KR20030080675A - Rna specifically binding to nfatc - Google Patents

Abstract

PURPOSE: RNA specifically binding to NFATc is provided, thereby inhibiting the activity of NFATc, so that the RNA can be useful as a immunosuppressive drug without toxicity to human. CONSTITUTION: RNA specifically binding to NFATc from NFATs(nuclear factor of activated T cells) which induces transcription of genes relating to immune reaction contains the nucleotide sequence specifically binding to the DNA binding region of NFATc in order to inhibit the DNA binding ability of NFATc, wherein the nucleotide sequence specifically binding to the DNA binding region of NFATc is selected from the nucleotide sequences represented by SEQ ID NO: 1 to 6.

Description

RNA Specific Binding to NFATc

(Technology)

The present invention relates to RNA that specifically inhibits the activity of NFAT (Nuclear factor of activated Tcells) acting on immune response regulation, and more specifically to other members by specifically binding to NFATc among the members of NFAT By specifically inhibiting only the DNA-binding activity of NFATc without affecting, it is to provide RNA that can be used as an immunosuppressant without toxicity.

(Background)

NFAT (Nuclear factor of activated T cells), a transcription factor composed of various components expressed in immune cells, plays a pivotal role in the expression of various cytokines or cell surface ligands, which are important for the regulation of immune responses, and are present in immune cells. It is known that activation is caused by stimulation of receptors involved in calcium changes, such as antigen receptors (see references 1 and 2 below).

The receptor stimulation and calcium changes promote the migration of NFAT into the nucleus by producing an activated phosphatase calcineurin, which removes phosphate groups of NFAT proteins (see Refs. 3-5).

NFAT consists of five members, four of which (NFAT1 / p, NFAT2 / c, NFAT3, NFAT4 / x) are regulated by calcineurin, and their function in each knockout mouse model It has already been analyzed (see Ref. 6).

Cyclosporin A and FK506, the immunosuppressive agents currently used to prevent tissue rejection during transplantation, inhibit phosphatase activity of calcineurin, preventing the phosphorylation of NFAT protein and migration into the nucleus. Notes are made by mechanism (see Ref. 7).

However, since these drugs have side effects that cause kidney, nerve, and gastrointestinal toxicity, diabetes and a high incidence of cancer, their use is very limited given other medical conditions (see references 8 to 10). Toxicity can lead to various physiological side effects by inactivating calcineurin in various organs or cell types (see Refs. 1 and 6). Therefore, finding a component targeting only NFAT directly without affecting the phosphatase activity of calcineurin would be one solution to the development of non-toxic immunosuppressive therapies.

Recently, drugs have been isolated that inhibit NFAT activity by only inhibiting calcineurin-NFAT binding without reducing phosphatase activity of calcineurin (see references 11 and 12). Also known are small molecules that interfere with NFAT activity by mechanisms other than CsA (cyclosporin A) or FK506 (see references 13 and 14).

Despite the potential immune response to other pathways, targeting each NFAT protein may be a more selective solution to control immunity (see Ref. 6).

Isolation of small drugs such as short peptides with strong binding to the protein of interest is not easy due to their broad specificity and unstable folding structure (see Ref. 15). However, short RNA molecules are capable of forming a fairly stable and unique tertiary structure by intramolecular base bonds (see Refs. 15 and 16); Systemic evolution of ligands by exponential enrichment (SELEX) technology allows short RNA detection molecules with high binding and specificity to RNA-binding proteins, etc., from random RNA libraries (see references 17 and 18); RNA detection of autoantibodies selected in vitro can interfere with the attachment of antigens to their antibodies and thus can be used as a potent drug against autoimmune diseases (see references 19-21); And in other groups, it has been reported that certain RNA molecules selected in vitro using animal models interfere with physiological functions known to be associated with the target protein (see Ref. 22); In view of the above, RNA molecules involved in NFAT may be strong candidates for specific and efficient immunosuppressive agents.

An object of the present invention is to provide a novel RNA that specifically inhibits only the DNA binding activity of NFATc without affecting other members of the NFAT, so that it can be applied as a non-toxic immunosuppressive therapy It is.

1 is a base sequence for the change site of RNA according to the present invention

Figure 2 is a secondary structure predicted from the base sequence of the RNA according to the present invention,

Figure 3 shows specific binding of NFATc DBD of RNA according to the present invention,

4 is a view showing the binding force between the RNA and the NFATc DBD of the present invention,

Figure 5 shows that the RNA of the present invention inhibits the DNA binding activity of NFATc without affecting NFATp,

Figure 6 shows that the RNA of the present invention specifically inhibits NFATc activity against human cells.

According to the present invention, a base sequence that specifically binds to the DNA binding domain (DNA binding domain, hereinafter referred to as DBD) of NFATc among the components of NFAT that serves to induce transcription of genes involved in immune response Thus, RNA that specifically inhibits only the binding ability of the DNA of the NFATc is provided.

Preferably the RNA according to the present invention comprises one nucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 6.

RNA according to the present invention, in connection with the development of selective immunosuppressants, uses RNA combinatorial libraries to detect RNA probes that specifically bind to the DNA binding site of NFATc involved in Th2 type of immune response (see Ref. 23). It comes from a new discovery.

RNA according to the present invention binds to NFATc with high specificity and avidity, and specifically inhibits only DNA binding ability of NFATc without affecting NFATp. Moreover, the RNA of the present invention may act as a decay in the cell and efficiently and selectively inhibit the expression of a detection gene induced only by NFATc and not by NFkB or NFATp.

Example

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

First, a description will be given of the screening and analysis process applied in connection with the present invention.

1. Recombinant Protein

Recombinant fragments of human NFATc DBD (amino acids 392-699) and human NFATp DBD (amino acids 390-694), that is, the N-terminal 2/3 (amino acids 392-583) portion of NFATc DBD and the C-terminal 1 / of NFATp DBD The chimeric NFAT protein, made by combining 3 (amino acids 581-694), was cloned into the pET21 expression vector and expressed as a recombinant protein with six histidines attached to the C-terminus.

Proteins are expressed in E. coli BL21DE3 in large quantities and isolated with nickel chelate resin (Ni-NTA agarose, Qiagen) as described (see Ref. 24). NFATc DBDs present between amino acids 392 and 699 have been reported to be highly conserved between NFAT families (see Ref. 1).

2. Screening

Screening is performed by known methods (see Refs. 18-20).

A random RNA oligomer population consisting of SEQ ID NO: 5′-GGGAGAGCGGAAGCGUGCUGGGCCN ₄₁ CAUAACCCAGAGGUCGAUGGAUCCCCCC-3 ′ is obtained by in vitro transcription from a DNA template synthesized using NTPs and T7 RNA polymerase. Where N ₄₁ is 41 nucleotides having the same insertion of A, G, C or U at each position.

To remove RNA that specifically binds to agarose beads, 10 ml of RNA library and 20 ul of Ni-NTA agarose beads are combined with 100 ml of binding buffer solution (30 mM Tris-HCl, pH 7.5, 150 mM NaCl). , 1.5mM MgCl 2, 2mM dithiothreitol (1% BSA), put into the precipitate after the shaking reaction for 30 minutes in advance at room temperature for each cycle to discard the precipitate.

The supernatant from which the sediment is removed is transferred to a new tube and allowed to react with NFATc DBD with 2 mg of histidine for 30 minutes at room temperature. The RNA-NFATc conjugate is precipitated using beads, and the precipitate is washed three times with 0.5 ml of binding buffer solution. RNAs are obtained again and amplified by RT-PCR, in vitro transcribed and the above procedure repeated.

From the eighth cycle of the screening, the NFATc DBD concentration was reduced by five times and the RNA-NFATc precipitate was washed five times. After the tenth cycle, the amplified DNA is cloned and sequenced from the clones.

3. Analysis of Selected RNA

The interior of the selected RNA of the present invention is labeled with an isotope and separated as directed (see references 18-20). The isolated RNA is reacted with the protein as usual. RNA-NFAT conjugates precipitate with Ni-NTA agarose beads and extract RNA from the precipitate. The extracted RNA is analyzed by 6% urea polyacrylamide gel. RNA is also reacted with NFATc DBD in 40 ml of binding buffer containing 1 mg of tRNA. RNA-NFAT conjugates were subjected to gel transfer analysis on 6% nondenaturing polyacrylamide gel with 2% glycerol.

4. Analysis of migration change by electrophoresis of NFAT-RNA (EMSA)

The oligomer probe corresponding to the NFAT binding site terminus derived from the IL-2 promoter of the mouse is 33mer, and its base sequence is 5'-GATCGCCCAAAGAGGAAAATTTGTTTCATACAG-3 '(see Reference 25). Two strands of oligomers are obtained by combining one strand of oligomer with its complement and labeled with [-32P] dATP (3000 Ci / mmol, Amersham) using T4 DNA kinase. The binding reaction for EMSA analysis is carried out in the usual manner with minor modifications (see Ref. 26). To explain this briefly, NFAT was added to a binding buffer solution (30 mM Tris-HCl, pH7.5, 150 mM NaCl, 1.5 mM MgCl2, 2 mM dithiothreitol, 1% BSA) with 1 mg of tRNA as an unlabeled RNA competitor. Reaction at room temperature. After reacting for 10 minutes, an isotopically labeled NFAT oligomer probe is added and the reaction is maintained for 20 minutes. DNA-NFAT conjugates are analyzed on 6% unmodified polyacrylamide gels.

5. Intracellular RNA Injection

HeLa cells are cultured in DMEM (Gibco BRL) medium supplied with 10% fetal bovine serum and 1% penicillin / streptomycin at 37 ° C. NFATc. The expression of the portion encoding the full length uses a previously known NFATc expression vector to be achieved by SR promoter regulation (see Ref. 24).

The NFATp expression vector produced by modifying pLGP3 was produced by Harvard Medical School. A. Rao may be provided (see Ref. 27).

Dr. PXIL2-Luc, a detectable plasmid from T. Hoey (Tularik Inc.), expresses a firefly luciferase induced by three NFAT moieties located at IL-2 promoter 45 in mice (see See Document 28).

The NFkB promoter-luciferase plasmid is used to control the firefly luciferase gene under minimal fos promoter control with three copies of a major histocompatability complex (MHC) class I kB element. P3XkB-Luc to express is Dr. University of Wisconsin B. Available from Sugden (see Ref. 29).

PRLTK, which expresses renilla luciferase regulated by the Herpes simplex virus thymidine kinase promoter, is used as an internal means to measure the efficiency of intracellular input.

In exponentially grown HeLa cells (1x106 cells), 1 mg of NFAT expression vector, 2 mg of pXIL2-Luc, and 0.2 mg of pRLTK are injected with 6 ml of DMRIE-C (Gibco BRL) depending on the presence of RNA. In the NFkB experiment, 2 mg of p3XkB-Luc and 0.2 mg of pRLTK were injected into the cells as lipofectamine with or without RNA, respectively. After 8 hours of treatment, the cells were collected and the activity of the detection gene was determined using a luminometer TD-20 / 20 (Turner Designs Instrument) and a dual-luciferase reporter assay system (Promega). Measure the relative amount of light.

Example 1 RNA Screening

RNAs of the present invention that specifically bind to NFATc were selected as described above. After producing an RNA library consisting of approximately 1014 different molecules of 5'-GGGAGAGCGGAAGCGUGCUGGGCCN ₄₁ CAUAACCCAGAGGUCGAUGGAUCCCCCC-3 'with ₄₁ random sequences (N ₄₁ ) linked by clear sequences, 10 screening operations were performed. The cDNA was amplified, cloned, and finally 13 RNAs were selected and analyzed for nucleotide sequence.

The selected RNA (hereinafter, referred to as 'SE RNA') is an RNA such as that shown in FIG. 1 in the base sequence (# 25-65) at the 41 position among the total 90 base sequences of FIG. 2. In FIG. 1, the numbers in parentheses indicate the number present, and the part indicated by a line instead of the base means that the base shown at each position is the same as the base of # 1. As shown in FIG. 1, 13 clones showed 6 different sequences consisting of very similar sequences.

The secondary structure of the RNA predicted from # 1 of the SE RNA of FIG. 1 is shown in FIG. 2. The secondary structure of the safest RNA of FIG. 2 was obtained via the MULFOLD program (see Ref. 30). In the folded structure, 41 nucleotide sequences selected from the RNA library are formed by a stem loop having two long internal loops formed by binding themselves or fixed bases together. It can be seen. Other selected RNA clones showed similar morphology to the secondary structure predicted in clone # 1.

Example 2. Binding Properties of Selected RNA to NFATc DBD

In order to determine the binding specificity of the SE RNA selected through Example 1, the inside of the RNA was labeled with an isotope, and the sedimentation experiment was performed.

As shown in FIG. 3A, an RNA library labeled with an isotope of 1 nM, an RNA population obtained after 10 selections (10th SE RNA), and SE RNA # 1 were present with NFAT DBD (100 nM) presence (+ E). And (-E), and the RNA-protein conjugate precipitated with Ni-NTA beads. Precipitated RNA was extracted and analyzed on 6% polyacrylamide gel with urea. The first lane shows 20% of the labeled RNA injected into each. Both SE RNA # 1 and the RNA population obtained after 10 iterations showed strong and specific binding to NFATc DBD without reacting to agarose beads, whereas the first RNA library showed little binding to protein. . Other RNA clones that were selected also bound specifically to the NFATc DBD without any reaction to the beads.

Histidine-binding NFATp DBD protein and SE RNA do not bind to each other, which means that the binding between NFATc and SE RNA is a conservative DNA binding site where SE RNA is also present in other NFAT members or as a marker for protein expression. It does not show non-specific binding to the fragment (not shown).

NFATp is known to be involved in the immune response activity of Th1 type and down-regulation of late cytokine late transcription of Th2 type (see Ref. 6). Among the selected clones, SE RNA # 1 was the most obtained. The following experiments were performed with SE RNA # 1.

In order to confirm that the SE RNA specifically binds to the NFATc DBD protein, a gel retardation assay was performed and the result is shown in FIG. 3B. Isotopically labeled SE RNA # 1 (50pM) was reacted with and without NFATc DBD (50nM), respectively (+ E). RNA-protein conjugates were distinguished from unbound RNA using a 6% unmodified polyacrylamide gel. To investigate the binding specificity of NFATc DBD and SE RNA, the amount of unlabeled RNA or SE RNA # 1 (lanes 3, 6, 5nM; lanes 4,7, 5nM; lanes 5, 8, 500nM) was determined. The binding reaction was performed while increasing. SE RNA # 1 effectively combined with NFATc DBD to form a conjugate on the gel. The formation of conjugates between the nucleic acid proteins was completely inhibited by the addition of excess unlabeled SE RNA # 1 by concentration. However, no inhibition was made on the first RNA library, which was a nonspecific inhibitor.

Example 3. Binding Ability of Selected RNA to NFATc

The binding of selected RNA to NFATc was tested. The binding force of SE RNA # 1 to the NFATc DBD was measured by the settling experiment between the increased amount of protein and the trace isotopically labeled RNA, the results are shown in Figures 4A to 4C.

4A and 4B are reactions of isotopically labeled library RNAs (A, 200pM) and SE RNA # 1 (B, 200pM) with increasing amounts of NFATc DBD (0-960nM). RNA-protein conjugates were precipitated with Ni-NTA beads. Precipitated RNAs were extracted and electrophoresed on 6% polyacrylamide gel with urea. The amount of isotope present in the RNA-NFATc conjugate was measured to calculate the RNA binding ratio for the NFATc DBD and graphed in FIG. 4C. The maximum binding ratio of RNA to NFATc was shown when the protein concentration was 960 nM. Numbers shown are normalized to mean values measured in three experiments.

As can be seen from the graph, the RNA library of 41 random sequences used cannot be so strongly bound to very high concentrations of NFATc DBD protein. However, SE RNA # 1 had a dissociation constant (kd) of 30 nM and showed high binding force. Eventually, it can be seen that SE RNA binds strongly to the NFATc DBD protein.

Example 4. Inhibition of DNA Binding Activity of NFATc Without Affecting NFATp of Selected RNA

It was tested whether SE RNA affected the DNA binding ability of NFATc DBD. DNA binding was measured by gel retardation analysis after labeling the oligomer corresponding to the NFAT binding site in the IL-2 promoter of the mouse (see reference 25), the results are shown in Figures 5A to 5C. The IL-2 promoter region in mice can directly bind to NFAT even in the absence of Fos and Jun proteins, which can be used to test whether SE RNA binding capacity to NFAT affects the binding between NFAT and DNA. (See Ref. 31).

The oligomer corresponding to the NFAT binding site of the IL-2 promoter (300pM) terminal of the mouse was labeled with 32P and reacted with (+ E) free (-E) with NFATc DBD (800nM), and 5% unmodified polyacrylic. Electrophoresis was performed on amide gels to distinguish DNA-NFATc conjugates from unbound RNA, and the amount of unlabeled SE RNA # 1 and tRNA as competitors to determine the inhibitory function of SE RNA (lines 3, 7, 3nM; 4, Line 8, 30 nM; line 5, 9, line 300 nM; line 6, 10; 3mM) to increase the binding reaction was shown in Figure 5A. As shown in FIG. 5A, it can be seen that, while the selected RNA effectively inhibited NFATc DBD protein attaching to DNA depending on the concentration, tRNA hardly inhibited even under the highest concentration used in this experiment. .

EMSA was performed with 20ng of purified recombinant NFATp DBD. To assess the inhibitory function of SE RNA, 5-, 10-, 100-, and 200-fold large amounts of unlabeled SE RNA # 1 and tRNA were added to the reaction sample and the results are shown in FIG. 5B. . In addition, the binding reaction was carried out with 10ng of chimeric NFAT protein (Nc-Np) consisting of 1/3 of the C-terminus of NFATp DBD and 2/3 of the N-terminus of NFATc DBD, and measuring the inhibitory effect of SE RNA. To this end, excess unlabeled SE RNA # 1 and tRNA were added to the reaction sample and the results are shown in Figure 5C.

It can be seen from the EMSA experiment using NFATp DBD in the presence of excessive unlabeled competitive RNA from FIG. 5B that SE RNA specifically inhibits the DNA binding ability of NFATc DBD. In addition, it can be seen from FIG. 5C that neither SE RNA nor tRNA can prevent NFATp DBD from binding to the NFAT binding site. This may be because the SE RNA specifically binds only to NFATc DBD without affecting NFATp DBD as described with reference to FIG. 1. In contrast, SE RNA effectively reduces the DNA-binding ability of the chimeric NFAT protein made by combining the N-terminal 2/3 (amino acid 392-583) portion of NFATc DBD and the C-terminal l 1/3 (amino acid 581-694) of NFATp DBD. Inhibition (FIG. 5C). These results show that SE RNA specifically binds to the N-terminal 2/3 site (amino acids 392-583) of the NFATc DBD.

Example 5. Inhibition of NFATc Activity in Human Cells of Selected RNA

Since it was found from the above examples that SE RNA specifically binds to NFATc DBD and can inhibit NFATc from binding to DNA binding sites, induction of gene expression in which SE RNA is regulated by NFATc DBD transcription factors See if you can suppress it.

To this end, NFATc expression vector and NFAT promoter-luciferase reporter construct (temporal promoter-luciferase reporter construct) were temporarily put together in HeLa cells to analyze the expression level, and the results are shown in FIG. 6. As a control, cells to which no RNA was added were used.

Figure 6A is a pXIL2-Luc reporter construct was injected into HeLa cells with NFATc expression vector, Figure 6B is a p3XkB-Luc reporter construct, Figure 6C is a pXIL2-Luc reporter with NFATp expression vector Constructed.

At this time, in order to measure the inhibitory function of SE RNA, RNA was not added to the cell (w / o RNA), tRNA (50nM), SE RNA # 1 (50nM) was carried out the experiment. In addition, luciferase activity was measured by treatment (black bars) or not (white bars) with PMA and ionomycin for 8 hours after transient intracellular injection. The result was expressed as the ratio of luciferase activity obtained in cells treated with PMA and ionomycin without any RNA addition, and was shown through three independent experiments.

In addition, in order to examine the quantitative dependence of SE RNA on NFATc activity regulated by SE RNA, the amount of tRNA is increased with NFATc expression vector, pXIL2-Luc reporter construct, or the amount of SE RNA # 1 is increased. HeLa cells were injected and the results are shown in FIG. 4D. At this time, after treatment with PMA and ionomycin in the cells, the measured luciferase value was determined by the same method as in FIGS. 4A to 4C.

As can be seen in Figure 6A, the activity of luciferase was increased approximately 2-fold after treatment with PMA and ionomycin. Whereas nonspecific RNA competitors, such as tRNA, were not used to prevent increased detection gene expression, SE RNA effectively interfered with NFATc, suggesting that NFATc dependent gene expression was observed in HeLa cells not treated with PMA and ionomycin. It was suppressed to the level of degree.

In FIG. 6B, which was introduced into transient cells using a detection vector derived by NFkB having a nucleotide sequence with little similarity to the Rel homology site in NFAT, both tRNA and SE RNA were treated differently from NFATc. It can be seen that the induction of NFkB regulation detection gene expression in one cell is almost impeded, so that SE RNA specifically blocks gene expression regulated by NFATc in human cells.

In addition, as can be seen from FIG. 6C, NFAT-luciferase reporter expression regulated by NFATp was hardly affected in the cells treated with SE RNA.

In addition, as can be seen in FIG. 6D, gene activity inhibition induced by NFATc regulated by SE RNA occurred depending on the amount of SE RNA with an IC 50 value of about 4 nM.

From the above embodiments, it can be seen that the RNA of the present invention specifically binds to NFATc specifically among members of NFAT and specifically inhibits only the DNA binding activity of NFATc without affecting other members. In particular, the RNA of the present invention inhibits the DNA binding ability of the chimeric NFAT protein consisting of the N-terminal 2/3 of the NFATc DBD and the C-terminal 1/3 of the NFATp DBD, from which the RNA of the present invention is NFATc DBD It can be seen that it specifically binds to the N-terminal 2/3 site of.

Given that the N-terminal 2/3 portion of the NFATc DBD is the portion with the most conservative sequence in the NFAT protein member (see Reference 32), the RNA of the present invention is a DNA binding site that is structurally different from NFATp and / or It can be predicted that it can bind to the N-terminus of the NFATc DBD containing nearby, and from the present invention will be the basis for the development of specific inhibitors between NFAT types from the structural differences between NFAT members.

That is, the RNA of the present invention specifically inhibits only the gene expression by NFATc without affecting the expression of genes regulated by NFkB or NFATp, thereby inhibiting the phosphatase activity of calcineurin without distinguishing NFAT members, thereby NFAT protein. In view of the fact that existing immunosuppressive agents that prevent phosphate removal and migration into the nucleus can cause various physiological side effects by inactivating calcineurin in various organs or cell types, immunosuppressive therapeutic agents without toxicity from RNA of the present invention Is expected to be developed.

As described above, the RNA according to the present invention specifically binds to NFATc among members of NFAT and thus specifically inhibits only the DNA binding activity of NFATc without affecting other members. Inactivation of calcineurin in non-immune cells leads to a variety of physiological side effects leading to kidney, nerve, and gastrointestinal toxicity, diabetes and high incidence of cancer, whereas the RNA of the present invention has been developed to develop a non-toxic immunosuppressive drug. It will be useful for the research and will also contribute to NFAT functional studies.

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Claims (2)

Among the components of the NFAT that plays a role in inducing the transcription of genes involved in the immune response specific binding to the DNA binding site of NFATc comprises a base sequence that specifically inhibits only the binding capacity of the DNA of the NFATc RNA specifically binding to NFATc. According to claim 1, RNA specifically binding to NFATc, characterized in that it comprises one base sequence selected from SEQ ID NO: 1 to SEQ ID NO: 6.