JP2009165394A - Aptamer against peroxiredoxin 6 (Prx6) - Google Patents

Abstract

To obtain an aptamer that specifically binds to peroxiredoxin 6 (Prx6) protein. A peroxiredoxin 6 (Prx6) protein detection / quantification reagent or a new detection / quantification method for the protein is provided.
An RNA aptamer capable of binding to peroxiredoxin 6 (Prx6) protein is obtained using the SELEX method. The obtained RNA aptamer has a property that the binding affinity for reduced Prx6 is higher than that of oxidized Prx6 protein.
[Selection figure] None

Description

  The present invention relates to an RNA aptamer for peroxiredoxin 6 (Prx6) protein, detection of peroxiredoxin 6 (Prx6) protein comprising the aptamer, quantitative reagent, and peroxiredoxin 6 (Prx6) using the detection and quantitative reagent The present invention relates to protein detection and quantification methods.

  Reactive oxygen species such as hydrogen peroxide, peroxide, and nitric oxide are very reactive, and easily react with biological components such as proteins, nucleic acids, and lipids to change their structures and functions. Therefore, when reactive oxygen species are generated in the living body and cannot be completely treated (reduced), a mechanism for maintaining a redox state called “oxidation stress” is broken. In diseases such as cancer, Parkinson's disease, and Alzheimer's disease, the living body is in an oxidative stress state, and reactive oxygen species are considered to be involved in these diseases. On the other hand, it is considered that cells have a reactive oxygen species elimination system and an oxidative stress response system to suppress the concentration of these reactive oxygen species, cause apoptosis, and control the cell cycle. Several enzymes and small molecules are involved in the reactive oxygen scavenging system, but it is clear that a group of new peroxidases called peroxiredoxin (Prx) plays an important role in oxidative stress response in recent years. (Fig.1).

  In recent years, peroxiredoxin (Prx) has been found in various cells such as bacteria, yeast, higher plants, and mammals, and it has been revealed that more than 50 families are formed from the homology of the primary sequence.

  On the other hand, in the field of aptamer (nucleic acid ligand) development that has recently advanced, it is possible to separate nucleic acids that bind with high affinity and specificity to target molecules by in vitro selection methods. . The strategy is to separate a rare nucleic acid molecule with high affinity for the target molecule from a random pool of nucleic acids and then repeat the cycle of selection and amplification. To date, this method has proved extremely useful for separating aptamers that bind tightly to a variety of targets such as metal ions, sugars, peptides, proteins, or whole cells. The binding affinity of various aptamers to their recognition molecules is comparable or superior to that of antibodies and antigens. Detection / quantification of analytes and protein functions / activity Aptamers have been developed for a variety of in vitro and in vivo applications, such as inhibition of. Furthermore, it is known that aptamers may have a higher ability to discriminate between closely related molecules than antibodies, and that only a smaller area is required for binding. For example, theophylline aptamer can discriminate caffeine, which differs from theophylline only in the methyl group at the N7 position, with an efficiency of 14,000 times (see Non-Patent Document 1). Under such circumstances, the present inventors have so far developed RNA aptamers with high affinity for viral proteins of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) (non-patented). References 2-4). Of the aptamers developed, anti-Tat aptamers bind to HIV Tat protein with a KD value of 120 pM, whereas anti-NS3 aptamers bind to HCV NS3 protein with a KD value of 10 nM. Anti-Tat aptamers and anti-NS3 aptamers have been found to inhibit the function of these proteins both in vitro and in vivo (see Non-Patent Document 2, Non-Patent Document 5, and Non-Patent Document 6).

Jenison, R.D., et al., “Science” (USA), published on March 11, 1994, 263, 5152, p1425-1429 Yamamoto et al., Genes to Cells, 2000 May, 5 (5): 371-88 Kumar et al., Virology, 1997 Oct 27, 237 (2): 270-82 (See Fukuda et al., Eur. J. Biochem. 2000 June, 267 (12), 3685-94) Kakiuchi et al., Combinatorial Chemistry & High throughput screening, 2003, 6, 155-160; See Nishikawa et al., Nucleic Acids Res. 2003 Apr 1, 31 (7), 1935-43

  As described above, reactive oxygen species are generated in the living body due to the process of energy metabolism in mitochondria, drugs, ultraviolet rays, etc., but if these endogenous and exogenous reactive oxygen species increase too much, the redox state in the living body The mechanism that maintains the state fails, and a state called “oxidative stress” occurs. In diseases such as cancer, diabetes, arteriosclerosis, Parkinson's disease, and Alzheimer's disease, the living body is in an oxidative stress state, and reactive oxygen species are considered to be involved in these diseases. A series of peroxidases, including peroxiredoxin (Prx), are antioxidant enzymes that reduce these reactive oxygen species and may be used as markers for diseases that are thought to involve oxidative stress. A method capable of distinguishing and detecting oxidized Prx is a very important issue.

  An antibody using Prx as an antigen is also commercially available, but it is a polyclonal antibody. In addition, in the case of an antibody, a 6 to 8-residue fragment or a larger surface area is required as a recognition site for the target protein. It is difficult to detect very slight differences such as the oxidized form.

Moreover, although it has been confirmed that a commercially available antibody using Prx as an antigen can be used for detection by immunoblotting, it cannot be confirmed that it can be used for detection under non-denaturing conditions using Biacore. For this reason, there is no assay system for quantitatively detecting Prx extracted from cells. Accordingly, an object of the present invention is to provide a useful aptamer capable of specifically recognizing peroxiredoxin (Prx), and to detect peroxiredoxin (Prx) using this, a quantification reagent, and a detection and quantification method. It is in the point to provide newly.

  As a result of diligent research, the present inventors succeeded in obtaining an aptamer that can specifically recognize peroxiredoxin 6 (Prx6) protein using the SELEX method, and the aptamer is Although it is not perfect, it has been found that the oxidized form and reduced form of peroxiredoxin 6 (Prx6) protein can be distinguished to some extent, and the present invention has been completed.

That is, the present invention is as follows.
(1) The base sequence shown in SEQ ID NO: 1 or 2, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and peroxiredoxin 6 (Prx6) An RNA aptamer characterized by having a binding ability to a protein.
(2) consisting of the base sequence represented by any of SEQ ID NOs: 3 to 7, or including a base sequence in which one or several bases are deleted, substituted or added, and peroxiredoxin 6 (Prx6) An RNA aptamer characterized by having a binding ability to a protein.
(3) Peroxiredoxin 6 comprising the nucleotide sequence shown in SEQ ID NO: 8 or 9, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence, and transcription. (Prx6) DNA that can be converted into an aptamer that has the ability to bind to a protein.
(4) consisting of a base sequence represented by any of SEQ ID NOs: 10 to 14, or including a base sequence in which one or several bases are deleted, substituted or added, and peroxiredoxin 6 ( Prx6) DNA that can be converted into an aptamer capable of binding to protein.
(5) The DNA according to (3) or (4) above, wherein a T7 promoter sequence is added to the 5 ′ end.
(6) DNA having a base sequence complementary to the DNA according to any one of (3) to (5) above.
(7) Double-stranded DNA obtained by hybridizing the DNA according to any one of (3) to (5) above and its complementary DNA.
(8) A reagent for detection and quantification of peroxiredoxin 6 (Prx6) protein comprising the RNA aptamer of (1) or (2) above.
(9) Peroxiredoxin 6 (Prx6) comprising contacting the detection and quantification reagent according to (8) above with a sample containing or possibly containing peroxiredoxin 6 (Prx6) protein ) Protein detection and / or quantification method.

The aptamer RNA of the present invention has a specific recognition ability for peroxiredoxin 6 (Prx6) protein and is useful as a reagent for identifying peroxiredoxin 6 (Prx6) protein.
In general, aptamers have fewer amino acid residues at the recognition site in a target protein than antibodies, and high affinity motifs of aptamers can distinguish proteins with high homology with high sensitivity. In addition, since aptamers recognize target molecules under non-denaturing conditions and have binding ability, peroxiredoxin 6 (Prx6) protein molecules can be detected quantitatively under non-denaturing conditions by binding assays using aptamer molecules. Peroxiredoxin can be detected in a state similar to that in vivo. Quantitative detection of peroxiredoxin 6 (Prx6) protein in the living body and monitoring increase / decrease in the amount of secretion open the way to diagnosis whether or not the living body is in an oxidative stress state.

  On the other hand, the aptamer of the present invention has a higher binding affinity for the reduced peroxiredoxin 6 (nPrx6) protein than the oxidized peroxiredoxin 6 (oxPrx6) protein, and the oxidized form is not perfect. And the ability to discriminate between reduced forms of peroxiredoxins. Thus, for example, by using nPrx6-only samples and oxPrx6-only samples as controls, comparing the affinity or amount of binding between the peroxiredoxin 6 (Prx6) protein sample extracted from the living body and the aptamer to it. Thus, the ratio of reduced peroxiredoxin 6 (nPrx6) protein to oxidized peroxiredoxin 6 (oxPrx6) protein can be grasped qualitatively to some extent.

The aptamer of the present invention is derived from a clone obtained by the SELEX method and has an ability to specifically bind to peroxiredoxin 6 (Prx6) protein. The gene sequence and amino acid sequence of peroxiredoxin 6 (nPrx6) protein are shown in SEQ ID NOs: 21 and 22 in the sequence listing.
The first aptamer of the present invention includes RNA containing at least the base sequence shown in SEQ ID NO: 1. Specific aptamers thereof include, for example, RNA shown in SEQ ID NO: 3 or 6, and these RNAs have the sequence shown in SEQ ID NO: 1 in common. The second aptamer includes RNA containing at least the base sequence shown in SEQ ID NO: 2, and specific aptamers thereof include RNA shown in SEQ ID NO: 4 or 7, and these RNAs are shown in SEQ ID NO: 2. It has the base sequence shown in common. Other aptamers include RNA having the base sequence shown in SEQ ID NO: 5.

On the other hand, in the present invention, not only the RNA represented by the above SEQ ID No. but also a DNA having the sequence corresponding to the RNA sequence represented by the above SEQ ID No. 1 or 2 and comprising the base sequence represented by SEQ ID No. 8 or 9, respectively. To do. More specifically, for example, a DNA consisting of the base sequence shown in SEQ ID NO: 10 or 12, and 11 or 13, and a DNA shown in SEQ ID NO: 14 corresponding to the RNA shown in SEQ ID NO: 7 can be mentioned. . Further, in the present invention, it includes DNA complementary to these DNAs, or dsDNA in which these complementary DNA strands constitute a double strand. These can be easily converted into the aptamer RNA of the present invention by conventional genetic manipulation means, and can be used as an intermediate for producing the aptamer of the present invention. These DNAs may have a sequence such as a T7 promoter (SEQ ID NO: 22) added to the 5 ′ end.
In the present invention, even if the base sequence of the aptamer of the present invention is modified by deleting, replacing, or adding one or several nucleotide residues, the peroxiredoxin 6 (Prx6) protein This includes any binding.

The aptamer of the present invention is obtained by a well-known SELEX (systematic evolution of ligands by exponential enrichment) method.
The SELEX method starts with the creation of a random-sequence nucleic acid library, selects the binding to the target protein as an index, and repeats the PCR amplification cycle multiple times. Only nucleic acids having high binding ability can be selected. The above selection and PCR amplification correspond to “spider” and “growth” in nature, respectively, and reproduce the evolution in nature in a short time in a test tube.

In the present invention, a DNA library (SEQ ID NO: 18) having 30 bases in the middle of the sequence as a random region is synthesized, amplified by PCR, reverse transcribed to form an RNA pool, and the above-mentioned normal type is used for this RNA pool. (Reduced) Peroxiredoxin 6 (nPrx6) and oxidized peroxiredoxin 6 (oxPrx6) RNA is selected and amplified, and this cycle is repeated multiple times to be specific for peroxiredoxin 6 protein. The RNA aptamers shown in SEQ ID NOs: 3 to 5 are recognized in the present invention. In the present invention, the RNA aptamers shown in SEQ ID NO: 2 and SEQ ID NO: 5 are further shortened and shown in SEQ ID NOs: 6 and 7. RNA aptamers have been obtained, and these shortened RNA aptamers also have binding ability to peroxiredoxin 6 (Prx6). The aptamer of the present invention is obtained not only when it is reduced but also when oxidized peroxiredoxin is targeted, and it binds to both oxidized and normal types, but the measured dissociation constants. Indicates that any aptamer has an equivalent or higher binding property (up to 10 times) to the normal type than the oxidized type.

As described above, the aptamer of the present invention is basically obtained by the SELEX method. However, in the present invention, the base sequence of the aptamer is clarified, and the base sequence is not determined by the SELEX method. The aptamer of the present invention can be synthesized. For example, the following method known per se can be used.
In vitro transcription method: The above DNA corresponding to the aptamer RNA to be synthesized is chemically synthesized, PCR amplified, and an RNA aptamer is synthesized from the amplified DNA by a transcription reaction with RNA polymerase. For example, the above DNA is linked to the downstream side of the T7 promoter and PCR amplified to obtain double-stranded DNA. Using the double-stranded DNA as a template, 5′-end primer, T7 RNA polymerase, and ATP, GTP, CTP RNA aptamers can be obtained by performing an RNA elongation reaction in a reaction solution containing the RNA polymerase substrate comprising UTP and UTP.

  In this method, a plasmid containing the T7 promoter sequence linked to the above dsDNA, or a chemically synthesized DNA can also be used as a template. The DNA synthesis can be performed using a DNA synthesizer (for example, 334 DNA synthesizer (Applied Biosystems)).

  Chemical synthesis method: RNA synthesis requires protection of the 2 'hydroxyl group of the ribose moiety, and various amidites have been developed as protecting groups. Using the recently developed 2-cyanoethoxymethyl (CEM) group The efficiency of the chain length extension reaction at the time of RNA synthesis is increased, and it becomes possible to synthesize a long chain RNA exceeding 100 mer. The RNA aptamer of the present invention can also be synthesized using such a chemical synthesis method.

The RNA aptamer of the present invention is used as a reagent for detecting and quantifying peroxiredoxin 6 (PrX6).
For example, the RNA aptamer of the present invention is labeled with a fluorescent dye such as fluorescein, rhodamine, Texas red, etc., contacted with a test sample, subjected to a binding reaction, removed unbound, and then detected or measured for fluorescence or its intensity By doing so, it is possible to detect peroxiredoxin 6 (PrX6) protein or measure the amount thereof. In this case, for example, it can be performed using a substrate on which either a labeled RNA aptamer or a test sample is immobilized.
The label is not limited to the fluorescent dyes described above, and may be a radioactive label, or modified with avidin or streptavidin. Biotin is used for detection and quantification of the aptamer peroxiredoxin 6 (PrX6) protein complex. That's fine.
Thus, since aptamers are nucleic acids, they are easy to synthesize and can be easily added with various modifications and labels. Those skilled in the art usually perform a method for detecting the binding between a target molecule and an aptamer in the art. Can be used as appropriate.

In the present invention, peroxiredoxin 6 (PrX6) protein can be detected and quantified without labeling with a fluorescent dye or the like. This includes, for example, a detection method using surface plasmon resonance (SPR). That is, the intermolecular binding reaction can be measured by adopting an optical phenomenon called surface plasmon resonance, which is a fine mass change at the time of binding of both molecules occurring on the sensor surface. As a device using the SPR method, there is Biacore T100 manufactured by Biacore. The measurement is performed by immobilizing either the RNA aptamer of the present invention or the test sample on the gold thin film surface of the sensor chip by a well-known method, supplying the test sample or the RNA aptamer thereto, and monitoring the binding reaction in real time.
Examples of the present invention are shown below, but the present invention is not limited to these Examples.

Example 1
The RNA pool (30N) used for in vitro selection of aptamers that specifically bind to the Prx protein was prepared as previously reported (Fukuda et al., Eur. J. Biochem., 267, 3685-3694 ( 2000)). This RNA pool contains a random 30 base core sandwiched between primer binding regions as follows:
5′-AGTAATACGACTCACTATAGGGAGAATTCCGACCAGAAG-N30-CCTTTCCTCTCTCCTTCCTCTTCT-3 ′ (SEQ ID NO: 18). Primers used for amplification of the RNA pool were: 5′-AGTAATACGACTCACTATAGGGAGAATTCCGACCAGAAG-3 ′ ((SEQ ID NO: 19), hereinafter referred to as 39.N30)
And 5′-AGAAGAGGAAGGAGAGAGGAAAGG-3 ′ ((SEQ ID NO: 20), hereinafter referred to as 24.N30). In the selection cycle, E. coli tRNA (Boehringer-Mannheim) was used as a non-specific competitive inhibitor.

In vitro selection is basically performed as previously reported (Urvil, PT et al., Eur. J. Biochem. 248, 130-38 (1997); Kumar, PK et al., Virology, 237, 270- 282 (1997)).
Specifically, a nickel chelate-coated microplate (Xenopore) was washed twice with 200 ul of buffer (10 mM Tris-HCl; 7.5, 50 mM NaCl), and then reduced peroxiredoxin as the target protein. 200 ul each of 6 (nPrX6) protein (1 uM) and oxidized peroxiredoxin (oxPrx) 6 protein (1 uM) were added and incubated at 4 degrees for 10 hours. Then wash three times with the above 200 ul buffer.

  A 20 uM RNA pool containing a random sequence (N30) having a length of 30 bases obtained as described above was added to a plate on which the above target protein was immobilized, and incubated at room temperature for 5 minutes. Wash three more times with 200 ul of buffer and then elute with 100 ul of elution buffer (10 mM HEPES; 7.4, 0,15 M NaCl, 0.35 M EDTA, 0,005% Tween-20). The obtained elution sample was purified by RNA precipitation using ethanol precipitation method, 50 mM Tris-HCl (pH 8.0), 40 mM KCl, 6 mM MgCl2, 0.4 mM dNTPs, 2.5 μM primer (24.N30) and Reverse transcription was carried out in a 20 μl reaction containing 5 U of AMV reverse transcriptase (WAKO). Nucleotides and enzymes were added after the annealing step (treatment at 95 ° C. for 5 minutes, then at room temperature for 10 minutes). Reverse transcription was performed at 42 ° C. for 90 minutes.

For amplification by polymerase chain reaction (PCR), 20 μl of the reverse transcription mixture (cDNA reaction solution) is mixed with 80 μl of PCR mixture (10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM) Diluted with MgCl2, 0.1% Triton X-100, 5U Taq DNA polymerase (Takara), 0.4 μM each of 24.N30 primer and 39.N30 primer). The reaction was repeated at 94 ° C for 1.15 minutes; -60 ° C for 0.45 minutes; -72 ° C for 1.15 minutes until the product band was obtained at the correct size. The PCR product was precipitated with ethanol and used for transcription. The in vitro transcription reaction was performed at 37 ° C. for 3 hours using a T7 Ampliscribe kit (Epicentre Technologies). After RNA synthesis and DNase I treatment, the reaction solution was fractionated on an 8% non-denaturing polyacrylamide gel. RNA was extracted from the gel and used for the next selection and amplification cycle.
Each sorting condition is shown in Tables 1 and 2 below.

  Using the RNA pool obtained in the 12th cycle, the binding affinity for nPrx6 protein and oxPrx6 was measured using a surface plasmon resonance apparatus BIAcore T100 (Biacore). The results are shown in FIGS.

The RNA pool obtained in the 12th cycle was cloned, and the sequence of each of 24 clones of RNA was decoded. Of these, 5 clones with high occupation rates were found, and they were named as follows.
Clone A (SEQ ID NO: 15), Clone B (SEQ ID NO: 16), Clone C (SEQ ID NO: 5), Clone D (SEQ ID NO: 17), Clone E (SEQ ID NO: 3), Clone F (SEQ ID NO: 4).
Among these, the secondary structures of clone C, clone E and clone F are shown in FIGS.
The occupation ratio of each clone is shown in Table 3 below.

Example 2
Binding specificity of the isolated aptamer for Prx6 protein The binding affinity of the six RNA aptamers of the isolated clone AF to Prx protein was measured using a surface plasmon resonance apparatus (BIAcore T100 (Biacore)). . The results are shown in FIGS.
According to this, clones C, E and F have higher binding ability to both oxidized peroxidexin 6 (oxPrx6) and reduced peroxidexin 6 (nPrx6) than clones A, B and D I understand. Clone E is obtained by targeting reduced peroxidexin 6 (nPrx6), clone F is obtained by targeting oxidized peroxidexin 6 (oxPrx6), and clone C is obtained by reducing reduced peroxidexin 6 (nPrx6) And oxidized peroxide 6 (oxPrx6).

Example 3
Measurement of aptamer dissociation constant by surface plasmon resonance method Since surface plasmon resonance method is expected to be more sensitive than dissociation constant by filter binding measurement and to be able to measure dissociation constant with higher accuracy, it was judged that affinity was high by preliminary experiments. The dissociation constants of the binding between the three types of RNA aptamers (clone C, E, F) and nPrx6 protein and oxPrx6 protein were measured with a surface plasmon resonance apparatus BIAcore T100 (Biacore).

  An RNA having a 24 nucleotide long oligo-adenine (A) extended to the 3 ′ end of the RNA aptamer was prepared and immobilized on a sensor chip for surface plasmon resonance as follows. First, 0.3 μM biotinylated oligo dT (24 nucleotides long) [5 '-() dissolved in HBS-EP ++ buffer (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.5) dT) 24-3 '], 20 μl was immobilized on a sensor chip coated with streptavidin (Sensor Chip SA, Biacore) at a flow rate of 2 μl / ml, and the buffer was 20 at a flow rate of 20 μl / ml. Unbound oligonucleotide was removed by running for a minute. Dissolve RNA aptamer containing oligo A (clone C, E, F) in HBS-EP buffer at a concentration of 0.3 μM, and flow 20 μl onto the chip with oligo dT immobilized at a flow rate of 2 μl / ml on the chip surface. Of oligo dT. The intensity of the response detected by the surface plasmon resonance device upon binding of the aptamer to the chip was about 5,000 response units (RU).

  Run 60 μl of nPrx6 protein or oxPrx6 protein solution in a certain concentration range to the above SA chip at a flow rate of 20 μl / ml, measure the binding reaction curve with the immobilized RNA aptamer, and measure each aptamer and reduced peroxidexin 6 The dissociation constant between the (nPrx6) protein and oxidized peroxidexin 6 (oxPrx6) protein was determined. The Prx6 protein to be added as an analyte was prepared by preparing samples of at least 5 different concentrations. The results are shown in Table 4 below together with the results of performing the same test on the shortened aptamer obtained in Example 5 described later.

  According to this, all of the aptamers described above have the same or higher affinity for reduced peroxiredoxin 6 than oxidized peroxiredoxin 6, and in particular clone F and clone Fmini are reduced. It is clear that the affinity is about 10 times higher than that of the oxidized form.

Example 4
Inhibition of Prx6 Enzyme Activity by RNA Aptamer The isolated RNA aptamer binds to and interacts with Prx6 protein to test whether the Prx6 protein originally inhibits the enzyme activity as follows.
A variable amount of peroxiredoxin was added to 50 mM Hepes-NaOH (pH 7.0) containing 1 mM EDTA, 1.5 μM thioredoxin, 0.3 μM thioredoxin reductase, 0.2 mM NADPH, and reacted at 30 ° C. for 3 minutes. After the reaction, 100 μM hydrogen peroxide was added as a substrate to start the reaction, and NADPH consumption was measured over time using UV-2450 (Shimadzu Corporation) as a decrease in absorbance at 340 nm. The NADPH extinction coefficient was 6.22 mM-1 cm-1, and the enzyme reaction rate was determined. Controls without sample were measured and subtracted as spontaneous activity due to the added reagent. When examining the inhibitory activity of an enzyme by an RNA aptamer, a similar experiment was conducted in the presence of peroxiredoxin.

The results are shown in FIGS. According to the results of FIG. 8, it was shown that the aptamer of clone C has about 50% enzyme inhibitory activity against the nPrx6 protein. This enzyme inhibitory activity was confirmed for all aptamers Clone C (Aptamer C), E (Aptamer E), and F (Aptamer F). Clone C was the highest, followed by Clone F and C. It was.
FIG. 9 shows the results of examining the effect of RNase in the assay of FIG. 8. Although there was a concern about degradation of RNA aptamer during the assay with RNase, there was a difference in the difference between the case where RNase inhibitor was added and the case where RNase inhibitor was not added. As a result, it was considered that there was almost no degradation of the RNA aptamer during this assay.
FIG. 10 shows the result of assaying whether the enzyme inhibitory activity of clone C RNA aptamer is dependent on the aptamer concentration. The concentration dependence of the aptamer to be added is up to 2 μM, but it is moderate at higher concentrations. Concentration dependence was confirmed.
When the inhibition constant of the clone C aptamer with respect to Prx6 protein was calculated | required based on the said series of enzyme inhibitory activity measurement, ki value was set to 1.4 micromol.

Example 5
Minimization of the isolated RNA aptamer The primer region common to all clones was deleted from the sequence of the isolated RNA aptamer to shorten the aptamer molecule. From the secondary structure prediction, it was confirmed that the minimized aptamer was not structurally significantly different from the full-length one, and it was assumed that the minimized aptamer contained a specific binding region with the target molecule. The adjustment method is as follows.
A DNA corresponding to the minimized RNA aptamer sequence was chemically synthesized, and a T7 promoter region was added upstream of the DNA, which was then PCR amplified. An RNA aptamer was synthesized from the amplified DNA by a transcription reaction using RNA polymerase.

The nucleotide sequences and predicted secondary structures of the obtained minimized RNA aptamers, clone Emini and clone Fmini, are shown in SEQ ID NOs: 6 and 7, and FIGS. 11 and 12, respectively.
For these minimized aptamers, the dissociation constants of these minimized aptamers, reduced peroxidexin 6 (NPrx6) and oxidized peroxidexin 6 were determined using a surface plasmon resonance apparatus in the same manner as in Example 3. The results are listed in Table 3 in Example 3.

FIG. 6 is a diagram showing the results of measuring the binding properties of a selected RNA pool to nPrx, which is a diagram showing the predicted secondary structure of clone E. It is a figure which shows the result of having measured the binding property of the selected RNA pool with respect to oxPrx using the surface plasmon resonance apparatus BIAcoreT100 (Biacore). It is a figure showing the predicted secondary structure of the clone C. 2 is a diagram showing a predicted secondary structure of clone E. FIG. 2 is a diagram showing a predicted secondary structure of clone F. FIG. It is a figure which shows the binding curve with respect to nPrx of clone AF measured using the surface plasmon resonance apparatus BIAcoreT100 (Biacore). It is a figure which shows the binding curve with respect to oxPrx of clone AF measured using the surface plasmon resonance apparatus BIAcoreT100 (Biacore). It is a graph which shows the result of having tested the inhibitory activity with respect to nPrx6 of aptamer C, E, and F. It is a graph which shows the result of having tested the influence of RNase with respect to the said inhibitory activity of aptamer C. It is a graph which shows the result of having tested the relationship between each density | concentration of clone C, and inhibitory activity. It is a figure showing the predicted secondary structure of clone Emini. It is a figure showing the predicted secondary structure of clone Fmini.

Claims (9)

  Against the peroxiredoxin 6 (Prx6) protein, including the base sequence shown in SEQ ID NO: 1 or 2, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence An RNA aptamer characterized by having binding ability.   It consists of a base sequence represented by any of SEQ ID NOs: 3 to 7, or contains a base sequence in which one or several bases are deleted, substituted or added, and peroxiredoxin 6 (Prx6) An RNA aptamer characterized by having a binding ability to a protein.   Peroxiredoxin 6 (Prx6) containing the nucleotide sequence shown in SEQ ID NO: 8 or 9, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence, and transcription DNA that can be converted into an aptamer that has the ability to bind to proteins.   Peroxiredoxin 6 (Prx6) protein comprising the base sequence represented by any of SEQ ID NOs: 10 to 14 or including a base sequence in which one or several bases are deleted, substituted or added in the base sequence DNA that can be converted into an aptamer having the ability to bind to.   The DNA according to claim 3 or 4, wherein a T7 promoter sequence is added to the 5 'end.   DNA having a base sequence complementary to the DNA according to any one of claims 3 to 5.   Double-stranded DNA obtained by hybridizing the DNA according to any one of claims 3 to 5 and its complementary DNA.   A reagent for detection and quantification of peroxiredoxin 6 (Prx6) protein comprising the RNA aptamer according to claim 1 or 2.   9. Detection of peroxiredoxin 6 (Prx6) protein comprising contacting the detection, quantification reagent according to claim 8 with a sample containing or possibly containing peroxiredoxin 6 (Prx6) protein And / or quantitative methods.