KR101090982B1 - Bioanalysis using quantum dot-aptamer conjugate - Google Patents

Abstract

The present invention provides a new method of biochemical analysis using quantum dot-aptamer conjugates. The analytical method of the present invention allows the identification of targets using conjugates of aptamers and quantum dots to targets such as DNA, RNA, proteins, peptides or small molecules.

Quantum Dots, Aptamers, Quantum Dot-Aptamer Conjugates

Description

Bioanalysis using quantum dot-aptamer conjugates

The present invention relates to a new bioanalytical method using conjugates of quantum dots and aptamers. More specifically, the present invention relates to an analytical method using a quantum dot-aptamer conjugate, which can replace Southern, Northern, and Western blotting, which have been the main biochemical and molecular biological analysis methods. .

Aptamers are non-naturally occurring structured oligonucleotides that can bind DNA, RNA, small molecules, peptides, and proteins. In general, aptamers are produced by an in vitro selection process called systemic evolution of ligands by exponential enrichment (SELEX). Aptamers that bind with high affinity to proteins present on the cell surface have potential utility as therapeutic antagonists, agonists, and diagnostic agents. If the target protein required the presence of a cell membrane or co-receptor to properly fold, it was difficult or impossible to program the Cellex experiment with purified soluble protein targets. However, recent advances in the useful range of Selex have been extended from relatively simple purified forms of soluble proteins to complex mixtures of proteins in membrane preparations or protein mixtures in situ on the surface of living cells. It makes it possible to discover aptamers for targets that could not be tracked. In addition, applying aptamer selection techniques to complex protein mixtures is possible even without detailed knowledge and characterization of the target. Complex target Selex has enabled the isolation of potent and selective aptamers for disease determinants in pathogenic organisms as well as a variety of cell-surface proteins, including receptors and markers of cell differentiation, and thus have extensive therapeutic and diagnostic utility.

On the other hand, the quantum dot (QD) is a semiconducting nanoparticle, has a low attenuation effect due to external light energy, has a high quantum efficiency, and thus improves measurement sensitivity. Their unique optical advantages, including excitation spectra and access to various functionalizations to enable site-specific targeting, have been useful and selective materials for biomolecular labeling. The photo-physical properties of these quantum dots provide great advantages over the fluorescent organic dyes conventionally used.

Southern, Northern, and Western blotting assays, on the other hand, are the most frequently used and indispensable methods in biological research for the detection and quantification of specific DNA, RNA and proteins, respectively. Confirm the formation of the conjugates using DNA, RNA and antibodies, each of which selectively binds to the intended target.

The present inventors have used conventional Southern, Northern, and Western blotting analysis methods using conjugates that combine quantum dots and aptamers, which are attracting attention as useful and selective materials for biomolecular labeling because of their unique optical advantages. The present invention has been completed by developing a new analysis method that enables faster and more effective biochemical and molecular biological analysis.

The present invention aims to provide a new bioanalytical method that can replace the Southern, Northern, and Western blotting assay methods. More specifically, the present invention aims to provide a new method of biochemical analysis using quantum dot-aptamer conjugates.

The present invention provides a new method of biochemical analysis using quantum dot-aptamer conjugates. The analytical method of the present invention enables the identification of targets using conjugates of aptamers and quantum dots that bind to targets such as DNA, RNA, proteins, peptides or small molecules.

More specifically, the present invention

i) manufacturing a quantum dot,

ii) preparing an aptamer that binds to the target to be identified,

iii) preparing a quantum dot-aptamer conjugate,

iv) separating the mixture comprising the target by gel electrophoresis and transferring to a polymer membrane,

v) hybridization by adding the quantum dot-aptamer conjugate prepared in step iii) to the polymer membrane, and

vi) identifying hybrids by imaging analysis

It provides a bioanalytical method using a quantum dot-aptamer conjugate.

Prior to the detailed description of the method of the present invention, a brief review of conventional analytical methods such as Southern blotting, Northern blotting and Western blotting, first, Southern blotting is performed by a specific DNA in a DNA mixture separated by gel electrophoresis. To confirm the presence of, transfer from the gel to the nitrocellulose membrane and hybridize with a single strand of DNA probe labeled ³² P on the preserved nitrocellulose membrane. The probe hybridizes to the DNA sequence with the complementary sequence, which can then be identified by autoradiography to locate the DNA fragment with the base sequence complementary to the base sequence of the probe.

Northern blotting is an application of Southern blotting to confirm the presence of RNA with a specific base sequence. Hybridization of the RNA molecules by gel electrophoresis, transfer them to nitrocellulose membranes, and hybridization with probes with complementary sequences Specific RNA can be identified by doing so.

Western blotting is an immunoassay that detects specific proteins using specific antibodies, wherein the protein mixture is separated by gel electrophoresis and then transferred to a polymer membrane such as nitrocellulose or polyvinylidene difluoride (PVDF) and Antibodies specific for the protein are added to the polymer membrane to react with the protein of interest. The protein-antibody complex formed on the membrane is then allowed to form the complex with the first antibody by washing the membrane with a second antibody with a radiolabel specific to the first antibody and then identified by its location on autoradiography to identify the protein of interest. Done.

The above three methods have a common point in principle, such as blotting and using autoradiography, but for analyzing DNA, RNA, or protein, each of which has a radiolabeled DNA, RNA, or antibody. The difference is that you use. In contrast, the present invention distinguishes the formation of selective conjugates to DNA, RNA, or proteins by using quantum dot-aptamer conjugates in place of radiolabeled DNA, RNA or antibodies. .

That is, for example, in order to identify a specific DNA, an aptamer having a sequence complementary thereto is conjugated to a quantum dot, and then the conjugate is added to the polymer membrane to hybridize and confirmed through imaging analysis using an imaging system. Know the location of the DNA. Moreover, the use of aptamers that selectively bind to a particular small molecule can be used, thus the scope of the application is much broader.

In addition, when blotting requires identification of a specific protein, Western blotting requires two antibodies, whereas the method of the present invention utilizes a quantum dot-aptamer conjugate that selectively binds to that particular protein and is used for imaging analysis of quantum dots. Since the protein can be identified, there is an advantage that the position of a specific protein can be confirmed by forming a single complex.

While antibodies are often difficult to produce, they are expensive and often impossible to purchase. On the other hand, an in vitro selection process called Celex makes aptamers that selectively bind to almost any protein. It can be reproduced very easily using PCR or oligonucleotide synthesis, and conjugation to quantum dots has great advantages in terms of cost and application.

Also, since the quantum dots have different radiation ranges depending on the size, for example, if aptamer A is bonded to a small size quantum dot and aptamer B is bonded to a large size quantum dot to prepare two quantum dot-aptamer conjugates, The presence of two proteins can be confirmed at one time.

Thus, the analytical method of the present invention makes it possible to effectively identify targets in a short time and at low cost compared to conventional Southern blotting, northern blotting, and western blotting.

In the method of the present invention, the preparation of the quantum dots of the first step is synthesized nanomaterials such as CdS, ZnS, PbS, CdTe, etc. according to the conventional quantum dot manufacturing method, and then, such as SH, -OH, -COOH, -NH ₂ It is made by coating with an organic material having a functional group, or may be purchased from a chemical company such as Sigma-Aldrich, Invitro, etc., and there is no particular limitation on the quantum dots that can be used in the present invention. Those skilled in the art can appropriately select and use.

Preparation of the aptamer of the second step of the method of the present invention, according to the general aptamer production method, after determining and synthesizing the sequence of the oligonucleotide having a selective and high binding ability to the target to be confirmed, and then 5 ' It can be made by modifying to -SH, -COOH, -OH or -NH ₂ so that the end or 3 'end can be covalently bonded to the functional group of the quantum dot portion.

If the target to be identified is DNA or RNA, DNA or RNA having a sequence complementary to the target sequence can be used as an aptamer. If the target is a protein, peptide, or small molecule, SELEX, an in vitro selection method, is used. ) Can be used as an aptamer by selecting oligonucleotides that bind to the target.

The Selex method is an in vitro selection method for finding single-stranded DNA or RNA oligonucleotides that perform various types of selected functions.The Selex method allows ligands that selectively recognize various types of target molecules such as small molecules, peptides, and proteins by the Selex method. Aptamer). By the Selex method, aptamers with the desired function can be found from oligonucleotides of about 10 ¹⁵ random populations with different sequences, each oligonucleotide having a unique three-dimensional structure, For example, repetitive selection of oligonucleotides with selective recognition of the target molecule, and amplification of the selected oligonucleotides by traditional molecular biology methods. Through this repetitive selection and amplification process, ultimately, oligonucleotides that have a selective binding to the desired molecule or transition states to its chemical process occupy the majority of the population. The sequence of each aptamer obtained at the end of this process is identified. Kits that automate this Selex are also available (eg, Biomek 2000 pipetting robot, Beckman Coulter, USA).

Aptamers require modification at the 3 'end or 5' end for coupling with the quantum dots, and for such modifications, solid-phase oligonucleotide synthesis or conventional molecular biology methods can be used. In brief, first, the 5'-end modified nucleoside unit is in monophosphate form for molecular biological methods and the protected phosphamidate form for solid phase synthesis with 3'- or 5'-end modified. To synthesize. Such nucleoside units are commercially available from companies such as Sigma-Aldrich, Trilink Biotechnologies.

Subsequently, for nucleoside units (monophosphate forms) with modified 5 'ends, oligonucleotides are prepared under normal transcription conditions, provided that other normal triphosphates (i.e., ATP, CTP, GTP, And UTP) in an amount of 50 to 100 times as much as 5 'terminal modified nucleoside units (monophosphate form). This is because, under these conditions, when the 5 'terminal modified nucleoside unit enters the middle of the RNA, no further extension reaction occurs because it is monophosphate, not triphosphate. The longest RNA is isolated from the resulting mixture to isolate aptamers with modified 5 'ends.

In the case of the protected phosphoramidate form in which the 3 'end or 5' end is modified, an aptamer in which the 3 'end or 5' end is modified can be obtained by a solid-state oligonucleotide synthesis method.

In the present invention, a target may be any DNA, RNA, protein, peptide, or small molecule (eg, antibiotics, hormones, monosaccharides, cocaine, etc.), etc., to be determined for its presence, and the app for it. There is no limit to the target as long as it is possible to obtain a timer. In the current state of the art it is possible to manufacture aptamers having selective binding to almost all proteins and peptides and small molecules, as well as DNA, RNA, by Selex et al. In fact, there is no limit to the target of the present invention.

Junction of the quantum dot and the aptamer of the third step of the method of the present invention can be made using a general coupling method. That is, the coupling between -COOH and -OH, -SH or -NH ₂ is EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) or DCC (dicyclohexylcarbodiide). Through the formation of ester or amide bonds using a coupling agent such as mead) and the coupling between -SH and -SH can be obtained by the formation of disulfide bonds between two thiol groups. To summarize the general method, an aqueous solution of quantum dots with thiol surface and thiolated oligonucleotides is stirred under helium for about a day, then the NaCl concentration of the solution is changed to 0.24 M and the concentration of phosphate buffer is about 0.1 M. Dialysis of the mixture with 0.2 M NaCl, 0.1 M phosphate buffer (pH 7.4), 0.01% sodium azide solution allows coupling of quantum dots with thiolated aptamers and thiol surfaces. Chemicals other than unreacted quantum dots can be easily removed by methods such as membrane filtration.

Separating the mixture comprising the target, which is the fourth step of the method of the present invention, by gel electrophoresis and transferring to the polymer membrane is performed by separating the DNA, RNA, or protein, and transferring the polymer membrane into a polymer membrane such as nitrocellulose. It can be easily carried out by those skilled in the art by a general method. Such gel electrophoresis and the like are described, for example, in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.

Hybridization by adding the quantum dot-aptamer conjugate prepared in step 3, which is the fifth step of the method of the present invention, to the polymer membrane may be performed by using a target molecule in the polymer membrane in general Southern blotting, northern blotting, western blotting, and the like. For identification, the same process as hybridization by adding a DAN probe or an antibody to the polymer membrane, for example, hybridization between the conjugate and the target by contacting the polymer membrane with a solution containing the quantum dot-aptamer conjugate for a predetermined time. May be used, and may be easily made by those skilled in the art.

Identifying hybrids by imaging analysis, a sixth step of the method of the present invention, can be accomplished using conventional imaging systems used for imaging analysis of quantum dots, an example of such an imaging system being the LAS-3000 imaging system ( Fuji Film), Macro-imaging system (Lightools Research, USA).

In addition, the method of the present invention can identify two or more targets in one analysis by differentiating the size of the quantum dots bonded to the aptamer for each target when two or more targets to be identified. Therefore, the number of targets which can be confirmed by one analysis by the analysis method of the present invention is not particularly limited.

The bioassay method using the quantum dot-aptamer conjugate of the present invention enables identification of DNA, RNA, proteins, peptides as well as small molecules, and the quantum dots without the need to use two antibodies to identify specific proteins. It is possible to identify proteins by using only aptamer conjugates, and also to identify two or more targets in one analysis using two or more quantum dots having different radiation ranges, and thus, the conventional analysis method in terms of efficiency and application range. Superior to

Hereinafter, the present invention will be described in more detail with reference to Examples. It is apparent to those skilled in the art that these examples are only for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  One. Quantum dot  Produce

To prepare quantum dot nanomaterials, a water-in-oil microemulsion of AOT / n-heptane was mixed with 4 mL of water and 200 mL of n-heptane, 14.0 g of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) After preparing, divided into 120 mL and 80 mL. 0.48 mL of 1.0 M heavy metal salt solution (Zinc, Cadmium, Gallium, Iridium, Lead, or Mercury AA-grade standard solutions available from Sigma-Aldrich) was added to 120 mL prepared above, and 0.32 mL of 1.0 M Na ₂ S solution. Was added to the 80 mL prepared above, and the two solutions were mixed. Stir under helium for 1 hour.

After obtaining the quantum dot nanomaterial as described above, referring to Bioconj. Chem. 2007, 18, 829-835, 0.66 mL of 0.32 M cystamine and 0.34 mL 0.32 M 2-sulfanylethane sulfone After mixing with acid, the mixture was stirred under helium for one day, the solvent was removed under vacuum, and then washed with pyridine, n-heptane, acetone, and methanol to prepare a quantum dot having a thiol surface. And 50 mM borate of 5 microM thiolated quantum dots, 45 microM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 9 microM poly (ethylene glycol) (DAPEG) After mixing in a buffer (pH 8.5) solution, the mixture was stirred for 2 hours, and then DAPEG-polymer coated quantum dots (DAPEG-PC-QD) were prepared by removing EDC and DAPEG which did not occur by membrane filtration.

Example  2. Aptamer  Produce

Aptamers prepared aptamers I and II which selectively bind to histidine tags, but differ in binding strength, with reference to Nucleic Acids Research, 2003, 31, e110. After preparing a guanine monophosphate (Mod-GMP) modified at 5'-end with-(CH ₂ ) ₅ COOH, adding 100-fold excess of the amount of other normal nucleotides for transcription reaction, and then 5 ' Aptamers with modified ends were prepared.

Example  3. Quantum dots Aptamer  Coupling

1.5 nmol of DAPEG-PC-QD, 13.5 nmol of EDC, and 13.5 nmol of aptamer were mixed in a 50 mM solution of borate buffer (pH 8.5), stirred for 2 hours, and then no reaction occurred by membrane filtration. EDC and DAPEG were removed and washed with n-heptane, acetone and methanol to prepare a quantum dot-aptamer conjugate.

Example 4. Quantum dots - Aptamers  With conjugate Of target  Confirm

In order to examine the specificity and selectivity of the obtained quantum dot-aptamer conjugate and the protein having His-tag, E. E. coli were incubated (see J. Cell Biol. 2003, 163, 71-81).

The cultured cells were totally obtained by general molecular biology methods, namely cell lysis, repeated centrifugation, resuspension, and sonication in cold phosphate-buffered saline (PBS) with 1 mM phenylmethanesulfonyl fluoride. Lysates were separated by 12% SDS-PAGE. Protein present in the gel is transferred to a polyvinylidene difluoride (PVDF) membrane using a semi-minor electroblotter (Trans-Blot SD) and the polymer membrane is transferred to a blocking buffer (10 mM Tris). Incubated with 5% skim milk, 0.15 M NaCl, and 0.1% Tween 20 solution in HCl (pH 7.0) at room temperature for 1 hour, the polymer membrane was 10 mM Tris-HCl (pH 7.0), 0.15 M NaCl, and After washing with 0.1% Tween 20 solution, the obtained polymer membrane was mixed at 5% bovine serum albumin, 0.15 M NaCl, and 0.1% Tween 20 solution in quantum dot-aptamer conjugate and 10 mM Tris-HCl (pH 7.0) at room temperature. Gently shake for 30 minutes at. The polymer membrane was then washed with 10 mM Tris-HCl, pH 7.0, 0.15 M NaCl, and 0.1% Tween 20 solution. Imaging was taken after exposure for two seconds using the LAS-3000 imaging system (Fujifilm). The results are shown in FIG.

The left side of FIG. 1 is a photograph stained with Coomassie blue after SDS-PAGE of a cell lysate, and the middle photograph is a photograph obtained by hybridizing with a quantum dot-aptamer I conjugate and imaging. Pictures obtained by hybridization with quantum dot-aptamer II conjugates. Both conjugates containing aptamer I and conjugates containing aptamer II can also identify proteins with a small amount of 500-ng His-tag, and that aptamer II binds to histidine tags as compared to aptamer I. This strength can be seen well in FIG.

1 is a photograph showing an analysis result by the analysis method of the present invention using a quantum dot-aptamer conjugate.

Claims (5)

i) manufacturing a quantum dot, ii) preparing an aptamer that binds to His-tag, iii) preparing a quantum dot-aptamer conjugate, iv) separating the mixture comprising the protein with His-tag by gel electrophoresis and transferring to the polymer membrane, v) hybridization by adding the quantum dot-aptamer conjugate prepared in step iii) to the polymer membrane, and vi) identifying hybrids by imaging analysis A method of bioanalyzing a protein having a His-tag using a quantum dot-aptamer conjugate. delete delete The method of claim 1, wherein the aptamer is selected by the SELEX method using the quantum dot-aptamer conjugate to bioanalyze a protein with His-tag. delete

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