KR101515723B1 - Composition for detecting or quantitative analysis comprising aptamer tagged by fluorescent substance with fluorescence resonance energy transfer - Google Patents

Abstract

The present invention relates to: a composition including an aptamer labeled with a fluorescent material inducing FRET for quantitatively analyzing a target material; a kit including the same composition for quantitatively analyzing a target material; a method for quantitatively analyzing a target material using the same composition and the same kit; a composition for including an aptamer labeled with a fluorescent material inducing FRET for detecting a target material; a kit including the same composition for detecting a target material; and a method for detecting a target material using the composition and the kit. When the composition of the present invention is used, existence of the target material in a sample or a level of the target material included in the sample can be identified at a fmol level. Due to characteristics of the aptamer, the composition can be produced and stored in an easy manner. The composition is expected to be applied to detect or to quantitatively analyze the target material more effectively.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for detecting or quantitatively analyzing a target substance comprising aptamer having a fluorescent substance that induces a fluorescence resonance energy transfer phenomenon.

The present invention relates to a composition for detecting or quantitatively analyzing a target substance containing a fluorescent substance-labeled aptamer that causes a fluorescence resonance energy transfer (FRET) phenomenon. More specifically, , A kit for quantitative analysis of a target substance comprising the composition, a method for quantitatively analyzing a target substance using the composition or kit, a method for quantitatively analyzing a target substance using FRET phenomenon A kit for detecting a target substance comprising the composition, and a method for detecting a target substance using the kit or the composition.

Aptamers are a special class of single-stranded nucleic acids (DNA, RNA or modified nucleic acids) that, by themselves, have a stable tertiary structure and are capable of binding to target molecules with high affinity and specificity. Aptamers can be obtained through the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process, a random sequence nucleic acid library. The aptamer is thought to be a good alternative to antibodies, and many aptamers are known to bind specifically to metal ions and small chemical molecules, proteins, and even cells to have a dissociation constant at the nanomole level of picomoles . In addition, aptamers have the following advantages over specific antibodies in comparison to antibodies: The first feature is that the aptamer can be obtained from a nucleic acid library that is designed to target certain molecules (from small inorganic ions to cells). This feature makes it possible to overcome limitations of antibodies that must be obtained from cells or animals. The second feature is that the selected aptamer can be amplified through a PCR (Polymerase chain reaction) process or can be transcribed to obtain a large amount of aptamers having high purity. The third feature is that the aptamer has a relatively simple chemical structure and is therefore susceptible to change its functionalities when it is intended to be used for other purposes such as immobilization on a solid surface. Finally, because the aptamer is much more stable than the antibody, it can be used in chemical applications requiring tougher conditions (even at higher temperatures or extremes of pH). In addition, aptamers can be chemically synthesized in large quantities, which are economical, have a target affinity comparable to that of antibodies, and are significantly smaller in size (about 1 to 2 nm) than antibodies.

Due to the superior effect of the aptamer over such antibodies, many studies have been conducted to develop a biosensor for a specific molecule using an aptamer. For example, Korean Patent Laid-Open Publication No. 2008-0084029 discloses a method of detecting a target protein using a fluorescently labeled aptamer and a kit used therefor. In Korean Patent Laid-Open Publication No. 2011-0126942, There is disclosed a biochip capable of detecting an electrochemical signal including a substrate provided with a working electrode on which an aptamer that is bonded to a target substance can be immobilized, and a method of using the same, and Korean Patent Laid-Open Publication No. 2013-0087274 Discloses a method for detecting a target protein using an aptamer and a coumarin blue solution bound to a substrate.

However, these techniques have been disadvantageous in that they use only aptamer as a simple binding medium and can not utilize an aptamer which has a distinct advantage over conventional antibodies. For example, in the case of an ELISA kit, which is a representative assay kit using an antibody, a label is bound to an antibody. When a labeling substance is directly bound to the antibody, there arises a problem that the structure or function of the antibody is altered to deteriorate detection sensitivity. Therefore, as a method of binding a labeling substance to an antibody, Is conjugated with streptavidin, and the antibody and the labeling substance are indirectly bound to each other through binding of biotin and streptavidin. When an ELISA kit is prepared by such a method, there is a problem that it takes a lot of time to manufacture and manufacture. This problem occurs because the antibody is essentially used. Therefore, a method of replacing such an antibody with an aptamer is being studied. However, there is no report yet on the method of using an aptamer as an alternative to an antibody.

Under these circumstances, the present inventors have made intensive researches to utilize the advantages of the aptamers distinguished from the conventional antibodies, and as a result, they have found that by using a fluorescence resonance energy transfer (FRET) phenomenon, When a composition including a fluorescent substance-labeled target substance and a fluorescent substance that serves as an acceptor is labeled and an aptamer capable of binding to the target substance is used, And the target substance can be detected or quantitatively analyzed from the sample. Thus, the present invention has been completed.

It is an object of the present invention to provide a composition for quantitative analysis of a target substance, which comprises an aptamer having a fluorescent substance labeled to cause a FRET phenomenon.

Another object of the present invention is to provide a kit for quantitative analysis of a target substance comprising the above composition.

It is still another object of the present invention to provide a method for quantitatively analyzing a target substance using the composition or kit.

It is still another object of the present invention to provide a composition for detecting a target substance comprising aptamer having a fluorescent substance labeled to cause a FRET phenomenon.

It is still another object of the present invention to provide a kit for detecting a target substance comprising the composition.

It is another object of the present invention to provide a method for detecting a target substance using the composition or kit.

In order to achieve the above object, the present invention provides a composition for quantitative analysis of a target substance comprising an aptamer having a fluorescent substance labeled to cause a FRET phenomenon.

Specifically, the composition for quantitative analysis of a target substance of the present invention comprises: (a) a target substance labeled with a first fluorescent substance that acts as a donor or an acceptor to cause a fluorescence resonance energy transfer (FRET) phenomenon; And (b) an aptamer to which a second fluorescent material capable of reacting with the first fluorescent material to cause a FRET phenomenon is combined, and capable of binding with the target substance.

The present inventors paid attention to a target substance detection method using fluorescence resonance energy transfer (FRET) phenomenon while performing various studies in order to utilize the advantages of aptamers different from conventional antibodies.

FRET refers to a phenomenon in which energy is transferred by resonance when two fluorescent materials are in close proximity. A fluorescent material that transfers energy among two fluorescent materials is called a donor, and a fluorescent material that receives energy is called an acceptor acceptor), the absorption and emission wavelength values of the donor are smaller than the absorption wavelength values of the acceptor, the emission wavelength values of the donor are similar to the absorption wavelength values of the acceptor, 10 nm. ≪ / RTI > When the donor and the acceptor are irradiated with light having an absorption wavelength of the donor, the donor absorbs the irradiated light and emits fluorescence. However, since the acceptor wavelength and the wavelength of the irradiated light are different The fluorescence is weakly emitted. Under such conditions, when the donor approaches the acceptor, the fluorescence of the emission wavelength generated from the donor acts as the light of the absorption wavelength of the acceptor and is absorbed by the acceptor, and the acceptor emits light of the wavelength emitted from the donor Absorbed and emit fluorescence again. Therefore, fluorescence of the donor is detected when the donor and the acceptor are isolated from each other under the condition of irradiating the light of the absorption wavelength of the donor. When the donor and the acceptor are approached or combined, fluorescence of the acceptor is detected.

The present inventors first developed a method for detecting and quantitatively analyzing whether a target substance and an aptamer are bound through a competitive binding by applying a FRET phenomenon to an aptamer that binds to a target substance and the target substance. That is, the donor or acceptor fluorescent substance is labeled on the target substance, the pair of the acceptor or the donor is labeled on the aptamer, and the target substance and the aptamer are immobilized under the condition of irradiating the light with the absorption wavelength of the donor When the fluorescence emitted from the donor is detected, it can be determined that the target substance and the aptamer are not bound. When the fluorescence emitted from the acceptor is detected, the target substance and the aptamer It can be judged to be combined.

The inventors of the present invention firstly proposed a method for quantitatively analyzing a target substance present in a sample through competitive binding with a target substance which is not labeled in the sample using a labeled target substance and an aptamer, . For example, in the condition of irradiating the light with the absorption wavelength of the donor, the target substance labeled with the acceptor fluorescent substance and the unlabeled target substance having the serial concentration are added to the aptamer labeled with the donor fluorescent substance, A standard curve is prepared by measuring the level of fluorescence emitted from the fluorescent material, and then the target substance labeled with the donor fluorescent substance and the target substance are included in the aptamer labeled with the acceptor fluorescent substance under the condition of irradiating the light with the absorption wavelength of the donor The level of emitted fluorescence of the donor fluorescent material is measured and the level of the measured emitted fluorescence is applied to a standard curve to quantify the target substance contained in the sample. Such a quantitation method can be performed because the target substance labeled with the donor fluorescent substance and the target substance contained in the sample competitively bind to the aptamer. The more the target substance contained in the sample, the more the concentration of the donor fluorescent substance By binding more to the aptamer than to the labeled target material, the rate of inhibiting the FRET phenomenon increases, resulting in an increase in the level of emitted fluorescence from the donor. Conversely, as the number of target substances contained in the sample decreases, the proportion of generating the FRET phenomenon is increased by bonding the donor fluorescent substance to the aptamer less than the target substance labeled with the constant concentration. As a result, . As a specific example, an HBV surface antigen (HBsAg) labeled with an acceptor fluorescent substance Cy5 and an anti-HBsAg aptamer labeled with a donor fluorescent substance Cy3 are reacted under the condition of irradiating light having a Cy3 absorption wavelength, When HBsAg was added at different concentrations and the change in the levels of Cy3 and Cy5 emission fluorescence was measured, the level of Cy3 emission fluorescence as a donor phosphor increased as the concentration of unlabeled HBsAg increased, and the acceptor fluorescence Cy5 The level of emitted fluorescence decreases. It was analyzed that the unlabeled HBsAg appeared to interfere with the binding between the donor fluorophore (aptamer) and the acceptor fluorophore (HBsAg), isolating Cy3 and Cy5.

Therefore, a target substance labeled with a first fluorescent substance that acts as a donor or an acceptor that causes a FRET phenomenon and a second fluorescent substance that reacts with the first fluorescent substance to cause a FRET phenomenon are bonded, The use of a composition for detecting a target substance including an aptamer capable of binding to a target substance enables to confirm whether or not the target substance is present in the sample and quantitatively analyze the target substance contained in the sample .

As described above, the composition for quantitative analysis of a target substance provided in the present invention uses an aptamer in place of an antibody, and a fluorescent substance capable of inducing FRET in a target substance capable of specifically binding to the aptamer and the aptamer, Lt; / RTI > In order to obtain a composition exhibiting the same effect using an antibody instead of an aptamer, a donor or an acceptor fluorescent substance should be labeled on the antibody. Usually, since the fluorescent substance is labeled by a chemical reaction, There is a risk that the structure of the antibody may be damaged by the chemical reaction, and the binding specificity with the antigen may be damaged. However, since the aptamer of the present invention is more stable than the antibody and has excellent resistance to the chemical reaction, the binding specificity to the target substance can be maintained even after labeling the fluorescent substance. It can be said that it is a new technology which can not be achieved by using it.

The term "aptamer " of the present invention refers to a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid having a stable tertiary structure per se and capable of binding to a target substance with high affinity and specificity ). ≪ / RTI > As described above, since the aptamer is composed of a polynucleotide which can specifically bind to a target substance and is more stable than the protein, has a simple structure, and is easy to synthesize, the fluorescent substance is labeled and the composition for detecting the target substance As shown in FIG.

In the present invention, the aptamer may be an aptamer capable of binding to a target substance, but is not limited thereto. For example, the aptamer may be an aptamer labeled with a fluorescent substance and capable of binding with a target substance, If the substance is a hepatitis B virus, it may be an aptamer labeled with a fluorescent substance and capable of binding to the surface antigen of the hepatitis B virus. For example, it could be the aptamer of SEQ ID NO: 1 labeled with Cy3, and the quantitative analysis effect of the aptamer labeled with Cy3 was confirmed in a specific example.

The term "target" of the present invention means a substance that exists in a sample and is subjected to quantitative analysis or detection by binding to an aptamer. Materials that can be quantitatively analyzed or detected by binding to an aptamer include, without limitation, proteins, nucleic acids, compounds, and the like.

In the present invention, the target substance is not particularly limited as long as it can bind to an aptamer, but it may be a proteinaceous substance capable of binding to an aptamer. HBsAg, a surface antigen of hepatitis B virus, was used as a target substance in a representative example of the present invention. As a result of applying the method of the present invention to quantitatively analyze the surface antigen of hepatitis B virus as a target substance, the present inventors have found that the surface antigen of hepatitis B virus can be quantitatively analyzed easily Respectively.

The term "fluorescent substance" in the present invention means a substance that emits light in a detectable region by absorbing light of a specific wavelength. The term " detectable region " means an area capable of quantifying the level of light emitted by the naked eye or using various measuring instruments.

In the present invention, the fluorescent material may be respectively labeled with a target substance and an aptamer capable of specifically binding to the target substance, and can be interpreted as a substance capable of causing a FRET phenomenon. The fluorescent substance to be induced is not particularly limited, but preferably fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxydodamine, carboxyrotamine 6G, carboxy (Cascade Blue), Cascade Yellow, Comarine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, Picoitrin, PerCP NED, ROX (5- (-) - N, N'-tetramethyluronium hexafluorophosphate), PerCP-Cy5.5, JOE 6-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Floor 430, Alexa Floor 488, Alexa Floor 532, Alexa Floor 546, Alexa Floor 568, Alexa Floor 594, Alexa Floor 633, Alexa Floor 647, Alexa Floor 660, Alexa Floor 680, 7-amino-4-methylcomarine-3-acetic acid, BODIPY FL, , Body pips 558/568, body pips 564/570, body pips 576/589, body pips 581/591, body pippers 630/650, body pippers 650/665, body pippers R6G, body pippers TMR, A pair of fluorescent materials having an absorption wavelength and an emission wavelength that are similar to each other may be selected from among the fluorescent materials. For example, fluorescent material pairs such as Cy3-Cy5 and Alexa Floor 488-Alexa Floor 532 can be used. A representative example is Cy3 as an donor fluorescent material and acceptor fluorescence It was confirmed that the method provided by the present invention by using a Cy5 as a quality can be carried out normally.

In addition, for more accurate quantitative analysis, BSA for use as an internal control group, and target substances by concentration necessary for preparing a standard curve may be included singly or in combination.

According to one embodiment of the present invention, a DNA polynucleotide (SEQ ID NO: 5) for obtaining an anti-HBsAg RNA aptamer (SEQ ID NO: 5) was prepared (Example 1), and RNA aptamers SEQ ID NO: 1) was synthesized (Example 2), Cy3 labeled with a donor fluorescent substance was labeled with the synthesized aptamer to obtain an aptamer labeled with Cy3 (Example 3). In addition, a hepatitis B virus surface antigen (HBsAg) labeled with an acceptor fluorescent substance Cy5 was obtained (Example 4). Then, the above-obtained Cy3-labeled aptamer, the obtained Cy5-labeled HBsAg and the unlabeled HBsAg / BSA (bovine serum albumin) were assayed by BSA by the labeled aptamer and HBsAg If the FRET phenomenon occurring is not interfered but the FRET phenomenon caused by HBsAg and HBsAg is interfered with the labeled aptamer and HBsAg and the labeled aptamer of the present invention and the labeled target substance are used, It was confirmed that the target substance could be detected at the fmol level (Fig. 2). Finally, using the obtained Cy3-labeled aptamer, the obtained Cy5-labeled HBsAg, and the unlabeled HBsAg, the donor fluorophore (aptamer) and the acceptor fluorophore (HBsAg). As a result, as the level of unlabeled HBsAg increases, the level of Cy3 emission fluorescence as a donor phosphor increases and the level of Cy5 emission fluorescence as an acceptor fluorescence decreases , It was found that the target substance could be quantitatively analyzed to the fmol level using a composition comprising the labeled aptamer of the present invention and the labeled target substance (Fig. 3).

In another embodiment, the present invention provides a kit for quantitative analysis of a target substance comprising the above composition.

The kit of the present invention can be used for quantitatively analyzing the level of a target substance from a sample containing a target substance, but is not limited thereto. However, the first fluorescent substance serving as a donor or an acceptor causing the FRET phenomenon, And a second fluorescent material that reacts with the first fluorescent material to induce a FRET phenomenon and is capable of binding to the target substance, as well as one or more other component compositions suitable for the assay method , Solutions or devices.

As a specific example, the kit for detecting a target substance of the present invention may be a kit in which the labeled aptamer is bound to a support such as a substrate or a bead.

The term "substrate" of the present invention means a support capable of attaching an aptamer. The type of the substrate is not particularly limited as long as it can fix an aptamer commonly used in the art, but it is preferably glass, alumina, ceramic, carbon, gold, silver, copper, aluminum, . The substrate can be surface treated to bind an aptamer, which can be functionalized to include a functional group for immobilizing the aptamer, and is preferably modified with an aldehyde group, a carboxyl group, an amine group, or a thiol group . For example, when a glass substrate or a semiconductor substrate is used, an amino group (-NH 3, -NH 2, or the like) or a thiol group can be formed by silane treatment. In order to effectively perform the silane treatment, (hydroxyl) (- OH) group.

The term "bead " of the present invention means a support which can form a spherical shape as a kind of a support capable of attaching an aptamer. The kind of the beads is not particularly limited as long as it can fix an aptamer commonly used in the art. Preferably, the beads include glass, alumina, ceramic, carbon, gold, silver, copper , Aluminum, compound semiconductors, and silicon as well as inorganic compound materials such as polyethylene, polypropylene, polyvinyl compounds and vinyl chloride compounds, or organic compound materials such as Sepharose and Sephadex. The beads may be surface treated to bind an aptamer as in the substrate described above.

The beads can increase the reactivity of the aptamer as compared with the substrate. Unlike the substrate fixed at one place, the beads actively move in the reaction solution to more effectively bind the target substance and the aptamer.

In addition, the kits of the present invention can be used in combination with test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), RNAse inhibitors, DEPC- water, sterile water, and the like. Further, for more accurate quantitative analysis, BSA for use as an internal control group, and target substances by concentration required for preparing a standard curve can be included singly or in combination.

In another embodiment, the present invention provides a method for quantitatively analyzing a target substance from a sample using the composition for quantitative analysis of the target substance or a kit for quantitative analysis of the target substance.

Specifically, the target substance quantitative analysis method of the present invention comprises the steps of: (a) simultaneously irradiating light of an absorption wavelength of a donor fluorescent substance with a target substance labeled with the labeled aptamer and a sample to be tested for the presence of the target substance ; And (b) measuring the level of emission fluorescence of the donor fluorescent material or the acceptor fluorescent material from the reactant. At this time, a standard curve is prepared by using the level of the emitted fluorescence measured from the target substance instead of the sample in the composition or kit, and the level of emitted fluorescence measured in the step (b) Curve to quantify the level of the target material contained in the sample. Further, for more accurate detection, as described in the above-described target substance detection method, the target substance labeled with the labeled aptamer is added while reacting with light having an absorption wavelength of a donor fluorescent substance, The level of the emitted fluorescence of the donor fluorescent material is used as an internal control, or the target substance labeled with the labeled aptamer and BSA not binding to the aptamer are irradiated while the light of the absorption wavelength of the donor fluorescent substance is irradiated And then using the level of emitted fluorescence of the donor fluorescent substance measured in the reaction as an internal control.

In another embodiment, the present invention provides a composition for detecting a target substance comprising aptamer with a fluorescent substance that causes a FRET phenomenon.

Specifically, the composition for detecting a target substance of the present invention comprises: (a) a target substance having a first fluorescent substance labeled to perform a donor or an acceptor function to cause a fluorescence resonance energy transfer (FRET) phenomenon; And (b) an aptamer to which a second fluorescent material capable of reacting with the first fluorescent material to cause a FRET phenomenon is combined, and capable of binding with the target substance. At this time, the fluorescent substance, the target substance, the aptamer, and the like are the same as described above.

If the target substance that generates the FRET phenomenon is labeled and the aptamer is used, it can be determined whether the target substance exists in the sample. For example, in the condition of irradiating the light of the absorption wavelength of the donor, the target substance labeled with the donor fluorescent substance and the sample expected to have the target substance are added to the aptamer labeled with the acceptor fluorescent substance, It is possible to determine that a target substance capable of binding to the aptamer competitively with the labeled target substance is present in the sample, and it is generally determined that the fluorescence emitted from the acceptor When fluorescence is detected, it can be determined that a target substance capable of binding to the aptamer competitively with the labeled target substance is not present in the sample. If fluorescence emitted from the donor and fluorescence emitted from the acceptor are detected together, It can be judged that a smaller amount of the target substance exists than the target substance labeled on the sample (FIG. 1). FIG. 1 is a schematic diagram showing a principle in which a fluorescence resonance energy transfer (FRET) phenomenon caused by binding of a labeled aptamer with a labeled target substance is changed by an unlabeled target substance.

As described above, the labeled target material and the unlabeled target material derived from the sample can be competitively bound to the labeled aptamer. If a target substance exists even in a small amount in the sample, the labeled target material can be competitively labeled The emitted fluorescent light of the donor fluorescent substance can be detected at a level higher than that of the emitted fluorescent light of the donor fluorescent substance which is not generated by binding of the target substance labeled with all the labeled aptamers have. Therefore, by using the composition for detecting a target substance of the present invention, it is possible to detect whether or not a target substance exists in the sample.

In another embodiment, the present invention provides a kit for detecting a target substance comprising the above composition.

The kit of the present invention can be used to detect a target substance from a sample to be inspected whether or not it contains a target substance, and includes, but is not limited to, a first fluorescent material that acts as a donor or an acceptor that causes the FRET phenomenon A target substance to which the substance is labeled and a second fluorescent substance which reacts with the first fluorescent substance to induce a FRET phenomenon are combined and the aptamer capable of binding with the target substance as well as one or more other Component compositions, solutions or devices may also be included.

As a specific example, the kit for detecting a target substance of the present invention may be a kit in which the labeled aptamer is bound to a support such as a substrate or a bead. At this time, the substrate and the bead are the same as those described above.

The kits of the present invention can be used in combination with test tubes or other appropriate containers, reaction buffers (pH and magnesium concentrations vary), RNAse inhibitors, DEPC-water, sterilized And the like. Also, for more accurate detection, it may include BSA that does not bind to the aptamer for use as an internal control and does not affect the level of the measured emitted fluorescence.

In another embodiment, the present invention provides a method for detecting a target substance from a sample using the kit for detecting a target substance or the kit for detecting a target substance.

Specifically, the target substance detection method of the present invention comprises the steps of: (a) simultaneously irradiating light of an absorption wavelength of a donor fluorescent substance with a target substance labeled with the labeled aptamer and a sample to be tested for the presence of a target substance, ; And (b) detecting emission fluorescence of the donor fluorescent substance or the acceptor fluorescent substance from the reactant. At this time, if the emission fluorescence of the donor fluorescent material is not detected, it is determined that the target substance does not exist in the sample. If the emission fluorescence of the donor fluorescent material is detected, it is determined that the target substance exists in the sample can do. In addition, for more accurate detection, the labeled target substance labeled with the labeled aptamer is reacted while irradiating light having an absorption wavelength of the donor fluorescent substance, and the level of emitted fluorescence of the donor fluorescent substance measured in the reactant is measured by an internal control Or a substance labeled with the labeled aptamer and a substance such as BSA that does not bind to the aptamer is added while irradiating light having an absorption wavelength of the donor fluorescent substance, and the donor fluorescent substance measured in the reaction Lt; RTI ID = 0.0 > fluorescence < / RTI > It is possible to determine whether the emission fluorescence level of the donor fluorescent substance obtained by treating the sample based on the level of the emitted fluorescence of the donor fluorescent substance measured as the internal control group is increased.

By using the composition for detecting or quantifying a target substance of the present invention, it is possible to confirm whether or not the target substance exists in the sample or the level of the target substance contained in the sample at the fmol level, and it is easy to manufacture and store due to the nature of the aptamer Therefore, it can be used to detect or quantitatively analyze a target substance more effectively.

FIG. 1 is a schematic diagram showing a principle in which a fluorescence resonance energy transfer (FRET) phenomenon caused by binding of a labeled aptamer with a labeled target substance is changed by an unlabeled target substance.
FIG. 2 is a graph showing that the FRET reaction of Cy5 bound to hepatitis B virus surface antigen (HBsAg) and Cy3 bound to an aptamer is specifically inhibited by unlabeled HBsAg.
FIG. 3 is a graph showing that the results of the FRET reaction of Cy5 bound to the hepatitis B virus surface antigen (HBsAg) and Cy3 bound to the aptamer are changed in a concentration-dependent manner by the unlabeled HBsAg.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: anti- HBsAg RNA AppTamer  For obtaining DNA  Production of polynucleotides

PCR was performed using the DNA polynucleotide of SEQ ID NO: 2 shown below as a template and the primers of SEQ ID NOS: 3 and 4 to obtain the DNA polynucleotide of SEQ ID NO: 5 for obtaining anti-HBsAg RNA aptamer. The PCR conditions were 95 ° C for 30 seconds, 30 cycles (95 ° C for 15-30 seconds, 45-68 ° C for 60 seconds, 68 ° C for 1 minute) and 68 ° C for 5 minutes. The PCR product thus obtained was purified by ethanol precipitation.

Template: 5'-GTATGTGGGC TGAACTCAAT CAGGTCCCAA TCCCCAACAT ACACATGACC CGTCGTTTAC GATCATTATA GACGGCCATG ATTGACACGC AATCAACCCC CTATAGTGAG TCGTATTA-3 '(SEQ ID NO: 2)

Forward primer: 5'-GTATGTGGGCTGAAC-3 '(SEQ ID NO: 3)

Reverse primer: 5'-TAATACGACTCACTA-3 '(SEQ ID NO: 4)

5'-TAATACGACT CACTATAGGG GGTTGATTGC GTGTCAATCA TGGCCGTCTA TAATGATCGT AAACGACGGG TCATGTGTAT GTTGGGGATT GGGACCTGAT TGAGTTCAGC CCACATAC-3 '(SEQ ID NO: 5)

Example  2: RNA  synthesis

RNA was synthesized from the PCR products obtained using GSMP (5'-deoxy-5'-thioguanosine-5'-monophosphorothioate) kit (Invitro transcription) according to a known method (Tetrahedron Letters 51: 3446-3448,2010 ). Approximately, 40 μl of the PCR product obtained, 16 μl of transcription buffer (10 ×) contained in the kit, 2 μl of rNTP mixture (20 mM), 100 μl of GSMP (40 mM), 2 μl of RNAse inhibitor and 20 μl of distilled water GSMP-RNA (SEQ ID NO: 1) was synthesized by adding 20 μl of T7 RNA polymerase and reacting at 37 ° C for 4 to 5 hours, and the GSMP-RNA thus synthesized was subjected to ethanol precipitation Lt; / RTI >

Aptamer: 5'-GGUUGAUUGC GUGUCAAUCA UGGCCGUCUA UAAUGAUCGU AAACGACGGG UCAUGUGUAU GUUGGGGAUU GGGACCUGAU UGAGUUCAGC CCACAUAC-3 '(SEQ ID NO: 1)

1 μl of ALK (Alkaline phosphatase), 10 μl of NBE buffer and 80 μl of DEPC water were added to 10 μl of GSMP-RNA, followed by reaction at 37 ° C for 1 hour. To the reaction mixture was added 2 μl of EGTA , And reacted at 65 DEG C for 10 minutes to obtain 5'-HS-RNA which is an aptamer capable of specifically binding to HBsAg, a surface antigen of hepatitis B virus.

Example  3: Cy3 in Labeled Of app tamer  purchase

50 nmole of sulfo-SMCC dissolved in DMSO and 500 nm of Cy3 dissolved in PBS buffer solution (pH 7.2) were added and reacted at room temperature for 2 hours to obtain a first reaction product. To the first reaction product, the aptamer obtained in Example 2 5 nmole was added and reacted at room temperature overnight to obtain a second reaction product. The secondary reaction product was added to a 30 k Armicon, centrifuged at 13,000 rpm for 10 minutes to remove the residue, 600 μl of washing buffer was added thereto, and centrifugation at 13,000 rpm for 30 minutes was repeated four times. Then, 300 μl of elution buffer was added and centrifugation at 13,000 rpm for 10 minutes was repeated twice to elute Cy3-labeled aptamers. The eluted sample was applied to a rotary evaporator, concentrated, and diluted with PBS buffer (pH 7.2) to obtain a Cy3-labeled aptamer.

Example  4: Cy5 in Labeled  Hepatitis B virus Of surface antigen (HBsAg)  purchase

The HBsAg surface antigen (HBsAg) was labeled with Cy5 using the NHS ester reaction.

Specifically, 1 nmole HBsAg was diluted with 0.1 M carbonate buffer (pH 9.3), and the diluted solution and Cy5-NHS ester dissolved in DMSO were mixed at 1: 8 (v / v). The mixture was stirred for 4 hours at room temperature and at room temperature. The mixture was applied to vivaspin, washed three times with 0.1 M sodium acetate pH 9.3, eluted with an elution solution (50 mM Tris-HCl pH 7.5) , And the absorbance of the eluted sample was analyzed using a UV scan. At this time, the ratio and mole concentration of Cy5 labeled on HBsAg were calculated based on Beer's law, and the molar extinction coefficient was estimated based on the amino acid sequence of the surface antigen. As a result, the average ratio of the fluorescent substance to the protein was 1.01, and the average concentration of the labeled protein was 0.8 mM.

Finally, HBsAg labeled with Cy5 was stored at 4 ° C in a dark room.

Example  5: FRET  Phenomenon HBsAg  detection

The Cy3-labeled aptamer (80 pmol / 200 μl) obtained in Example 3 was added to the reaction buffer (20 mM Tris-HCl, 140 mM NaCl and 5 mM MgCl 2) and reacted at 4 ° C. for 12 hours. Then, Cy5-labeled HBsAg (5 pmol / 600 μl) and 0 to 50 pM of unlabeled HBsAg or 15 to 45 pM of unlabeled BSA were added to the reaction mixture, followed by reaction at 4 ° C. for 12 hours, Was taken and measured with a spectrophotometer (Fig. 2). FIG. 2 is a graph showing that the FRET reaction of Cy5 bound to the hepatitis B virus surface antigen (HBsAg) and Cy3 bound to the aptamer is specifically inhibited by unlabeled HBsAg. As shown in FIG. 2, when the BSA used as the control group was treated, FRET was not affected at all. However, when the unlabeled HBsAg was used, as the unlabeled HBsAg concentration in the sample was increased, HBsAg contained in the sample could be detected at a concentration of 500 fmol (0.5 pmol) to 50 pmol as well as the FRET reaction of the combined Cy5 and Cy3 bound to the aptamer.

Thus, using a composition comprising the labeled aptamer of the present invention and the labeled target material, it was found that the target substance can be detected at the fmol level.

Example  6: FRET  Phenomenon HBsAg  Quantitative analysis

The Cy3-labeled aptamer (80 pmol / 200 μl) obtained in Example 3 was added to the reaction buffer (20 mM Tris-HCl, 140 mM NaCl and 5 mM MgCl 2) and reacted at 4 ° C. for 12 hours. Then, Cy5-labeled HBsAg (5 pmol / 600 μl) and 0 to 50 pM of unlabeled HBsAg were added to the reaction mixture, followed by reaction at 4 ° C. for 12 hours. Then, 200 μl of the reaction product was measured with a spectrophotometer (Fig. 3). FIG. 3 is a graph showing that the results of the FRET reaction of Cy5 bound to the hepatitis B virus surface antigen (HBsAg) and Cy3 bound to the aptamer are changed in a concentration-dependent manner by the unlabeled HBsAg. As shown in FIG. 3, it was confirmed that as the concentration of the unlabeled HBsAg increases, the level of the Cy3 emission wavelength of the donor phosphor increases and the level of the Cy5 emission wavelength of the acceptor phosphor decreases. It was analyzed that the unlabeled HBsAg interferes with the binding between the donor fluorophore (aptamer) and the acceptor fluorophore (HBsAg), isolating Cy3 and Cy5.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Composition for diagnostic or diagnostic          aptamer tagged by fluorescent substance with fluorescence          resonance energy transfer <130> PA130982 / KR <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 98 <212> RNA <213> Artificial Sequence <220> <223> aptamer <400> 1 gugugauugc gugucaauca uggccgucua uaaugaucgu aaacgacggg ucauguguau 60 guuggggauu gggaccugau ugaguucagc ccacauac 98 <210> 2 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> template <400> 2 gtatgtgggc tgaactcaat caggtcccaa tccccaacat acacatgacc cgtcgtttac 60 gatcattata gacggccatg attgacacgc aatcaacccc ctatagtgag tcgtatta 118 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 gtatgtgggc tgaac 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 taatacgact cacta 15 <210> 5 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> PCR product <400> 5 taatacgact cactataggg ggttgattgc gtgtcaatca tggccgtcta taatgatcgt 60 aaacgacggg tcatgtgtat gttggggatt gggacctgat tgagttcagc ccacatac 118

Claims (22)

(a) a hepatitis B surface antigen (HBsAg), which is a target substance with a first fluorescent substance labeled as a donor or acceptor that causes fluorescence resonance energy transfer (FRET) HBsAg); And
(b) an aptamer to which a second fluorescent material capable of reacting with the first fluorescent material to cause a FRET phenomenon is bound and capable of binding to the target substance HBsAg surface antigen (HBsAg) Composition for quantitative analysis of hepatitis B virus surface antigen (HBsAg).
The method according to claim 1,
Wherein the first fluorescent material and the second fluorescent material are selected from the group consisting of fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxydodamine, carboxyrotamin 6G, Carboxyhodamine 110, Cascade Blue, Cascade Yellow, comarine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, picoerythrin, PerCP (peridinin chlorophyll (a-6) -A protein), PerCP-Cy5.5, JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), NED, ROX Carboxy-X-Rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Floor (Alexa Fluor, trade name) 350, Alexa Floor 430, Alexa Floor 488, Alexa Floor 532, Alexa Floor 546, Alexa Floor 568, Alexa Floor 594, Alexa Floor 633, Amino-4-methylcomarine-3-acetic acid, BODIPY FL, body fat FL-Br2, body fat 530/550, body fat 558, 566, body pit 564/570, body pit 576/589, body pit 581/591, body pit 630/650, body pit 650/665, body pit R6G, body pit TMR and body pit TR, Wherein the fluorescent substance is a fluorescent substance capable of exhibiting a FRET phenomenon by interaction.
delete The method according to claim 1,
Wherein the aptamer is the RNA of SEQ ID NO: 1.
A kit for quantitative analysis of hepatitis B virus surface antigen (HBsAg) which is a target substance comprising the composition of any one of claims 1, 2, and 4.
6. The method of claim 5,
And wherein the aptamer further comprises a substrate or bead coupled thereto.
6. The method of claim 5,
Wherein the kit further comprises an internal control that does not bind to the aptamer and does not affect the level of the measured emitted fluorescence.
8. The method of claim 7,
Wherein the internal control is BSA (bovine serum albumin).
(HBsAg), which is a target substance to which a first fluorescent substance is labeled, which acts as a donor or an acceptor for causing a FRET phenomenon while irradiating light of an absorption wavelength of a donor fluorescent substance, and a target substance A second fluorescent substance which reacts with the first fluorescent substance to induce a FRET phenomenon is bound to a separated sample to be tested for the presence of the hepatitis B virus surface antigen (HBsAg), and the target substance, hepatitis B virus To an aptamer capable of binding to a surface antigen (HBsAg) at the same time; And
(b) measuring the level of fluorescence emitted from the donor fluorescent substance or the acceptor fluorescent substance from the reactant. The method of quantitatively analyzing a hepatitis B virus surface antigen (HBsAg) as a target substance.
10. The method of claim 9,
(HBsAg) is not present in the sample when the emitted fluorescent light of the donor fluorescent substance is not detected, and when the emitted fluorescent light of the donor fluorescent substance is detected, (HBsAg), which is a target substance, in the presence of the hepatitis B virus surface antigen (HBsAg).
10. The method of claim 9,
In the step (b), the HBsAg surface antigen (HBsAg), which is a target substance labeled with the aptamer, is added while reacting with light having an absorption wavelength of the donor fluorescent substance, and the donor fluorescent substance Lt; RTI ID = 0.0 &gt; of fluorescence &lt; / RTI &gt;
10. The method of claim 9,
In step (b), the HBsAg surface antigen (HBsAg), which is a target substance labeled on the aptamer, and a substance that does not bind to the aptamer are simultaneously added while irradiating the light having the absorption wavelength of the donor fluorescent substance And using the level of emitted fluorescence of the donor fluorescent substance measured in the reaction as an internal control.
13. The method according to claim 11 or 12,
Further comprising the step of determining whether the emission fluorescence level of the donor fluorescent substance measured in the step (b) is increased, based on the level of emitted fluorescence of the donor fluorescent substance measured as the internal control group.
(a) a hepatitis B virus surface antigen (HBsAg) which is a target substance with a first fluorescent substance labeled as a donor or an acceptor, which causes fluorescence resonance energy transfer (FRET) phenomenon; And
(b) an aptamer to which a second fluorescent material capable of reacting with the first fluorescent material to cause a FRET phenomenon is bound and capable of binding to the target substance HBsAg surface antigen (HBsAg) (HBsAg) detection of hepatitis B virus.
15. The method of claim 14,
An internal control that does not bind to the aptamer and does not affect the level of the measured emitted fluorescence, a hepatitis B virus surface antigen (HBsAg) that is a concentration-specific target substance required to prepare a standard curve, and combinations thereof &Lt; / RTI &gt;
16. The method of claim 15,
Wherein the internal control is BSA (bovine serum albumin).
A kit for quantitative analysis of hepatitis B virus surface antigen (HBsAg) which is a target substance comprising the composition of any one of claims 14 to 16.
(HBsAg), which is a target substance to which a first fluorescent substance is labeled, which acts as a donor or an acceptor for causing a FRET phenomenon while irradiating light of an absorption wavelength of a donor fluorescent substance, and a target substance A second fluorescent substance which reacts with the first fluorescent substance to induce a FRET phenomenon is bound to a separated sample to be tested for the presence of the hepatitis B virus surface antigen (HBsAg), and the target substance, hepatitis B virus To an aptamer capable of binding to a surface antigen (HBsAg) at the same time; And
(b) measuring the level of fluorescence emitted from the donor fluorescent material or the acceptor fluorescent substance from the reactant.
19. The method of claim 18,
Reacting the aptamer with a hepatitis B virus surface antigen (HBsAg) as a target substance in place of a sample, reacting the HBsAg with a concentration, and preparing a standard curve using the level of emitted fluorescence measured therefrom; And quantifying the level of the hepatitis B virus surface antigen (HBsAg), which is a target substance included in the sample, by applying the level of the emitted fluorescence measured in the step (b) to the standard curve / RTI &gt;
19. The method of claim 18,
The HBsAg surface antigen (HBsAg), which is a target substance labeled with the aptamer, is added to the donor fluorescent substance while the light having the absorption wavelength of the donor fluorescent substance is irradiated, and the level of the fluorescence emitted from the donor fluorescent substance measured in the reactant Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; as a control.
19. The method of claim 18,
The HBsAg surface antigen (HBsAg), which is a target substance labeled with the aptamer, and a substance which does not bind to the aptamer are added while irradiating light having an absorption wavelength of the donor fluorescent substance, and the donor Further comprising the step of using the level of fluorescence emission of the fluorescent substance as an internal control.
22. The method of claim 21,
Wherein said internal control is BSA (bovine serum albumin).

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