KR100828937B1 - Single-Stranded Nucleic Acid Aptamer Specifically Binding to Serum Protein - Google Patents

Abstract

The present invention relates to a single-stranded nucleic acid aptamer that specifically binds to a serum protein capable of diagnosing cardiovascular disease. According to the present invention, there is provided a single-stranded nucleic acid aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 19 that specifically binds to a serum protein.

Description

Single-Stranded Nucleic Acid Aptamer Specifically Binding to Serum Protein}

1 shows the capture of streptoavidin-magnetic beads in serum using a MARC column,

Figure 2 is a photographic picture of the electrophoresis by labeling with Cy-5 while amplifying the complex of RNA aptamer and serum sample by RT-PCR,

Figure 3 is a laser scanner image of the reacted aptamer-based biochip,

4 is an image similarity analysis diagram using a GeneSpring program for the image information of FIG. 3;

5 is a diagram hierarchically clustering the overall similarity of spots;

6 shows the sensitivity, selectivity, positive predictive value, and negative predictive value calculated from FIG. 5;

7 is a result of searching 21 efficient spots by the algorithm of k-means and SOM,

FIG. 8 is a result of classifying patients with cardiovascular disease into 21 spots of FIG.

9 shows the sensitivity, selectivity, positive predictive value, and negative predictive value calculated from FIG. 8;

(Technology)

The present invention relates to a single-stranded nucleic acid aptamer, and more specifically, it is possible to diagnose cardiovascular disease by analyzing specific binding patterns of a single-stranded nucleic acid aptamer that specifically binds to human serum proteins and human serum proteins. The present invention relates to a single-stranded nucleic acid aptamer that specifically binds to a serum protein.

(Background)

Ligands that can identify specific substances and analyze their quantitative changes continue to evolve with the development of physics and biochemistry.However, the development of more efficient ligands in terms of maintenance costs, ease of use, accuracy, sensitivity, test time, and automation The demand continues.

The method of analyzing a specific substance is not an ultimate goal in itself but a part of the whole process, but it is very useful for microbial and virus identification, cell, protein and organic substance analysis, and is widely used in medicine, veterinary medicine, environmental engineering, food engineering and agriculture. It is applied.

The incidence of cardiovascular disease is increasing not only in Western countries but also in Korea, and the most recent mortality rate from the National Statistical Office, 2001, is 121 per 100,000 people. In 1997, the standardized mortality rate of cardiovascular disease in Korea was 304 per 100,000, which is not much lower than that of 360 in the United States.

Aging of the population is inevitable, and given the dreaded increase in coronary artery disease, a 10-fold increase in mortality over the past 18 years, it is only a matter of time before the cardiovascular disease becomes more serious in the United States than in the United States.

Cardiovascular disease test methods include electrocardiogram and cardiac surgery.

For the patient, the heart is a muscle organ that acts as a pump that circulates the circulation medium called blood, and its movement is controlled by fine electricity. The electrocardiogram is recorded by amplifying and recording a small electric wave from each of the cardiomyocytes that make up the heart, and a method of measuring the change in the electrocardiogram (cardiomyocytes causing the electrocardiogram), that is, the change in heart motion. ECG test.

In-depth surgical examination measures the pressure and oxygen saturation of the atrium and ventricles in the heart after inserting a catheter shaped like a ballpoint pen with a diameter of about 2.0-3.0 mm and a length of about 120 cm to the blood vessels. X-ray contrast is injected to determine the shape of the heart.

However, the conventional cardiovascular disease test method has a problem that the patient is hospitalized or takes a long time to the test, as well as the patient's rejection due to the invasive test.

As is well known, nucleic acids are linear multimers in which nucleotides are covalently linked, and nucleotides are small organic compounds consisting of phosphoric acid, sugars and purines (adenine or guanine) or pyrimidine (cytosine, thymidine, uracil). .

Nucleic acids exist in single- or double-stranded strands, which bind together by hydrogen bonds and interactions between nucleotides under specific physical conditions to form unique conformations, which are determined by single-stranded nucleotide sequences. .

Generally, nucleic acids such as DNA and RNA are a storage of information for expressing proteins with cell structure and enzyme activity, but since 1982 reports that RNA has a specific structure as well as enzymes, the nucleic acid has been reported as having an enzyme. There are many reports of structural features and their specific function.

Nucleic acid is composed of four base repeats to maintain a high diversity to form a large number of three-dimensional structure, these three-dimensional structure is stabilized by interacting with a specific material to form a complex.

Nucleic acid can act as a ligand to a specific substance containing a protein, with high binding power and specificity through a constant screening process and sequencing from a library of single stranded nucleic acids arranged in various nucleotide sequences. Nucleic acids that bind to specific substances are being screened.

The method of screening nucleic acids for binding to a specific substance is called SELEX (Systematic Evolution of Ligand by Exponential enrichment), and the selected nucleic acid is usually called aptamer (Tuerk C. and Gold L .; Science , 249 , pp 505-510, 1990).

Through SELEX, molecules that can bind with a very high affinity to various biomolecules, including proteins that cannot bind nucleic acids in nature, as well as proteins that do not bind nucleic acids, are selected.

It is an object of the present invention to provide a single stranded nucleic acid aptamer specifically binding to serum proteins.

Disclosure of the Invention An object of the present invention is to detect a specific binding and binding pattern between a single-stranded nucleic acid aptamer and a serum protein that specifically binds to a serum protein, so that the cardiovascular disease can be diagnosed more quickly, easily and accurately. We want to be able to diagnose the patient.

According to the present invention there is provided a single-stranded nucleic acid aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 19 that specifically binds to a serum protein.

The aptamers according to the present invention selected through a method modified from SELEX standard method (Bock LC et al .; Nature , 355 (6360) , pp564-6, 1992) (see Korean Patent Publication No. 2005-0106759), Single-stranded nucleic acids having a structure capable of specific binding to serum proteins. For this purpose, a single-stranded structure having the highest stability after confirming secondary structure and structure free energy using the MFOLD program modeling the secondary structure of nucleic acid is identified. Select strand nucleic acid aptamers.

Preferably, the specific binding reaction between the aptamer and the target serum protein according to the present invention comprises a reaction solution (analytical reagent) containing a composition that prevents the aptamer from binding to the target serum protein and prevents nonspecific binding. To run.

Preferably, as the reaction solution, (1) 20 to 100 mM Tris hydrochloric acid (pH 7.4) for buffering against pH, (2) 1 mM to potassium chloride, sodium chloride, and magnesium chloride for secondary structure stabilization of the aptamer 200 mM, (3) sodium azide 0.05-0.2% for bacterial propagation inhibition, (4) bovine serum albumin 0.1-0.3% for nonspecific binding inhibition. The sodium azide may not be included. The reaction temperature is ideally carried out at a temperature lower than the SELEX operating temperature, preferably the reaction is carried out at a temperature of 20 to 30 ℃.

As the labeling material of the aptamers according to the present invention, a radioactive isotope, a fluorescein (Fluorescein), a fluorescent material such as Cy3 or Cy5, or a biotin may be used, and preferably a fluorescent material is used.

In order to prevent the nonspecific binding of the single-stranded nucleic acid aptamer according to the present invention, the protein can be treated in excess, and the conjugated and unbound aptamers of the target serum protein and the single-stranded nucleic acid aptamer are membrane filtration or centrifugation. And so on.

The aptamer and serum protein complexes according to the present invention are determined by the specificity and binding strength between them, from which specific substances can be identified and quantified. The specific binding force of the aptamer and serum protein of the present invention changes the degree of binding of the complex. Therefore, by analyzing the degree of binding can be confirmed whether the cardiovascular disease and the progression of the disease in humans.

In the diagnosis of cardiovascular disease using the aptamer according to the present invention, nucleic acid quantitative analysis or fluorescence measurement may be selected and used according to the type of the substance labeled on the aptamer. According to the present analysis method, angina pectoris and myocardial infarction may be included. Patients with cardiovascular disease can be diagnosed.

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When the aptamer according to the present invention is reacted with serum proteins and analyzed according to the analysis method of Korean Patent Laid-Open Publication No. 2005-0106759 (published on November 11, 2005), cardiovascular disease can be diagnosed.

As a method of searching for a label of the reaction result of the aptamer and the serum protein of the present invention, nucleic acid quantitative analysis or fluorescence measurement may be used, and preferably, nucleic acid quantitative analysis may be used.

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

Example 1 Screening of Single-stranded Nucleic Acid Specific to Serum Protein in Patients with Cardiovascular Disease

Serum was biotin attached to serum proteins from which excess protein was removed using MARC columns (Agilent Technologies Inc. USA) and captured with streptoavidin-magnetic beads (see FIG. 1).

After reacting with the Cellex RNA library and the modified serum protein in the Cellex buffer, using a magnetic force to select a RNA that binds to the serum proteins by separating the complex of serum protein and RNA.

RT-PCR of the isolated complex was amplified to prepare a DNA pool indicative of RNA binding to the serum protein. DNA pool was synthesized by in vitro transcription of RNA, followed by a selection and amplification process about 10 times, and then a DNA library for serum proteins was cloned into a plasmid of DNA, a PCR product obtained by RT-PCR of the obtained RNA pool. Was produced.

Example  2: screened Single-stranded nucleic acid With aptamers Serum protein  Specific reaction

Serum used was performed in the following experiment using a total of 80 samples including 20 samples of angina pectoris, 20 samples of myocardial infarction, 20 samples of liver cancer and 20 samples of healthy people.

PCR was performed by selecting 1,000 clones from the DNA library of serum proteins and using the microarray, a DNA array composed of 1,000 spots was used and used as an aptamer-based biochip.

10 μl of serum sample was added to 90 μl of PBS solution and reacted by shaking for 30 minutes in a nitrocellulose membrane disc.

1,000 RNA aptamers were prepared using the selected clones, and then reacted with a serum sample attached disk for 30 minutes. The RNA aptamer and serum sample-coated discs were treated with RT-PCR solution and labeled with Cy-5 while performing RT-PCR (see FIG. 2).

Cy-3-labeled standard solution was prepared and prepared in the same manner.

The two solutions were mixed in the same ratio, and treated with aptamer-based biochips, hybridized at 60 ° C. for 4 to 12 hours, and washed with 42 × 0.1 × SSC solution. The aptamer-based biochip was terminated using a laser scanner to produce an image. Three to four replicates were performed on the same sample to produce a total of 261 images.

From an image analyzed using a patient serum sample of cardiovascular disease (angina and myocardial infarction) and a healthy human serum sample (see FIG. 3), the protein corresponding to the red spot was found in the cardiovascular serum more than the healthy human serum. It can be confirmed that the protein is present, the blue spot is the reverse case, the yellow case is a protein similarly present on both sides.

Example  3: analysis of produced information

After constructing a database using the GeneSpring (Agilent Technologies Inc. USA) program, 261 image information were produced, and the similarity of the whole images was compared by using the manual of the program and the patient image of cardiovascular disease (VD 29-2) was compared. Similarity analysis of the images in the database showed that 99.4% and 99.3% of the other images (VD 29-3, VD 29-1) were similar to those of patients with cardiovascular disease until the 16th (Fig. 4). Reference).

5 is a result of hierarchical clustering by comparing 620 spots that are significant by ANOVA with respect to the density of spots in the image information, and then comparing and analyzing the overall similarity of the 620 spots with the program. The calculated sensitivity was 90%, the selectivity was 100%, the positive predictive value was 100.00%, and the negative predictive value was 90.90% (see FIG. 6). ).

In addition, as a result of searching the efficient spots that can distinguish each group by algorithms such as k-means and self-organizing map (SOM), 21 spots were identified (see FIG. 7).

FIG. 8 shows 21 spots for patients with cardiovascular disease. The calculated sensitivity is 95.00%, the selectivity is 97.50%, the positive predictive value is 97.44%, and FIG. The negative predictive value was 95.12% (see Figure 9).

As a result of determining nucleotide sequences by standard methods by separating plasmids from the aptamer clones corresponding to 21 spots, two clones determined a total of 19 different DNA nucleotide sequences using the same nucleotide sequence. Complementary RNA aptamer base sequence according to the present invention was the same as the base sequence of SEQ ID NO: 1 to SEQ ID NO: 19.

Aptamers of SEQ ID NO: 1 to SEQ ID NO: 19 according to the present invention specifically binds to human serum proteins to distinguish cardiovascular diseases including angina pectoris and myocardial infarction to a significant level to examine patients of cardiovascular disease through serum analysis It is expected to be used for the purpose.

According to the single-stranded nucleic acid aptamer specifically binding to other serum proteins in the present invention described above, it is possible to diagnose a cardiovascular disease patient by detecting the specific binding and binding patterns of the aptamer and the serum protein of the present invention, It is expected to be able to diagnose patients with cardiovascular diseases more quickly, easily and accurately.

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Claims (2)

A single-stranded nucleic acid aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 19 that specifically binds to a serum protein. delete

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