KR101069590B1 - Single-stranded DNA aptamers specifically binding to CEA - Google Patents

Abstract

The present invention relates to aptamers comprising single stranded DNA that specifically binds to CEA. In the present invention, the single-stranded DNA that specifically binds to CEA is thought to allow the three-dimensional structure of the single-stranded DNA of SEQ ID NO: 1 to SEQ ID NO: 3 to achieve specific binding to CEA.
Biosensor using the DNA aptamer of the present invention can maintain the activity for a long time compared to the conventional diagnostic reagent consisting of a DNA sequence consisting of a DNA sequence, and does not change even in environmental conditions such as heat can be stored stably for a long time.

Description

Single-stranded DNA aptamers specifically binding to CEA}

The present invention is a DNA aptamer that has a high affinity for CEA so that it can be used in a biosensor that detects CEA by specifically binding to a DNA aptamer used in a biosensor, specifically CEA ( Carcinoembryonic antigen), which is widely known as a tumor marker. It is about.

In general, for diagnosis of a disease, it is necessary to detect a target biomolecule such as a specific protein, and it is known that a biosensor for detecting such a target biomolecule is used.

Conventional biosensors use aptamers to detect target biomolecules. Such an aptamer is a substance capable of binding to a specific target biomolecule, preferably any one or more selected from the group consisting of nucleic acids, amino acids, peptides, proteins, enzyme substrates, ligands, cofactors, antisenses, organic small molecules and carbohydrates. have. At this time, as the target biomolecule, a tumor marker such as CEA, AFP, or PSA, or a protein or small organic molecule is preferably used.

However, for detection of a target biomolecule, an attempt has been made to use an antibody antigen reaction with a target biomolecule, and for this purpose, an antibody that specifically binds to the target biomolecule has been used.

In general, antibodies are made up of proteins by substances that the mammalian immune system makes to fight external stimuli. Therefore, in order to obtain an antibody against a specific target biomolecule, an antibody is produced by artificially stimulating an animal cell, and the antibody thus produced must be separated through a separate separation process.

However, since the antibody is a protein, it is difficult to maintain uniform properties (activity), and has disadvantages of being vulnerable to heat or other environmental conditions. And, without affecting the protein structure, it was not easy to directly immobilize the reporter or add a functional group.

Accordingly, there is an attempt to use a nucleic acid that has high affinity with a target biomolecule and can be stably preserved for a long period of time, instead of an antibody, which is such a protein, as an aptamer.

In addition, since it is easy to add a reporter such as a fluorescent substance and various functional groups to such a nucleic acid, its usefulness as an aptamer has been confirmed.

Meanwhile, CEA ( Carcinoembryonic antigen ) is a widely known tumor marker and is mainly distributed in the digestive system and is overexpressed in endothelial cancers such as digestive, lung, thyroid, breast, prostate, and uterine cancer. Therefore, the measurement of the CEA concentration can be usefully used to diagnose the cancer diseases.

Currently, the concentration of CEA in blood is measured using a monoclonal antibody, but a nucleic acid sequence that favorably binds to CEA has not yet been found, and a biosensor using a nucleic acid has not yet been developed.

In order to solve the problems of the prior art as described above, the present invention provides a DNA aptamer using a nucleic acid sequence having a high affinity for CEA, which is a tumor marker.

Polynucleotide according to one feature of the present invention for solving the above problems is composed of the DNA sequence of SEQ ID NO: 1.

In addition, the polynucleotide according to another feature of the present invention is composed of the DNA sequence of SEQ ID NO: 2.

In addition, the polynucleotide according to another feature of the present invention is composed of the DNA sequence of SEQ ID NO: 3.

In addition, the aptamer for CEA according to another feature of the present invention includes a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1.

In addition, the aptamer for CEA according to another feature of the present invention includes a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2.

In addition, the aptamer for CEA according to another feature of the present invention includes a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3.

As described above, since the DNA of the present invention has excellent affinity with CEA, which is a tumor marker, it can be used as an aptamer in a biosensor for detecting CEA by specifically binding to CEA.

Accordingly, the biosensor using the DNA aptamer of the present invention can replace the conventional diagnostic reagent using an antibody at a lower cost. In addition, the biosensor using the DNA aptamer of the present invention can maintain its activity for a long period of time compared to a conventional diagnostic reagent consisting of a DNA sequence and consisting of a protein sequence, and can be stably stored for a long time because it does not change even under environmental conditions such as heat. Therefore, the biosensor using the DNA aptamer of the present invention is excellent in marketability and economy.

1 shows the binding affinity of the ssDNA of Table 1.
Figure 2 shows the three-dimensional predicted structure of the ssDNA of Table 1.

In the present invention, the SELEX method was used to select a polynucleotide of a DNA sequence that specifically binds to CEA.

By this method, the present inventors have identified polynucleotides of the DNA sequences shown in SEQ ID NOs: 1 to 3, which have high affinity with CEA, which is a tumor marker, and can be used as an aptamer.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments and the accompanying drawings so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

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Step 1: random DNA Library and Of the primer making

PCR was performed using a 5'- GAATTC GGGAGACAAGAATAAACGCTC-3' forward primer and a 5'-GCCTGTTGTGAGCCTCCTGTCG AAGCTT- 3' reverse primer, respectively. Here, among the underlined portions, GAATTC indicates the location where the EcoR I restriction enzyme, and AAGCTT indicates the location where the Hind III restriction enzyme will bind.

Through such PCR and asymmetric PCR, a single-stranded DNA (hereinafter, simply referred to as ssDNA) library consisting of 115 nucleotides such as 5'-GAATTCGGGAGACAAGAATAAACGCTC-N60-CGACAGGAGGCTCACAACAGGCAAGCTT-3' was prepared. Here, GAATTC at the 5'end is EcoR I restriction enzyme, and AAGCTT at the 3'end is Hind It is a part recognized by III restriction enzyme and is included for subsequent sequencing, and N60 in the center is a random sequence corresponding to a candidate for aptamer. Here, 27 nucleotides at the 5'end and 28 nucleotides at the 3'end are each used as a sequence in which a primer is located in PCR.

And, for asymmetric PCR, reverse primers were prepared by labeling with fluorescein at the 5'end.

Step 2: CEA Coated magnetism Bead Ready

Magnetic beads (Dynabeads M-270 Epoxy (Dynal Biotech, Norway) ^{were dispersed in 0.1 M sodium phosphate buffer at a concentration of about 1×10 9} beads/ml. The beads were reacted with CEA for 24 hours to form CEA on the bead surface. The concentration of CEA used herein was about 100 mg/ml of CEA solution so that the final concentration was 20 mg protein/mg beads.

After that, the magnetic beads to which CEA was bonded were separated using a magnet. The separated CEA-magnetic beads were again stored in PBS (Phosphate Buffer Saline) at 4°C containing 0.1% BSA (Bovine Serum Albumin) protein and 0.02% sodium azide at pH 7.4.

Step 3: SELEX Fair performance

10 μl of the DNA fragment (100 pmol/ml) of the single-stranded DNA library prepared in step 1 was reacted with 10 μl of CEA magnetic beads dispersed in a binding buffer (a mixed solution of 100 mM NaCl and 20 mM Tris-HCl, pH 7.6). . At this time, the DNA fragment was reacted for at least 30 minutes at 37°C so that it could form a three-dimensional structure and bind to CEA.

Then, the ssDNA-CEA magnetic bead complex was separated from the supernatant using a magnet. Thereafter, the separated ssDNA-CEA magnetic bead complex was washed 5 or more times with PBS buffer to remove ssDNA not bound to the beads.

Next, the ssDNA bound to the CEA beads was separated from the CEA beads as follows and then amplified.

First, treatment was performed at 94° C. for 10 minutes using the two types of primers of Step 1, and then 15 PCR cycles were repeated at 94° C. for 30 seconds and 60° C. for 30 seconds to obtain double-stranded DNA.

Thereafter, asymmetric PCR was performed using a reverse primer labeled with fluorescein at the 5'end and EX Taq polymerase. In this case, treatment was performed at 94°C for 4 minutes, followed by amplification at 94°C for 30 seconds and at 60°C for 30 seconds in 30 cycles.

Step 4: single strand DNA of Cloning And sequence confirmation

After performing step 3 for 6 cycles, the amplified single-stranded DNA was cloned into a T easy vector to confirm the sequence of the random portion of N60.

After that, the sequence of the random portion of N60 was confirmed using software ClusterW (http://www.ebi.ac.uk/Tools/clustalw2).

In addition, the three-dimensional structure of single-stranded DNA was also analyzed using the software mfold (www.bioinfo.rpi.edu/applications/mfold/), which is a minimum free energy algorithm.

Table 1 below shows the isolated single-stranded DNA sequences.

designation DNA sequence 1(A) GGTGTAGGT GTCGATGCGTGTCGGTGGTCTTTAGTA 1(B) GTCGATGCGTGTCGGTGGTCTTTAGTACGTAGTGGGTA CACGCTTGTTCT 56 GACTTGGAAG GA A GCG A GTC AC TGGCTTTAGTA 3 A T TG A TGAGAT C T G G A GG CCCT T G GT A GAT G A G GTG 59(A) A C TG T TGAGAT G T C G G GG ATTC T T GTGAT A A T GTG 59(B) TG T C T G C A G G GG TGAGAT ATTC GTGAT T T A A T GTG TGTGGAATCTCTCGG 5 GA TA AT GA G A CGCCTG GCG GGT T A C T T T G CGGA C G GA TC G TGT T C C T C G

As can be seen from Table 1 above, a high similarity was confirmed to the nucleotide sequence of the isolated ssDNA (in bold).

Step 5: For aptamer DNA Sequence identification

CEA affinity of the ssDNA disclosed in Table 1 was confirmed.

First, the binding of the CEA-coated magnetic beads and the ssDNA of Table 1 was measured at 525 nm using a fluorescence spectrometer.

Here, since the fluorescein-labeled single-stranded DNA floating in the supernatant without binding to the CEA-coated magnetic beads can be detected by the fluorescence signal, the binding strength of each aptamer candidate sequence can be confirmed by the intensity of the fluorescence signal.

The results of CEA affinity confirmation through this fluorescence spectrometer are shown in FIG. 1.

As can be seen in Fig. 1, of ssDNA, 01(A) (hereinafter referred to as SEQ ID NO: 1) showed the highest affinity, and 01(B) (hereinafter referred to as SEQ ID NO: 2) and 56 (hereinafter referred to as SEQ ID NO: 2) It was confirmed that the number 3) shows about 50% affinity compared to 01(A). In contrast, it can be seen that the other four sequences (3, 5, 59(A), 59(B)) hardly bind to CEA.

Figure 2 compares the expected three-dimensional structure of each single-stranded aptamer. It can be seen from FIG. 2 that the two-dimensional predicted structure of ssDNA of SEQ ID NO: 1 to SEQ ID NO: 3 is similar, and it is assumed that such a similar three-dimensional structure allows specific binding to CEA.

In the above, preferred embodiments of the present invention have been described, but the present invention is not limited thereto, and it is natural that it is possible to implement various modifications within the scope of the technical spirit of the present invention, and this also falls within the scope of the appended claims. Do.

Attach electronic file of sequence list

Claims (2)

Polynucleotide consisting of the DNA sequence of SEQ ID NO: 2. Carcinoembryonic, characterized in that the polynucleotide consisting of the DNA sequence of SEQ ID NO: 2 aptamers that specifically bind to antigen ).

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