtamer  Pair Interaction Tendency and Prediction

Using the catRAPID graphical Web server ( http://service.tartaglialab.com/grant_submission/catrapid_graphic ), we predicted the protein-RNA pair interaction tendencies represented by discriminative power (DP). The interaction tendency was obtained by measuring the possibility of interaction between protein and RNA. The measurements are based on the observed propensity of the ribonucleic acid protein complex components to indicate the specific characteristics of the physical-chemical profile that can be used for prediction. The discrimination power is a statistical value whose interaction tendency is evaluated for the corresponding catRAPID training. This represents the reliability of the prediction.

Analysis of the binding tendency, discrimination power, and RNA protein intensity of APT1, APT2 and APT3 showed that for the reference set, the selected aptamer had the ability to bind at an appropriate level with the target MLL1. For further analysis, it was confirmed that all of the selected plastomer sequences had a 37% interaction tendency and an 85% discriminating power and had an interaction strength of about 100% (FIG. 5).

Experimental Example  6: Common motif analysis

Next, in order to obtain the actual motif of the library binding to the MLL1, it is necessary to use a multiple em for motif elicitation (MEME) and a gapped local alignment of motifs (GLAMS2) A common motif was confirmed (Fig. 6A). We chose MEME because we were able to find new, ungapped motifs (repetitive fixed length patterns) in the nucleotide sequence (the sample derived from the sequence). MEME separates patterns of various lengths into two or more motifs. GLAM2, on the other hand, looks for new, gapped motifs (repeating patterns of varying lengths) in DNA or protein sequences (samples derived from sequences).

In MEME, we chose the normal discovery mode for motif discovery, selected the location distribution of occurrences of 0 to 1 per sequence, and set the total motif number to three sequences. The most common motifs obtained with MEME (Motif No. 1 in FIG. 6A) had 72% sequence homology with APT1 and 60% sequence homology with APT2 in S16. In addition, APT1 and APT2 (31% and 26%, respectively, compared to the entire sequence) were 76% sequence homologous to each other. The result is to prove that after only a few screenings, only the platamers actually binding remain.

In GLAM2, a value obtained by subtracting the alignment score excluding the segment from the alignment score including the segment is expressed as a marginal score. The marginal score reflects how closely each segment corresponds to the other segment. As a result, APT1, APT2 and APT5 have the highest critical scores (24.9, 24.4 and 24.9, respectively) (Fig. 6B).

Experimental Example  7: Fluorescence microscopy observation

To further confirm the specific interaction of the MLL1 protein and the selected tamtamer, Ni-NTA-MLL1, Ni-NTA agarose (FAM) with FAM (Fluorescein Amidite) modified ssRNA tamtamer (APT1, APT2, APT3 and control RNA) Beads and Ni-NTA-TNFa. All of the three compressors showed strong fluorescence intensity against Ni-NTA-MLL1 (Fig. 7). No fluorescence was observed in the Ni-NTA agarose beads or Ni-NTA agarose beads in which the TNF alpha was bound. The data show that all selected platemers specifically bind to MLL1 and are not selected as false positives.

Experimental Example  8: FLAA  (Fluorescence linked Aptamer  Assay) to measure the dissociation constant (Kd)

In order to confirm the specificity of the MLL1-abatumer interaction, FLAA was performed on the tagged APT1, APT2 and APT3 fluorescent dye, FAM, to determine the equilibrium dissociation constant and binding specificity. The Kd values measured for the top three sequences, APT1, APT2 and APT3, were 8.28 nM, 98 nM and 36.61 nM, respectively (Fig. 8). APT1 was found to have the lowest Kd value for MLL1, which means that the most concentrated platamer, APT1, has the strongest affinity.

Experimental Example  9: Abtamer's  Binding affinity evaluation

The sequencing results and the dissociation constants are to prove that APT1 is the most potent binding pressure tamer for MLL1. To further confirm the above results, the affinity of the three platamers for the MLL1 protein was determined.

For this, qRT-PCR analysis confirmed that APT1 binds maximal binding to MLL1 protein (APT1, APT and APT3 are 281 ng, 177 ng and 79 ng, respectively). (S1, S3, S7, S11, and S16 were 0.63 ng, 11.2 ng, 4 ng, 141 ng, and 125 ng, respectively) as the cycle of SELEX increased ). The results clearly demonstrate that APT1 is the most potently binding platelet sequence for MLL1 and that APT1 has better binding ability than APT2 and APT3.

Experimental Example  10: MLL1 - APT1  For complex EMSA  ( Electrophoretic  Mobility Shift Assay)

The complex form of APT1 and MLL1 was further confirmed by EMSA analysis (Fig. 10). As the concentration of MLL1 increased, the APT-MLL complex appeared and the APT1 band was weakened. The data further demonstrate that an MLL1-APT1 complex is formed.

Experimental Example  11: MLL1 - APT1  Bond stability

In order to characterize MLL1-APT1 complex formation, binding strength and binding stability were evaluated at various pH and temperature stress conditions. As a result, it was confirmed that the APT1-MLL1 complex binds well at pH 7.5 and the change in pH value promotes dissociation thereof, because the secondary and tertiary structure of the protein changes under various pH conditions (FIG. 11).

Further, it was confirmed that the mutual action was weakened as the temperature rises (Fig. 12). This result implies that the specificity of the interaction between APT1 and MLL1 contributes to various weak interactions such as hydrogen bonding, ionic bonding and hydrophobic interaction.

Experimental Example  12: MLL1 - APT1  3D structure of complex

To predict the 3D structure of APT1, iFoldRNA was used. For docking, we chose the most spiral, low energy, and rigid structure. This structure was found to be very important for MLL1-protein recognition. Thus, macromolecular docking between the APT1 and MLL1 proteins was performed using the ZDOCK server (Fig. 13). The predicted structure shows that rA15, rC16, rA17, rG18, rA19, rG20, rG21, rG22 and rU23 are in close proximity to the enzyme active site S-adenosyl-homocysteine (AdoHcy) . Lysine 4 of histone H3 was inserted into the channel at the end of the AdoHcy binding site. Perhaps, such a structure appears to play a role in inhibiting enzyme activity by the MLLl SET domain.

Experimental Example  13: Abtamer's MLL1  Active inhibition confirmation

To confirm the effect of platemaster on MLL1 activity, chemiluminescence studies were performed on APT1, APT2 and APT3. Compared with the control (DMSO), APT1 and APT2 reduced MLL1 activity by about 70% (p < 0.005) (Fig. 9A). In contrast, the control libraries (SO) and APT3 failed to inhibit enzyme activity to significant levels. In particular, the inhibitory activity of APT1 (10 [mu] M) was similar to that of the known MLL1 inhibitor GS-13041 (Fig. 9B). The data show that APT1 and APT2 specifically interact with MLL1 and effectively inhibit it.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Gachon University of Industry-Academic cooperation Foundation <120> Aptamers that specifically bind to MLL1 and the use thereof <130> KPA151172-KR <160> 19 <170> Kopatentin 2.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> SELEX primer-F <400> 1 gctaatacga ctcactatag gctcgagg 28 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SELEX primer-R <400> 2 gctaggtacc gtaagcttcc 20 <210> 3 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> custom adapter sequence-F <400> 3 aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> custom adapter sequence-R-5 ' <400> 4 caagcagaag acggcatacg agat 24 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> custom adapter sequence-R-3 ' <400> 5 gtgactggag ttcagacgtg tgctcttccg atct 34 <210> 6 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> FAM APT1 <400> 6 cgaggaacgu acagagggug gagagugggu 30 <210> 7 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> FAM APT2 <400> 7 cgaggacgua acagagggag gcgaguggg 29 <210> 8 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> FAM APT3 <400> 8 cgaggaccga agucgagggg gacgugaggg 30 <210> 9 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> FAM control <400> 9 aucgaaguua auugagucau uucagucuga 30 <210> 10 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT1 <400> 10 ggcucgagga acguacagag gguggagagu ggguggaagc uuacgguacc uagc 54 <210> 11 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT2 <400> 11 ggcucgagga cguaacagag ggaggcgagu ggguggaagc uuacgguacc uagc 54 <210> 12 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT3 <400> 12 ggcucgagga ccgaagucga gggggacgug agggggaagc uuacgguacc uagc 54 <210> 13 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT4 <400> 13 ggcucgagga ccuaaguggg aaggugagcg ggugggaagc uuacgguacc uagc 54 <210> 14 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT5 <400> 14 ggcucgagga acguacagag ggcggagagu ggguggaagc uuacgguacc uagc 54 <210> 15 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT6 <400> 15 ggcucgagga aaugcaagga ggucggaggu cggaggaagc uuacgguacc uagc 54 <210> 16 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT7 <400> 16 ggcucgagga ccgacgacga gggggacgug agggggaagc uuacgguacc uagc 54 <210> 17 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT8 <400> 17 ggcucgagga uaagcauggg ggucgagagg ggacggaagc uuacgguacc uagc 54 <210> 18 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT9 <400> 18 ggcucgagga acguacagag ggaggcgagu ggguggaagc uuacgguacc uagc 54 <210> 19 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> APT10 <400> 19 ggcucgagga acguacagag gguggagagu ggguggaagc uuacgguacc uagc 54

Claims (11)

An aldosterone specifically binding to MLL1 (Mixed Lineage Leukemia 1) having a nucleotide sequence of any one of SEQ ID NOS: 10 to 12 and a complementary base sequence.
2. The platemaker of claim 1, wherein the aptamer is single stranded DNA or single stranded RNA.
An MLTl (Mixed Lineage Leukemia (MLL1)) gene comprising at least one nucleotide analogue modified in an extramammary having a nucleotide sequence of any one of SEQ ID NOS: 10 to 12, and a complementary base sequence, 1). &Lt; / RTI &gt;
The modified nucleotide analogue according to claim 3, wherein the modified nucleotide analogue is a nucleotide analogue in which the hydroxyl group at the 2 'position of the ribosome of the nucleotide is replaced by a hydrogen atom, a fluorine atom, an -O-alkyl group, Tamer.
[2] The composition of claim 1, wherein the aptamer is selected from the group consisting of PEG (polyethylene glycol), inverted deoxythymidine (IST), LNA (Locked Nucleic Acid), 2'-methoxy nucleoside, Wherein at least one member selected from the group consisting of 2'-amino nucleoside, 2'F-nucleoside, amine linker, thiol linker, and cholesterol is combined and modified.
A pharmaceutical composition for preventing or treating MLL1 mediated diseases comprising the platemer according to any one of claims 1 to 5,
Wherein said MLLl mediated disease is selected from the group consisting of leukemia, breast cancer, colon cancer, gastric cancer and cervical cancer.
7. A composition for the diagnosis of MLL1 mediated diseases comprising the platemer of any one of claims 1 to 5,
Wherein said MLLl mediated disease is selected from the group consisting of leukemia, breast cancer, colon cancer, stomach cancer and cervical cancer.
7. A composition for imaging an MLL1 mediated disease site comprising the platemer of any one of claims 1 to 5,
Wherein said MLL1 mediated disease is selected from the group consisting of leukemia, breast cancer, colon cancer, stomach cancer and cervical cancer.
The imaging composition of claim 8, further comprising a radiation isotope, fluorophores, quantum dots, super paramagnetic particles, or ultrasuper paramagnetic particles.
9. A nanoparticle contrast agent comprising a composition for imaging an MLL1 mediated disease site of claim 8.
(a) reacting the biotimer according to any one of claims 1 to 5 with the separated biological sample; And
(b) measuring the degree of binding of the platamer in the biological sample, and determining that the disease is MLL1-mediated when the degree of binding is higher than the degree of binding in the normal sample, As a method of providing,
Wherein said MLLl mediated disease is selected from the group consisting of leukemia, breast cancer, colorectal cancer, gastric cancer and cervical cancer.

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