JP2007129992A - Ribonucleic acid binding to runt domain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a substance useful for elucidating mechanism of pathogenesis of disease caused by relation to leukemia fusion protein having AML1, RUNX2 and RUNX3 being human transcription factors and a RUNT domain derived from AML1 such as leukemia-associated AML1-MTG8, AML1-MTG16, TEL-AML1, AML1-EVI1, AML1-EAP, AML1-MDS1, AML1-MDS1-EVI1, etc., and diagnosing and treating the diseases and a substance useful for research of an intracellular process such as transcription, etc. <P>SOLUTION: The RNA has an activity of binding to a RUN domain obtained by an SELEX method. The RNA aptamer is useful for functional analysis of a leukemia fusion protein having AML1, RUNX2, RUNX3 and a RUNX3 domain and diagnosing and treating diseases such as cancer, etc. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to ribonucleic acids that bind to the RUNT domain, such as leukemia fusion proteins derived from the transcription factors AML1, RUNX2, RUNX3 and AML1.

  In leukemia, there are many chromosomal translocations characteristic of the disease type, and new oncogenes are generated by gene fusion by translocation. Representative examples include t (9; 22) for chronic myeloid leukemia, t (8; 21) for acute myeloid leukemia, inv (16), t (15; 17), t ( 12; 21) and the like, and fusion genes of BCR-ABL, AML1-MTG8 (also known as AML1-ETO), CBFβ-MYH11, PML-RARα, and TEL-AML1 are generated, respectively. Chimeric proteins are produced from these fusion genes, and it is thought that canceration is induced by mutating the original functions of the two proteins. AML1 (also known as RUNX1) is an essential transcription factor for hematopoietic cell development and differentiation, and AML1-MTG8 is about 15% of acute myeloid leukemia, and TEL-AML1 is acute It accounts for about 20% of lymphocytic leukemia and is one of the most common chromosomal translocations. In addition, AML1-MTG8, AML1-MTG16, TEL-AML1, AML1-EVI1, AML1-EAP, AML1-MDS1, and AML1-MDS1-EVI1 are known as fusion genes related to AML1.

  The AML1-MTG8 protein is a chimeric protein in which the RUNT domain, which is the DNA binding region of AML1, and MTG8 in almost the entire region are linked. Since MTG8 is a protein with transcriptional repression activity that recruits N-CoR, mSin3A, SMRT, and HDACs, it is thought that differentiation is suppressed by suppressing dominantly genes that should be transcriptionally activated by AML1.

It has been clarified so far that the expression of transcription factors and differentiation plasma proteins involved in differentiation is actually suppressed. Furthermore, the expression of p14 ^ARF, a p53 stabilizing protein responsible for cell cycle arrest and apoptosis induced by cell differentiation, is also suppressed by AML1-MTG8. Therefore, the expression of AML1-MTG8 is considered to be a factor of canceration by promoting the self-renewal of stem cells and suppressing differentiation. Similarly, fusion proteins other than AML1-MTG8 are considered to cause leukemia by dominantly suppressing the gene expressed by AML1.

  Currently, for leukemias with chromosomal translocations, therapeutics targeting fusion genes have been developed and are actually being treated. Imatinib (Gleevec Novartis), a molecular targeted therapy for chronic myeloid leukemia, targets the BCR-ABL protein and inhibits the tyrosine kinase activity of ABL, resulting in abnormally activated cell proliferation signals in cancer cells Inhibits and suppresses the proliferation of leukemia cells, thereby increasing the therapeutic effect. In acute myeloid leukemia that expresses PML-RARα, transcription inactivation by PML-RARα is canceled by administration of a large amount of retinoic acid, and blocked transcription is reactivated and cell differentiation is resumed. Treatment is taking place. As nucleic acid drugs for leukemia fusion genes, suppression of fusion gene expression has been studied by ribozymes, antisenses, siRNAs, etc., but no drugs applicable as pharmaceuticals have been obtained so far. As an anti-sense agent for leukemia treatment, Genasense (Genta USA) targeting Bcl-2 is currently in progress to Phase III clinical trials.

  AML1 is an approximately 50 kDa protein factor having a RUNT domain and a transactivation (TA) domain, and forms a transcription factor called a core binding factor by forming a complex with CBFβ. The RUNT domain, the DNA-binding domain of AML1, binds to recognition sequences and transcribes various hematopoietic cell-specific genes by forming complexes with the transcriptional coactivator histone acetylase p300 / CBP. Activation of. In leukemia, AML1 is thought to be involved in the onset of AML1-mediated transcriptional activation that is essential for the differentiation of blood cells by the formation of fusion genes such as AML1-MTG8 and TEL-AML1 and decreased expression levels. Yes.

  As the AML1 family gene (RUNX family) having a RUNT domain, RUNX2 and RUNX3 have very high homology at the amino acid level, and in particular, the RUNT domain has 90% or more homology. These are all transcription factors, but AML1 is involved in the hematopoietic system, whereas RUNX2 is expressed in osteogenesis and RUNX3 is expressed in many tissues, but is particularly expressed in the stomach, small intestine, large intestine, etc. is doing. RUNX2 dysfunction results in clavicular dysplasia, and RUNX3 is known to be a major suppressor gene for gastric cancer. In addition, all currently known fusion proteins involving AML1 have the AML1 RUNT domain. Therefore, a molecule that recognizes and binds to the RUN1 domain of AML1 is considered to be very effective as a reagent for detecting leukemia. In addition, molecules that suppress the function of AML1 and fusion genes related to AML1 can be expected to be effective as therapeutic agents for leukemia. In addition, molecules that bind to the RUNX domain of RUNX2 and RUNX3 are expected to find applications in bone disease and cancer detection and treatment.

  By the way, a new method called SELEX method has been developed as a method to select and concentrate RNA that specifically binds to a target substance in vitro (C. Tuerk & L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249: 505-510, 1990). In the SELEX method, RNA having a random sequence of an appropriate length including a PCR primer sequence (including a T7 polymerase sequence at one end) is synthesized, and this is associated with a target protein and immobilized. After washing the unbound RNA, the bound RNA is recovered, amplified by RT-PCR, and used as an RNA template to be used in the next round. By repeating this around 10 rounds, an RNA aptamer that specifically binds to the target protein is obtained. In this method, RNA having physiological activity that promotes or inhibits the function of the target protein can be obtained. This suggests that the aptamers obtained can be applied as pharmaceuticals by performing SELEX targeting pathogenic proteins. The advantages of drug discovery using this SELEX method compared to conventional drug discovery are as follows: (1) Screening can be performed systematically from a larger population than conventional chemical screening. (2) A large amount can be easily synthesized in a test tube. (3) There is no immune exclusion. (4) It can be easily improved for the purpose. (5) It is possible to target proteins that are highly conserved and difficult to produce antibodies. Etc.

JP 2004-344008 A

  The present invention relates to AML1-MTG8, AML1-MTG16, TEL-AML1, AML1-EVI1, AML1-EAP, AML1-MDS1, AML1-MDS1-EVI1 associated with human transcriptional regulators AML1, RUNX2, RUNX3 and leukemia Substances that can be used to elucidate the pathogenesis of diseases caused by leukemia fusion proteins with the RUN1 domain derived from AML1, such as those that can be used for diagnosis and treatment, or to study intracellular processes such as transcription The purpose is to provide.

  The present inventors created an RNA aptamer against the RUN1 domain of AML1 as a candidate for such a substance. That is, SELEX was performed using the RUN1 domain of AML1 as a target protein, and the binding activity of the obtained aptamer to the target protein and RUNX2, RUNX3, and AML1-MTG8 was examined, and its usefulness was shown. The present invention has been completed based on these findings.

  The gist of the present invention is as follows.

(1) RNA represented by the base sequence described in any one of SEQ ID NOs: 1-12.

(2) An RNA having a nucleotide sequence capable of forming a secondary structure represented by the following formula (I) and having a binding activity to the RUNT domain.

(In the secondary structure, N1 is 3 or more nucleobases, N2 is 6 or more nucleobases, N3 is 1 or more nucleobases, N4 is 3 or more nucleobases, A is adenine, and G is cytosine. , G is guanine, U is uracil, and a circle connecting nucleobases represents a hydrogen bond between nucleobases.)

(3) RNA shortened by cutting part of the RNA according to (1) or (2), and having binding activity to the RUNT domain.

(4) RNA obtained by adding another RNA to the RNA described in (1) to (3), and having binding activity to the RUNT domain.

(5) RNA represented by a nucleotide sequence in which one or several bases are substituted, deleted or inserted in the nucleotide sequence of the RNA according to any one of (1) to (4), RNA with binding activity.

(6) RNA into which at least one modified nucleotide is introduced in the RNA according to any one of (1) to (5), and having binding activity to the RUNT domain.

(7) The RNA according to any one of (1) to (6), which is labeled.

(8) DNA having a base sequence complementary to the base sequence of RNA according to any one of (1) to (5).

(9) A vector into which the DNA according to (8) is inserted.

(10) A method for detecting a protein having a RUNT domain using the RNA according to (7).

(11) A method for quantifying a protein having a RUNT domain using the RNA according to (7).

(12) A method for isolating a protein having a RUNT domain using the RNA according to any one of (1) to (6).

  Hereinafter, the present invention will be described in detail.

  The RNA of (1) has binding activity to the RUNT domain. The RUNT domain is associated with transcription factors AML1, RUNX2, and RUNX3, and leukemia fusion proteins such as AML1-MTG8, AML1-MTG16, TEL-AML1, AML1-EVI1, AML1-EAP, AML1-MDS1, and AML1-MDS1-EVI1. included. “Having binding activity” means having higher binding activity than a random sequence RNA pool.

  The RNA of (2) contains a nucleotide sequence that can form the secondary structure shown in the above formula (I).

  In the above secondary structure, N1 is 3 or more nucleobases, preferably 3 to 7 nucleobases. N1 can take all nucleobases. N2 is 6 or more nucleobases, preferably 6 to 10 nucleobases. N2 can take all nucleobases. N3 is one or more nucleobases, preferably 1 to 3 nucleobases. N3 can take all nucleobases. N4 is 3 or more nucleobases, preferably 3 to 7 nucleobases. N4 can take all nucleobases. As an example of a preferable secondary structure, a secondary structure represented by the following formula (II) can be given.

  In addition, the nucleotide sequence which comprises this secondary structure respond | corresponds to the 11th-37th nucleotide sequence of Apt1 (sequence number 1) mentioned later.

  Two stem-loop structures can be cited as a feature of the secondary structure. In the biological world, the concept of `` molecular mimicry '' in which RNA mimics the protein's three-dimensional structure has been submitted (Ito K., et al., A tripeptide 'anticodon' deciphers stop codon in messenger RNA, nature, 403 : 608-684, 2000, Nakamura Y., et al., Mimicry grasps reality in translation termination, Cell, 101: 349-352, 2000). This concept indicates the possibility that RNA may produce protein-like functions by creating stable higher-order structures such as stem-loop structures, pseudoknot structures, and intramolecular quadruplex structures (G-quartets). In fact, some RNAs having such a higher-order structure bind to the functional domain of the protein and have physiological activity. The two stem-loop structures of the secondary structure described above are also considered to form such a stable structure and impart physiological activity.

  The RNA (3) is obtained by shortening the RNA (1) to (2) according to the purpose. Either the 5 ′ end or the 3 ′ end may be shortened, or both ends may be shortened. Moreover, what deleted the loop region which does not take base pairing on secondary structure may be used. Although the length to cut | disconnect is not specifically limited, It is preferable that it is 3-20mer, and it is still more preferable that it is 3-10mer. Specific examples of the RNA of (3) include RNA represented by the nucleotide sequence set forth in SEQ ID NO: 15 (Apt1-S1, RNA with 3mer ends of Apt1 shortened by 18 mer).

  In the RNA of (4), the length of RNA to be added is not particularly limited, but is preferably 100 mer or less, and more preferably 30 mer or less. Thus, RNA aptamers suitable for various purposes can be prepared by adding another RNA. As a specific example of the RNA of (4), RNA represented by the nucleotide sequence shown in SEQ ID NO: 16 (Apt1-S2, Apt1 5 ′ end was shortened by 8 mer, GGG was added, and 3 ′ end was 24 mer. After shortening, RNA to which CCC is added can be mentioned.

  The RNA of (5) can be obtained by introducing a mutation into the RNA of (1) to (4) and performing SELEX again. The evolved RNA obtained here may have higher binding activity and physiological activity than the RNAs (1) to (4). By introducing mutations into RNA in this way, RNA aptamers that meet various purposes can be prepared. The number of bases to be substituted, deleted or inserted may be within several, but is preferably 5 or less, and most preferably 1. Specific examples of RNA of (5) include RNA represented by the nucleotide sequence set forth in SEQ ID NO: 24 (RNA in which the 8th A of Apt1-S2 is replaced with U), and the nucleotide sequence set forth in SEQ ID NO: 25 RNA represented (RNA in which 9th C of Apt1-S2 is replaced with A), RNA represented by the nucleotide sequence described in SEQ ID NO: 26 (RNA in which 10th G of Apt1-S2 is replaced with A) RNA represented by the base sequence described in SEQ ID NO: 27 (RNA in which 28th A of Apt1-S2 is replaced with G), RNA represented by the base sequence described in SEQ ID NO: 28 (29 of Apt1-S2) RNA in which the th U is replaced with A), RNA represented by the base sequence set forth in SEQ ID NO: 29 (RNA in which the 30 th C in Apt1-S2 is replaced with G), and the base sequence set forth in SEQ ID NO: 33 RNA represented (RNA in which the 17th C of Apt1-S2 is replaced with A), RNA represented by the base sequence described in SEQ ID NO: 34 (RNA in which the 18th A in Apt1-S2 is replaced with C) Described in SEQ ID NO: 35 RNA represented by the base sequence (RNA in which 19th C of Apt1-S2 is replaced with A), RNA represented by the base sequence described in SEQ ID NO: 36 (20th G of Apt1-S2 is replaced with C) RNA).

  The RNA of (6) is obtained by modifying the ribose part, nucleobase part, 5 ′ end or 3 ′ end of the RNA of (1) to (5), and the stability of RNA of (1) to (5) , Binding activity and physiological activity can be enhanced. For example, fluorination or methylation of the ribose 2′-OH of pyrimidine nucleotides makes it ribonuclease resistant and dramatically improves stability. Modified nucleotides include deoxyribonucleotides.

  It can be used for detection and quantification of a protein having a labeled RUNT domain in (7). For example, by introducing an aptamer labeled with a fluorescent substance into a cell, the behavior of the protein in the cell can be observed. Proteins with RUNT domains include transcription factors AML1, RUNX2, and RUNX3, and AML1-MTG8, AML1-MTG16, TEL-AML1, AML1-EVI1, AML1-EAP, AML1-MDS1, and AML1-MDS1-EVI1 Examples include leukemia fusion proteins.

  By separating the 5 'end of RNA of (1) to (6) with biotin and immobilizing it, a protein separation column having a RUNT domain using an RNA aptamer can be prepared. Proteins with a RUNT domain can be isolated.

  The present invention provides RNA aptamers capable of binding to leukemia fusion proteins having AML1, RUNX2, RUNX3 and other RUNT domains. The RNA aptamer of the present invention can be used for functional analysis of leukemia fusion proteins having AML1, RUNX2, RUNX3 and RUNT domains and for diagnosis and treatment of diseases such as cancer.

  An outline of the SELEX method used in the present invention is shown in FIG.

  Selection of RNA aptamers for leukemia fusion proteins with AML1, RUNX2, RUNX3, and RUNT domains is achieved by highly expressing the AML1 RUNT domain fused with a histidine tag at the N-terminus in E. coli and using it in Ni-NTA agarose (Qiagen) In this manner, the product was subjected to primary purification using a Resource S column (Amersham Biosciences) and secondary purification using a target protein. Hereinafter, the AML1 RUNT domain used in the experiment refers to a fusion protein fused with a histidine tag. This was immobilized on Ni Sepharose HP (Amersham Biosciences), TALON metal affinity resin (Clontech) or Ni-NTA magnetic resin (Qiagen), and selection was performed on an RNA pool with a 30mer or 40mer random sequence. It was. After repeating the selection cycle up to 9 rounds, the cDNA corresponding to the concentrated RNA was cloned into pGEM-T Vector (Promega), and the nucleotide sequence was determined. As a result, 8 types of converging sequences were obtained from the RNA pool having a 30-mer random sequence and 4 types from the RNA pool having a 40-mer random sequence (FIG. 2). These are Apt1 (SEQ ID NO: 1), Apt2 (SEQ ID NO: 2), Apt3 (SEQ ID NO: 3), Apt4 (SEQ ID NO: 4), Apt5 (SEQ ID NO: 5), Apt6 (SEQ ID NO: 6), Apt7 (SEQ ID NO: 7). ), Apt8 (SEQ ID NO: 8), Apt9 (SEQ ID NO: 9), Apt10 (SEQ ID NO: 10), Apt11 (SEQ ID NO: 11), and Apt12 (SEQ ID NO: 12).

The binding activity of the converged sequence was measured by surface plasmon resonance analysis using BIACORE X system (Biacore AB). The RNA obtained by immobilizing poly-A to sensor Chip SA (Biacore AB) immobilized with poly-dT pre-biotinylated and injecting the AML1 RUNT domain has binding activity to the resulting RNA. (FIG. 3A, B, C, D) (The figure shows a graph when the AML1 RUNT domain was injected). All RNAs obtained by selection had binding ability to the AML1 RUNT domain. Among the RNA obtained by selection from RNA pool with a random sequence of 30 mer, the dissociation constant K _d for Apt1 frequency was high was calculated by experiments with BIACORE X system. The calculated K _d of Apt1 was about 4 nM. The K _{d of} double-stranded DNA (hereinafter referred to as AML1-binding dsDNA, SEQ ID NO: 13 and SEQ ID NO: 14) containing a DNA sequence recognized by AML1 was calculated in the same manner. Its value was about 70 nM. Furthermore, it was confirmed that the RNA aptamer obtained by competition experiments between Apt1 and AML1-binding DNA actually has stronger binding activity than AML1-binding dsDNA. AML1-binding dsDNA was immobilized on the sensor chip, and an RNA aptamer or a solution in which AML1-binding dsDNA and AML1 RUNT domain were 1: 1 was injected. As a result of the experiment, the RNA aptamer completely inhibited the binding of AML1 RUNT domain and AML1 binding dsDNA (Fig. 4). (The figure shows the injection of a solution containing RNA aptamer or AML1 binding dsDNA and AML1 RUNT domain at a ratio of 1: 1. (Shows time graph). From the above results, it was shown that RNA aptamers with higher binding ability than the DNA sequence originally recognized by the AML1 RUNT domain were obtained by selection.

  In humans, there is a protein with a RUNT domain that is very homologous to the AML1 RUNT domain. They are classified as RUNX family proteins and are named RUNX2 and RUNX3. We examined whether Apt1 has binding activity against these RUNX family proteins. RUNX2 RUNT domain and RUNX3 RUNT domain were highly expressed in E. coli with N-terminal fused histidine tag and purified in the same manner as AML1 RUNT domain. Apt1 had binding activity equivalent to that of AML1 RUNT domain for RUNX2 RUNT domain and RUNX3 RUNT domain (Figure 5) (Figure shows graph when protein is injected)

Of the obtained RNA aptamers, the secondary structure of Apt1, which had a high frequency of selection, was predicted by Enzyme Probing. CIP (Phosphatase, Alkaline from calf intestine, Roche) was used, and the 5 ′ end of Apt1 was converted to an OH group by incubating at 50 ° C. for 2 hours. The RNA was labeled with ³² P at the 5 ′ end using [γ- ³² P] ATP (Amersham Biosciences) and T4 RNA Ligase (TAKARA BIO INC.). The labeled RNA was incubated in Buffer A for 5 minutes at 85 ° C. and then rapidly cooled on ice. Alkaline degradation was performed by incubating 15,000 cpm ³² P-labeled RNA at 90 ° C. for 10 min under conditions of 50 mM Na carbonate (pH 9.0) and 1 mM EDTA. Digestion of RNase T1 (Ambion) under denaturing conditions was performed by incubating 15,000 cpm ³² P-labeled RNA under conditions of 20 mM Na citrate (pH 5.0), 7 M Urea, 1 mM EDTA at 50 ° C. for 30 min. For digestion under non-denaturing conditions, 15,000 cpm ³² P-labeled RNA was converted to 20 mM Na phosphate (pH 6.5), 2 mM Mg (OAc) ₂ , 50 mM KOAc, enzyme (1 U / μl RNase T1 or 0.002 U / μl RNase V1 ( Ambion), 1 U / μl Mung Bean nuclease (TOYOBO)), 0.5 μg / μl Yeast RNA (Ambion), and incubated at 25 ° C. for 10 min. After the reaction, the sample was ethanol precipitated and then electrophoresed on a 15% polyacrylamide gel (7M Urea, 1 × TBE). The gel after electrophoresis was transferred to an imaging plate (BAS-MS2040, Fujifilm) and analyzed with a fluoro image analyzer (FLA-3000G, Fujifilm). FIG. 6 shows the result of electrophoresis, the cleavage site of RNA, and the secondary structure of Apt1 predicted from the pattern.

From the predicted secondary structure, it was investigated whether it can be miniaturized with the ability to bind to AML1b Runt domian. Apt1-S1 (SEQ ID NO: 15) with two stem structures and two loop structures left intact and the 3 'end shortened by 18mer, and the 5' end shortened by 6mer, the 3 'end shortened by 4mer, and T7 RNA polymerase. Apt1-S2 (SEQ ID NO: 16) was prepared by substituting the base at the 5 ′ end with G for 3 bases so that transcription was possible (FIG. 7). As a result of investigating the binding activity of the miniaturized RNA, both of the two miniaturized RNAs retain the ability to bind to the AML1 RUNT domain, and the calculated binding constant K _d is approximately 10 nM for Apt1-S1. Apt1-S2 was about 5 nM (FIG. 8) (the figure shows a graph when the AML1 RUNT domain was injected).

Whether fluorination of 2′-OH of pyrimidine nucleotides affected the binding activity of Apt1, Apt1-S1, and Apt1-S2 with the AML1 RUNT domain was examined. The binding constants K _d of these modified RNA aptamers were about 7 nM for Apt1, about 16 nM for Apt1-S1, and about 9 nM for Apt1-S2. These results indicate that Apt1, Apt1-S1, and Apt1-S2 have little effect on the binding activity of pyrimidine nucleotide 2'-fluorination with the AML1 RUNT domain and can acquire nuclease resistance while retaining the binding ability. It was done.

  In order to examine the necessary elements of the RNA aptamer when binding to the AML1 RUNT domain, mutants were prepared in which the bases of the loop region of Apt-S2 were replaced with other bases one by one (SEQ ID NOs: 24-40). The binding activity to these mutant RNAs and AML1 RUNT domain was examined. When the 14th to 16th and 23rd bases were replaced with other bases, the binding activity to the AML1 RUNT domain was completely lost. Substitution of the 20th, 21st, and 24th bases resulted in a significant decrease in binding activity. Substitution of other bases had little effect on the binding activity against the AML1 RUNT domain (Table 1). Furthermore, the binding activity of the mutants to the RUNX2 RUNT domain and RUNX3 RUNT domain was examined. The result was exactly the same as the trend of AML1 RUNT domain. This can be inferred that RNA aptamers bind to the three types of RUNT domains in a similar manner.

  From the results of RNA aptamer point mutation experiments, the elements that the RNA aptamer binds to the RUNT domain were determined. In the following secondary structure, N1 is 3 or more nucleobases, preferably 3 to 7 nucleobases. N1 can take all nucleobases. N2 is 6 or more nucleobases, preferably 6 to 10 nucleobases. N2 can take all nucleobases. N3 is one or more nucleobases, preferably 1 to 3 nucleobases. N3 can take all nucleobases. N4 is 3 or more nucleobases, preferably 3 to 7 nucleobases. N4 can take all nucleobases.

  The obtained RNA aptamer was immobilized on a resin and examined to function as an AML1 separation column. Mix well E. coli lysate with high expression of AML1 RUNT domain and Streptavidin Sepharose High Performance (Amersham Biosciences) with 5'-end biotinylated Apt1-S2 (synthesized by Dharmacon), and gently mix at 4 ° C for 30 minutes Was stirred. Apt1-S2 used here has 2'-fluorinated part of the nucleotide as follows.

5'-Biotin-GGGA U GGA C GA CCC A CC A C GG C GAGG U A UCCC A UCCC -3 '
(Underline represents 2′-fluorinated nucleotides.)

  The resin was washed thoroughly with Buffer A, and 4M Urea was added to elute the adsorbed protein. The collected elution fractions were electrophoresed using NuPAGE 4-12% Bis-Tris Gel (using 1 × NuPAGE MES SDS Running Buffer, Invitrogen). After electrophoresis, it was stained with SYPRO Orange (Molecular Probes), and the protein bands were analyzed with a fluoro image analyzer (FLA-3000G, Fuji film). As a result, it was confirmed that the AML1 RUNT domain was bound only to the resin on which the RNA aptamer was immobilized (FIG. 9). This suggests that the resin on which the RNA aptamer is immobilized can function as an affinity column for proteins having a RUNT domain such as AML1.

  An experiment similar to the case of the above-mentioned Escherichia coli lysate was also performed on a cultured cell lysate expressing AML1 or AML1-MTG8 fusion protein. Using pDEST26-AML1 {inserted the entire coding region sequence of AML1b} and pDEST26-AML1-MTG8 (inserted the entire coding region sequence of AML1-MTG8) as an expression vector, AML1b and AML1 in Hela cells -MTG8 protein was expressed. Lysates were obtained from these cells. Western blotting using specific peptide antibodies was used to examine whether AML1 or AML1-MTG8 fusion protein could be separated from the lysate by RNA aptamer. As a result, both AML1 and AML1-MTG8 fusion proteins were confirmed to have specific bands in the resin on which the RNA aptamer was immobilized (FIG. 10). This suggested that the resin immobilized with RNA aptamer can function as a separation column even in lysates of human cells. Furthermore, this experiment proved that the obtained RNA aptamer also has binding activity to the full-length AML1 and AML1-MTG8 fusion proteins.

  It was verified whether it was possible to distinguish between AML1 and AML1-MTG8 fusion proteins. As a method for identifying AML1 and AML1-MTG8 fusion proteins or cells containing them, surface plasmon resonance analysis using BIACORE X system was used. The experiment was performed under the same conditions as the measurement of the RNA aptamer binding activity by BIACORE. First, 20 μl of pyrimidine nucleotides were 2′-fluorinated and Apt1 (4 ng / μl) added with 3′-polyA was injected to immobilize RNA on the sensor chip. Three minutes later, samples (100 nM AML1 RUNT domain, 100 nM AML1-MTG8-like fusion protein (a protein containing RUNT domain and TAF domain around the fusion site of AML1-MTG8) or 40 ng / μl cultured cell lysate) were injected. The protein was immobilized with an RNA aptamer on the sensor chip. Three minutes later, 10 μg / μl of an antibody against the TAF domain of MTG8 was injected. When the AML1 RUNT domain was immobilized on the sensor chip, no increase in response was observed even when the antibody was injected. However, in the case of the AML1-MTG8-like fusion protein, an increase in response was observed (FIG. 11A). Furthermore, in the case of lysates of cultured cells, only in the case of lysates that highly expressed the AML1-MTG8 fusion protein, the increase in response was clearly higher than in other lysates (FIG. 11B). Thus, it was shown that AML1 and AML1-MTG8 fusion protein can be easily distinguished by using an RNA aptamer and an antibody against the TAF domain of MTG8.

  Hereinafter, the method of the experiment of the present invention will be specifically described with reference to examples. In addition, these Examples illustrate the present invention and do not limit the scope of the present invention.

[Example 1] Protein purification Human and AML1 RUNT domain, RUNX2 RUNT domain, RUNX3 RUNT domain, AML1-MTG8-like fusion protein used in the present invention (a protein comprising a RUNT domain and a TAF domain around the fusion site of AML1-MTG8) Is overexpressed in Escherichia coli as a fusion protein of AML1 RUNT domain and MTG8 TAF domain with histidine tag at the N-terminus, roughly purified using Ni-NTA agarose (Qiagen), and further cation exchange column Resource S (Amersham Biosciences).

[Example 2] Acquisition of RNA aptamer
In vitro RNA selection using the SELEX method was performed by Ellington et al. (Ellington AD and Szostak JW, In vitro selection of RNA molecules that bind specific ligands, Nature, 346: 818-822, 1990) and Tuerk et al. (Tuerk C and Gold L., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science, 249: 505-510, 1990). The RNA pool used for in vitro selection was prepared from the following chemically synthesized primers and a random sequence template (composed by SIGMA GENOSYS). A double-stranded DNA was prepared by Klenow Fragment (Large Fragment E. coli DNA polymerase I, TAKARA BIO INC.), And this was used as a template for transcription. By the promoter of T7 RNA polymerase contained in primer P1, AmpliScribe T7-Flash Transcription Kit (EPICENTRE Biotechnologies) was transferred and purified by phenol extraction and gel filtration.

P1: 5'-TAATACGACTCACTATAGGGACACAATGGACG-3 '(SEQ ID NO: 17)
30N random sequence template: 5'-CTCTCATGTCGGCCGTTA (30N) CGTCCATTGTGTCCCTATAGTGATCGTATTA-3 '(SEQ ID NO: 18)
40N random sequence template 5'-CTCTCATGTCGGCCGTTA (40N) CGTCCATTGTGTCCCTATAGTGATCGTATTA-3 '(SEQ ID NO: 19)
Binding of RNA and protein was increased with buffer A [20 mM sodium phosphate (pH 6.5), 2 mM magnesium acetate, 50-200 mM potassium acetate (increased stepwise as the selection cycle progressed (Table 2)), 5% (v / v) Glycerin, 0.05% (v / v) Triton-X100, 5 mM 2-mercaptoethanol]. After reaction with RNA, pull-down with histidine tag affinity resin (Ni Sepharose HP (Amersham Biosciences) or TALON metal affinity resin (Clontech), Ni-NTA magnetic resin (Qiagen), Table 2) After purification and reverse transcription using primer P2, cDNA was amplified by PCR.

  This cDNA was used for in vitro transcription with AmpliScribe T7-Flash Transcription Kit (EPICENTRE Biotechnologies) to obtain the next round of RNA.

P2: 5'-CTCTCATGTCGGCCGTTA-3 '(SEQ ID NO: 20)
After 9 rounds of selection, the PCR product was subcloned into pGEM-T vector (Promega) and then transformed into E. coli strain XL1 Blue (STRATAGENE) to obtain a single clone. After extracting the plasmid, the DNA sequencer (model 3100, ABI) was used to determine the base sequence using the following sequence primer.

sequence primer: 5'-GTAAAACGACGGCCAGT (SEQ ID NO: 21)

[Example 3] Evaluation of binding activity The binding activity of RNA obtained after 9 rounds to the AML1 RUNT domain was examined by surface plasmon resonance analysis using a BIACORE X system (Biacore AB). For measurement, a sensor chip SA (Biacore AB) flow cell 1 to which about 100 RU of biotinylated primer P3 was added was used.

P3: 5'-biotin-TTTTTTTTTTTTTTTT-3 '(SEQ ID NO: 22)
In order to immobilize RNA on the sensor chip, RNA with 16 A added to the 3 ′ end was prepared in advance. CDNA was amplified by PCR using as a template a vector in which the RNA aptamer sequence obtained at the time of sequencing was incorporated. At this time, P1 (SEQ ID NO: 17) was used as the 5′-side primer, and P4 was used as the 3′-side primer.

P4: 5'-TTTTTTTTTTTTTTTTCTCTCATGTCGGCCGTTA (SEQ ID NO: 23)
Using the cDNA amplified by this PCR as a template for transcription, RNA having 16 A added to the 3 ′ end was obtained in the same manner as in [Example 2].

  The binding activity to RNA obtained by Selection was as follows: flow rate 20 μl / min, buffer condition (20 mM Na phosphate (pH 6.5), 2 mM Mg (OAc) 2, 200 mM KOAc, 0.1% (v / v) TWEEN 20 , 1 mM DTT). First, 20 μl of RNA added with poly-A at the 3 ′ end (4 ng / μl) was injected in Normal Injection mode. Four minutes later, 500 nM AML1 RUNT was injected in 180 seconds of Delayed Injection mode.

The dissociation constant _Kd was obtained by adding about 10 RU of biotinylated primer P3 to flow cell 1 of sensor chip SA (Biacore AB). The measurement was performed under a flow rate of 50 μl / min and buffer conditions (20 mM Na phosphate (pH 6.5), 2 mM Mg (OAc) ₂ , 300 mM KOAc, 0.1% (v / v) TWEEN 20, 1 mM DTT). 20 μl of RNA added with poly-A at the 3 ′ end (4 ng / μl) was injected in Normal Injection mode. Four minutes later, 1-100 nM AML1 RUNT domain was injected in 180 seconds Delayed Injection mode. _Kd was calculated using data analysis software BIA evaluation version 4.1 (Biacore AB). The K _{d of} double-stranded DNA (AML1-binding dsDNA) originally containing a DNA sequence recognized by the AML1 RUNT domain was also calculated in the same manner.

[Example 4] Competition experiment
In order to compare the strength of binding of Apt1 to the AML1 RUNT domain with AML1-binding DNA, competition experiments were performed using the BIACORE X system. For measurement, a sensor chip SA (Biacore AB) flow cell 1 to which about 100 RU of biotinylated primer P3 was added was used. The measurement was performed at a flow rate of 20 μl / min and buffer conditions (20 mM Na phosphate (pH 6.5), 2 mM Mg (OAc) ₂ , 200 mM KOAc, 0.1% (v / v) TWEEN 20, 1 mM DTT). First, AML1-binding dsDNA was immobilized on a sensor chip, and a solution prepared by making 50 nM Apt1 or poly-A-free AML1-binding dsDNA, 30N random RNA 1: 1 with the AML1 RUNT domain was injected.

[Example 5] Binding activity of RNA aptamer to RUNX family proteins In humans, there is a protein having a RUNT domain that is highly homologous to AML1 RUNT domain binding. They are classified as RUNX family proteins and are named RUNX2 and RUNX3. It was investigated whether Apt1 has binding ability to these RUNX family proteins. RUNX2 RUNT domain and RUNX3 RUNT domain were highly expressed in E. coli with N-terminal fused histidine tag and purified in the same manner as AML1 RUNT domain. The binding activity between these proteins and the RNA aptamer was examined in the same manner as when the binding activity with the AML1 RUNT domain was examined.

[Example 6] Prediction of secondary structure of RNA aptamer Among the obtained RNA aptamers, the secondary structure of Apt1 with a high frequency of selection was predicted by Enzyme Probing. Enzyme probing is a method for predicting secondary structure from the pattern of RNA cleaved with a ribonuclease (RNase) having specific cleavage activity. CIP (Phosphatase, Alkaline from calf intestine, Roche) was used, and the 5 ′ end of Apt1 was converted to an OH group by incubating at 50 ° C. for 2 hours. The RNA was labeled with ³² P at the 5 ′ end using [γ- ³² P] ATP (Amersham Biosciences) and T4 RNA Ligase (TAKARA BIO INC.). The labeled RNA was incubated in Buffer A for 5 minutes at 85 ° C. and then rapidly cooled on ice. Alkaline degradation was performed by incubating 15,000 cpm ³² P-labeled RNA at 90 ° C. for 10 min under conditions of 50 mM Na carbonate (pH 9.0) and 1 mM EDTA. Digestion of RNase T1 (Ambion) under denaturing conditions was performed by incubating 15,000 cpm ³² P-labeled RNA under conditions of 20 mM Na citrate (pH 5.0), 7 M Urea, 1 mM EDTA at 50 ° C. for 30 min. For digestion under native conditions (Enzyme Probing), 15,000 cpm ³² P-labeled RNA was converted to 20 mM Na phosphate (pH 6.5), 2 mM Mg (OAc) ₂ , 50 mM KOAc, enzyme (1 U / μl RNase T1 or 0.002 U / μl RNase V1 (Ambion), 1 U / μl Mung Bean nuclease (TOYOBO)), 0.5 μg / μl Yeast RNA (Ambion) was incubated at 25 ° C. for 10 min. After the reaction, the sample was ethanol precipitated and then electrophoresed on a 15% polyacrylamide gel (7M Urea, 1 × TBE). The gel after electrophoresis was transferred to an imaging plate (BAS-MS2040, Fujifilm) and analyzed with a fluoro image analyzer (FLA-3000G, Fujifilm).

[Example 7] Miniaturization of RNA aptamer It was investigated from the predicted secondary structure whether it could be miniaturized while having binding activity with the AML1 RUNT domain. Apt1-S1 (SEQ ID NO: 14) with two stem structures and two loop structures left intact, and the 3 'end shortened by 18mer, and then the 5' end shortened by 6mer and the 3 'end shortened by 4mer, and T7 RNA polymerase Apt1-S2 (SEQ ID NO: 15) was prepared by substituting the base at the 5 ′ end with G for 3 bases so that transcription was possible (FIG. 7). DNA having an Apt-S1 or Apt-S2 sequence with a T7 promoter on the 5 'side was synthesized (requested by SIGMA GENOSYS). 5'-side primer is P5 (SEQ ID NO: 41), 3'-side primer is Apt-S1 is P6 (SEQ ID NO: 42), and Apt-S2 is P7 (SEQ ID NO: 43). When transcription was performed in the same manner as Apt1, RNA was obtained with polyA added to the 3 ′ side. The binding activity of these miniaturized RNAs was tested by the method described above using the BIACORE X system.

[Example 8] Determination of necessary elements of RNA aptamer when binding to AML1 RUNT domain
In order to examine the necessary elements of the RNA aptamer when binding to the AML1 RUNT domain, mutants were prepared in which the bases of the loop region of Apt-S2 were replaced with other bases one by one (SEQ ID NOs: 24 to 40). The numbering of bases in the sequence was performed in ascending order on the 3 ′ side, with the base at the 5 ′ end being 1. A DNA having a mutant sequence in which a T7 promoter was arranged on the 5 ′ side was synthesized (synthetic request from SIGMA GENOSYS). P5 (SEQ ID NO: 41) for all 5′-side primers, P7 (SEQ ID NO: 43) for SEQ ID NOS: 24-34, P8 (SEQ ID NO: 44) for SEQ ID NO: 35, SEQ ID NO: 36 is P9 (SEQ ID NO: 45), SEQ ID NO: 37 is P10 (SEQ ID NO: 46), SEQ ID NO: 38 is P11 (SEQ ID NO: 47), SEQ ID NO: 39 is P12 (SEQ ID NO: 48), SEQ ID NO: For 40, P13 (SEQ ID NO: 49) was used to enhance the synthetic DNA by PCR, and when transcription was performed in the same manner as Apt1, RNA was obtained with polyA added to the 3 ′ side. The binding activity to these mutant RNAs and AML1 RUNT domain was examined. The experiment was performed using the BIACORE X system under the conditions described above.

[Example 9] Examination of influence on binding ability of RNA aptamer by fluorination of pyrimidine nucleotide 2'-OH
Fluorination of the ribose 2-OH of the pyrimidine nucleotides of RNA makes it ribonuclease resistant and dramatically improves stability. RNA prepared by 2'-fluorination of Apt1 and its miniaturized Apt1-S1 and Apt1-S2 pyrimidine nucleotides was prepared and examined whether there was a change in the binding ability before modification. RNA obtained by 2′-fluorination of pyrimidine nucleotides was obtained using DuraScribe T7 Transcription Kit (EPICENTRE Biotechnologhies). The binding activity was measured in the same manner as in [Example 3].

[Example 10] Pull-down experiment of Escherichia coli lysate The obtained RNA aptamer was immobilized on a resin and examined to function as an AML1 separation column. Escherichia coli with high expression of AML1 RUNT domain was transformed into Buffer A (20 mM Na phosphate (pH 6.5), 2 mM Mg (OAc) ₂ , 200 mM KOAc, 0.1% (v / v) TWEEN 20, 1 mM DTT, Protease inhibitor (Protease Inhibitor Cocktails for General, SIGMA) and suspended by sonication, centrifuged at 15,000 rpm for 30 minutes, and the supernatant was recovered to obtain an E. coli lysate, 100 pmol of synthetic 5'-biotin-S2 (Requested synthesis from DHARMACON) and 20 μl Streptavidin Sepharose High Performance (Amersham Biosciences) were mixed well and left for 10 minutes at 4 ° C. After thoroughly washing the resin with Buffer A, 10 μg of E. coli lysate and Yeast RNA (Ambion) ) 20 μg of Buffer A was added to a total volume of 200 μl, which was gently stirred for 30 minutes at 4 ° C. The resin was washed thoroughly with Buffer A, and then 4 M Urea was added to elute the adsorbed protein. Concentrate the solution with an ultrafiltration tube (Microcon-10, MILLIPORE) and use NuPAGE 4-12% Bis-Tris Gel (1 x NuPAGE MES SDS Running Buffe). After electrophoresis, staining was performed with SYPRO Orange (Molecular Probes), and protein bands were analyzed with a fluoro image analyzer (FLA-3000G, Fujifilm).

[Example 11] Experiments for producing HeLa cells that transiently express AML1 or AML1-MTG8 fusion protein
HeLa cells were cultured in a medium containing RPMI1640 medium (Nissui Pharmaceutical) with 10% fetal calf serum (HyClone) and penicillin streptomycin (GIBCO) at 37 ° C. in the presence of 5% carbon dioxide gas. The day before the expression vector was introduced, 2 × 10 ⁶ HeLa cells were seeded in a 10 cm petri dish. As an expression vector, pDEST26-AML1 {pDEST26 (an expression vector for mammalian cells expressing a protein having a histidine tag at the N-terminus, Invitrogen) with the sequence of the entire coding region of AML1b} and pDEST26-AML1-MTG8 ( pDEST26 with the entire coding region sequence of AML1-MTG8) was used. PDEST26 (nothing inserted) was used as a negative target. 30 ml of the cationic lipofection reagent DMRIE-C (Invitrogen) was mixed with 5 ml of OPTI-MEM medium (Invitrogen) for 20 mg of each expression vector, and left at room temperature for 45 minutes to form liposomes. The expression vector / DMRIE-C was added to the HeLa cells washed once with OPTI-MEM medium immediately before, and treated for 4 hours at 37 ° C. in the presence of 5% carbon dioxide gas. 5 ml of RPMI1640 medium containing 20% fetal bovine serum was added, and further cultured at 37 ° C. in the presence of 5% carbon dioxide gas. After 24 hours, the cells were washed once with PBS, 1 ml of PBS was added, the cells were scraped with a rubber policeman, and transferred to a 1.5 ml centrifuge tube. Cells were collected by centrifugation (800 xg, 1 min) and the supernatant removed.

[Example 12] Pull-down experiment of cultured human cell lysate
Hela cells (human uterine cancer-derived cell line) expressing AML1 or AML1-MTG8 fusion protein were suspended in Buffer A and disrupted with ultrasound. After centrifugation at 15,000 rpm for 30 minutes, the supernatant was recovered and used as a cultured cell lysate. The cultured cell lysate was used in an amount of 20 μg, and other operations were performed in the same manner as in the case of E. coli lysate, and electrophoresis was performed using NuPAGE 4-12% Bis-Tris Gel (using 1 × NuPAGE MOPS SDS Running Buffer, Invitrogen). After electrophoresis, the protein was transferred to Sequi-Blot PVDF Membrane (BIO-RAD). After the transfer, the membrane was placed in TBS-T (20 mM Tris, 500 mM NaCl, 0.1% (v / v) TWEEN 20, pH 7.5) containing 5% (w / v) skim milk, and shaken for 1 hour. The operation of washing the membrane by shaking with TBS-T for 10 minutes was performed three times. Next, put the membrane in Anti-human AML1 / RHD Domain (Ab-2) (CALBIOCHEM) prepared to 2.5 μg / ml with 0.5% casein-containing TBS-T and infiltrate for 1 hour, then with TBS-T for 10 minutes The washing and washing operation was performed 3 times. Furthermore, anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (Amersham Biosciences) diluted 1000 times with TBS-T, infiltrate for 1 hour, shake for 10 minutes with TBS-T, and wash for 3 minutes. I went twice. For detection, ECL plus Western Blotting Detection System (Amersham Biosciences) was used and analyzed with a lumino image analyzer (LAS-1000, Fujifilm).

[Example 13] Identification of AML1 and AML1-MTG8 fusion proteins using RNA aptamers
It was verified whether it was possible to distinguish between AML1 and AML1-MTG8 fusion proteins. The experiment was performed using the BIACORE X system under the same conditions as the measurement of the binding activity of RNA aptamer ([Example 3]). First, 20 μl of pyrimidine nucleotide was 2'-fluorinated and Apt1 (4 ng / μl) with polyA added to the 3 'end was injected in Normal Injection mode to bind polydT and polyA on the chip, and RNA on the sensor chip. Immobilized. After 3 minutes, in Normal Injection mode, sample (100 nM AML1 RUNT domain, 100 nM AML1-MTG8-like fusion protein or 40 ng / μl cultured cell lysate (AML1 or AML1-MTG8 expressed or nothing expressed) )) Was injected, and the protein was immobilized with an RNA aptamer on the sensor chip. Three minutes later, 10 μg / μl of anti-P3X (an antibody recognizing the TAF domain of MTG8) was injected in the Delayed Injection mode for 180 seconds.

The figure which shows the outline of the SELEX method. A DNA containing a T7 RNA polymerase promoter sequence and a random region is synthesized, and PCR is performed using P1 and P2 as primers to obtain a template. The synthesized RNA is associated with the target substance, and the bound RNA is recovered. A template for the next round is obtained from the recovered RNA by RT-PCR. In the present invention, this was repeated 9 rounds. The RT-PCR product in the final round is used for cloning and sequencing. The figure which shows the arrangement | sequence of RNA selected by SELEX method. When the nucleotide sequence of the selected RNA pool of the ninth round was determined, RNA derived from the 30N random pool converged to 8 types of sequences, and RNA derived from the 40N random pool converged to 4 types of sequences. It was. The frequency of each selection is shown in the figure. These RNAs were designated as Apt1, Apt2, Apt3, Apt4, Apt5, Apt6, Apt7, Apt8, Apt9, Apt10, Apt11, and Apt12 from the top of the sequence in the figure. The figure which shows the result of the binding experiment using the BIACORE X system. The increase in RU when AML1 RUNT domain was injected was clearly greater for all RNAs with converged sequences than when 30N or 40N random RNA was immobilized on the sensor chip. Therefore, all of Apt1 to 12 were shown to have binding activity against the AML1 RUNT domain. The figure shows a graph when the AML1 RUNT domain is injected. (A) shows the experimental results of Apt1 and pyrimidine 2′-F-modified Apt1, (B) shows the experimental results of Apt2,4,5,7, (C) shows the experimental results of Apt3,6,8, (D ) Shows the experimental results of Apt9-12. The figure which shows the result of the binding competition experiment using the BIACORE X system. To the sensor chip on which AML1-binding dsDNA was immobilized, a solution prepared by making 50 nM Apt1 or poly-A-free AML1-binding dsDNA, 30N random RNA 1: 1 with the AML1 RUNT domain was injected. In the case of AML1-binding dsDNA without 30N random RNA or poly-A, an increase in RU was observed, but in the case of Apt1, no increase in RU was observed. The figure shows a graph when the solution is injected. The figure which shows the binding experiment result with respect to the RUNX2,3 RUNT domain of Apt1 using the BIACORE X system. Apt1 was immobilized on the sensor chip, and 200 nM AML1 or RUNX2 or RUNX3 RUNT domain protein was injected. The figure shows a graph when the protein is injected. The figure which showed the cleavage pattern by RNase in Enzyme Probing experiment to the secondary structure of Apt1. The RNase cleavage site used is indicated by a symbol in the figure. The figure which shows the secondary structure of Apt1, Apt1-S1, Apt1-S2. The right shows Apt1, the middle shows Apt1-S1, and the left shows Apt1-S2. The figure which shows the binding experiment result with respect to AML1 RUNT domain of Apt1-S1, pyrimidine 2'-F-ized Apt1-S1, Apt1-S2, pyrimidine 2'-F-ized Apt1-S2 using BIACORE X system. RNA was immobilized on the sensor chip, and 200 nM AML1 RUNT domain was injected. The figure shows a graph when the protein is injected. The figure which shows the electrophoresis result of Escherichia coli lysate Pull down experiment. The AML1 RUNT domain shows the lane in which the protein purified with Ni-NTA agarose was run. E. coli lysate indicates a lane in which the lysate was allowed to flow. Apt1-S2 indicates a sample using a resin in which 5′-terminal biotinylated Apt1-S2 is immobilized on Streptavidin Sepharose High Performance, and negative control indicates a sample using a resin that is not immobilized at all. “Supernatant” indicates the lane in which the resin did not bind, and “Elution” indicates the lane in which the bound (4M Urea elution fraction) was passed. The band of the AML1 RUNT domain is indicated by an arrow. The figure which shows the result of the western blotting of the lysate Pull down experiment of a cultured cell. The AML1 RUNT domain shows the lane in which the protein purified with Ni-NTA agarose was run. Hela, AML1, and AML1-MTG8 cell lysates indicate lanes in which the lysate was allowed to flow. “+” Indicates a sample using a resin in which 5′-terminal biotinylated Apt1-S2 is immobilized on Streptavidin Sepharose High Performance, and “−” indicates a sample using a resin that is not immobilized at all. The AML1 and AML1-MTG8 bands are indicated by arrows. The figure which shows the result of the discrimination experiment of the AML1-MTG8 fusion protein by the Apt1 and MTG8 specific antibody using the BIACORE X system. The figure shows a graph when the antibody was injected. (A) shows the result of analyzing the recombinant protein, and (B) shows the result of analyzing cultured cells.

Claims (12)

  RNA represented by the base sequence described in any one of SEQ ID NOs: 1-12. An RNA comprising a nucleotide sequence capable of forming a secondary structure represented by the following formula (I) and having a binding activity to a RUNT domain.
(In the secondary structure, N1 is 3 or more nucleobases, N2 is 6 or more nucleobases, N3 is 1 or more nucleobases, N4 is 3 or more nucleobases, A is adenine, and G is cytosine. , G is guanine, U is uracil, and a circle connecting nucleobases represents a hydrogen bond between nucleobases.)   3. RNA shortened by cleaving a part of the RNA according to claim 1 or 2, wherein the RNA has binding activity to the RUNT domain.   An RNA obtained by adding another RNA to the RNA according to claim 1, wherein the RNA has a binding activity to the RUNT domain.   An RNA represented by a base sequence in which one or several bases are substituted, deleted or inserted into the base sequence of the RNA according to any one of claims 1 to 4, wherein the RNA has a binding activity to a RUNT domain Having RNA.   6. RNA having at least one modified nucleotide introduced in the RNA according to claim 1, wherein the RNA has binding activity to the RUNT domain.   The RNA according to any one of claims 1 to 6, which is labeled.   DNA which has a base sequence complementary to the base sequence of RNA in any one of Claims 1-5.   A vector into which the DNA according to claim 8 is inserted.   A method for detecting a protein having a RUNT domain using the RNA according to claim 7.   A method for quantifying a protein having a RUNT domain, using the RNA according to claim 7.   A method for isolating a protein having a RUNT domain using the RNA according to claim 1.