KR20200145781A - Analysis methods using the constructs of aptamers and reporter - Google Patents

Abstract

The present invention relates to a quantitative analysis method using an aptamer-reporter gene fused body, wherein as a target recognition substance, an aptamer composed of nucleic acids is used instead of an antibody. The analysis method of the present invention enables a microscopic analysis through amplification with a maximized detection signal. A target is coupled by a capture aptamer, and a reporter protein capable of generating a signal through in vitro expression (ALIVE) using a reporter gene conjugated to a detection aptamer bound to another site of the target as a template is synthesized. Then, if the produced reporter protein is an enzyme, a substrate is added to amplify the signal. Thus, targets present in trace amounts of an aM level can be detected.

Description

Analysis methods using the constructs of aptamers and reporter}

The present invention relates to an analysis method using an aptamer-reporter gene fusion.

Immunoassays that rely on the unique target specificity and affinity of an antibody are widely used for qualitative, quantitative analysis and diagnosis of diseases. In particular, the enzyme-linked immunosorbent assay (ELISA) has become the main standard method of immunoassay due to well-established procedures and readily available reagents. However, there are some inherent limitations to the analysis and diagnosis methods using antibodies. First, preparation of an antibody requires a long time and a complicated procedure, and thus a lot of time is required, thus delaying the development of a diagnostic tool for a newly discovered target. Second, like other protein molecules, antibodies are easily denatured by factors such as high temperature and physical adsorption, so it is difficult to maintain their activity during storage and transport. Finally, antibodies generally only recognize large molecules (usually >600 Da), and binding with small molecule substances, including compounds, is less effective.

Compared to the antibody-based assay, the aptamer-based assay is considered an alternative option that can compensate for many of the shortcomings of the antibody. Aptamers can be synthesized using chemical procedures that are simpler, faster, and less expensive than antibodies, but also have a very stable structure, allowing long-term storage and transport without freezing and refrigeration facilities. In addition, optimal aptamers for various targets ranging from small compounds to large proteins can be selected from combinatorial libraries.

Thus, aptamers are increasingly being used to detect many targets in addition to or by replacing antibodies.

Like ELISA, the signal of an aptamer-based assay is usually generated by an enzyme bound to the detection aptamer. In this conventional configuration of enzyme-linked assay, the sensitivity of detection is largely determined by the number of rotations of the enzyme, and there is an inherent limitation in the amplification of the readout signal because only a limited number of enzymes can be conjugated to a given aptamer.

As for the aptamer-related technology, Korean Patent Publication No. 2012-0315669 discloses a technology related to an aptamer-based analyte analysis method, but in vitro expression using the reporter gene conjugated with the aptamer of the present invention as a template (aptamer- linked in vitro expression: ALIVE), an analysis method using an aptamer-reporter gene fusion that amplifies a signal through protein synthesis from target binding activity and reporter DNA has not been disclosed.

The present invention was derived from the above requirements, and the present invention provides an analysis method using an aptamer-reporter gene fusion, and in order to confirm the detection limit (LOD) of the quantitative analysis method of the present invention, a single strand A'readable' aptamer consisting of double-stranded DNA encoding anti-thrombin DNA, sfGFP and luciferase was prepared. After binding thrombin to the antithrombin region, it was confirmed using sfGFP that protein synthesis of the fusion construct was possible through protein synthesis reaction in vitro using a protein synthesis reaction solution extracted from E. coli, and luciferase as a reporter for ALIVE analysis. By confirming that the limit of detection (LOD) of thrombin can be lowered by using, the present invention was completed.

In order to achieve the above object, the present invention includes the steps of: (1) fixing a capture aptamer capable of capturing a target to a support;

(2) after step (1), adding a sample containing a target to react so that the capture aptamer can capture the target, and washing to remove unreacted residue;

(3) coupling the detection aptamer to which the reporter gene is conjugated to the target captured by the capture aptamer in step (2);

(4) after the step (3), synthesizing a reporter protein through in vitro expression (ALIVE) using the reporter gene conjugated to the detection aptamer as a template; And

It provides an analysis method using an aptamer-reporter gene fusion comprising; (5) analyzing the reporter protein synthesized by the in vitro expression (ALIVE) to detect the content of the target.

The present invention is derived from the above requirements, the present invention relates to an analysis method using an aptamer-reporter gene fusion, the present invention performs PCR to add a double-stranded reporter gene to the end of the single-stranded aptamer And, by using this, a reporter protein that generates an amplification signal through an in vitro expression reaction was synthesized. Therefore, a quantitative method using a reporter gene fusion conjugated with a'readable' binding aptamer is a quantitative analysis method that can significantly increase detection sensitivity. For example, when analyzing thrombin, the quantitative analysis method of the present invention may achieve a detection limit as low as 2.7 aM.

1 is a conceptual diagram of a quantitative analysis method in which a signal is amplified through catalytic conversion with a substrate after expression of an intracellular protein using a reporter gene to which an aptamer is conjugated as a template.
2 is a conceptual diagram for the design of a reporter gene conjugated with an aptamer of the present invention.
3 is a result of confirming that the reporter gene conjugated with Apt15-sfGFP and Apt29-sfGFP aptamer can induce expression in vitro at a level similar to that of the control sfGFP gene not conjugated with the aptamer.
4 is a result of comparing the in vitro expression levels of reporter genes to which Apt15-sfGFP and Apt29-sfGFP aptamers are conjugated according to biotinylation.
5 is a view confirming the expression level of sfGFP according to the concentration of the reporter gene to which Apt15-sfGFP and Apt29-sfGFP aptamer are conjugated.
Figure 6A confirms that the in vitro expression of Apt15-sfGFP increases fluorescence in proportion to the supplied amount of thrombin, and Figure 6B shows the capture aptamer as Apt15 by exchanging the capture aptamer and the detection aptamer with each other, and detection aptamer. This is the result of confirming that Apt29 was less sensitive to thrombin concentration.
7 is a result of confirming that the background signal is high when Apt15 is used as the capture aptamer and Apt29 is used as the detection aptamer.
8 is a result of confirming the expression level according to the type of luciferase, (A) firefly luciferase (Fluc) and (B) Nluc luciferase.
9 is a result of confirming the minimum detection limit (LOD) of thrombin by an aptamer-linked in vitro expression (ALIVE) assay.

The present invention comprises the steps of: (1) fixing a capture aptamer capable of capturing a target to a support;

(2) after step (1), adding a sample containing a target to react so that the capture aptamer can capture the target, and washing to remove unreacted residue;

(3) coupling the detection aptamer to which the reporter gene is conjugated to the target captured by the capture aptamer in step (2);

(4) after the step (3), synthesizing a reporter protein through in vitro expression (ALIVE) using the reporter gene conjugated to the detection aptamer as a template; And

It relates to an analysis method using an aptamer-reporter gene fusion comprising; (5) analyzing the reporter protein synthesized by the in vitro expression (ALIVE) to detect the content of the target.

The target is not limited from a low-molecular metabolite to which an aptamer can bind to a high-molecular protein, but preferably may be a protein, nucleic acid, or metabolite to which the aptamer can bind.

The capture aptamer or detection aptamer in the present invention is a single or double-stranded DNA; Alternatively, as an RNA nucleic acid strand, the molecular weight is preferably about 10 to 15 kDa, but is not limited thereto. Since the ALIVE analysis method of the present invention is used in a plug-and-play method, all analytes that can be used by the aptamer can be analyzed.

If the reporter protein is an enzyme that catalyzes the color development or luminescence reaction of a substrate, between steps (4) and (5), adding a substrate to obtain a reaction product to amplify a signal to be detected; further It may include, but is not limited thereto.

Enzymes that catalyze the color reaction of the substrate include horseradish peroxidase (HRP), alkaline phosphatase, glucosidase, tyrosinase, and GUS (β- glucuronidase), but is not limited thereto.

The enzyme that catalyzes the light-emitting reaction of the substrate is preferably luciferase, but is not limited thereto. The substitution, deletion, or insertion of one or more amino acid residues within a range in which the activity of the luciferase can be maintained or the activity can be better can be any number.

The reporter gene is preferably a gene encoding a fluorescent protein, but is not limited thereto. The fluorescent protein is preferably any one selected from Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), Red Fluorescent Protein (RFP), and Aequorin, but is not limited thereto.

In step (2), the sample is preferably any one selected from blood, plasma, serum, urine, or lymph, but is not limited thereto.

The capture aptamer in step (1) is preferably bound to the support by using biotin as a linker, but is not limited thereto. It is preferable to further include a spacer between the detection aptamer and the reporter gene, but is not limited thereto. In vitro expression (ALIVE) using the reporter gene conjugated to the detection aptamer as a template is preferably performed under conditions of a reaction temperature of 20 to 40°C and a reaction time of 2 to 6 hours, but is not limited thereto.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for describing the present invention in more detail, and it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

[Materials and methods]

PCR primers and anti-thrombin DNA aptamers were synthesized and used in Bioneer (Daejeon, Korea). ATP, GTP, UTP, CTP, creatine phosphate, creatine kinase and total tRNA were purchased from Roche Applied Science (Indianapolis, IN, USA). S12 extract was prepared from E. coli BL21Star (DE3) strain. Purified human α-thrombin was purchased from Hematology Technologies (Essex Junction, VT, USA). Polyclonal anti-thrombin antibodies were purchased from Thermo Fisher Scientific (Rockford, IL, USA).

Horseradish peroxidase (HRP)-conjugated monoclonal anti-thrombin antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Nano-Glo luciferase assay kit was purchased from Promega (Madison, WI, USA). Streptavidin was purchased from New England Biolabs (Ipswich, MA, USA). L-[U- ¹⁴ C] leucine (11.9 GBq/mmol) was purchased from Amersham Bioscience (Uppsala, Sweden). All other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification.

1. readable Aptamer Produce

Aptamer-forward primers were used to PCR amplify the gene of sfGFP or NLuc from plasmids pK7sfGFP and pK7NLuc, respectively. C ₂ A spacer was inserted between the aptamer and the primer sequence to maintain the structure of the aptamer as a single strand after PCR. Thus, the PCR product consists of a serially linked anti-thrombin aptamer and reporter protein expression cassette. The PCR product was purified using a Wizard PCR clean-up kit (Promega), and then the concentration was measured.

Plamid and primer sequences used in the present invention primer Sequence (5'->3') Sequence number T7 15up(Forward) 5'-TCGATCCCGCGAAATTAATACGACTCACTATAGG-3' One GTB(Reverse) 5'-CAAAAAACCCCTCAAGACCCGTTTAGA-3' 2 GTB-C12-Apt15
(Reverse) 5'-GGTTGGTGTGGTTGG-[C ₁₂ ]-CAAAAAACCCCTCAAGACCCGTTTAGA-3' 3 Apt15 5'-TTTTTTTTTTTTTTTGGTTGGTGTGGTTGG-3' 4 Apt29 5'-TTTTTTTTTTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' 5 pK7-FLuc Firefly luciferase cloned in the plasmid pK7 pK7-NLuc Nano-Glo luciferase with C-terminal His-tag cloned in the plasmid pK7

2. In vitro expression

The standard reaction mixture for in vitro expression of the reporter gene was a total volume of 30 μl, 57 mM HEPES-KOH, pH 8.2; 1.4mM ATP; 1.0 mM each of GTP, UTP and CTP; 80 mM ammonium acetate; 12 mM magnesium acetate; 90mM potassium acetate; 33 µg/ml of 1,5-formyl-5,6,7,8-tetrahydrofolic acid; 2.5mM each of the 20 amino acids; 2% polyethylene glycol 8000; 3.2 U/ml creatine kinase; 60mM creatine phosphate; And 27% (v/v) S12 extract. In vitro expression was performed in 96-well microtiter plates at 30° C. for 3 hours.

3. Aptamer Pair sandwich Assay

The aptamer-based sandwich analysis used a pair of aptamers that bind to different positions of the thrombin molecule represented by Apt15 and Apt29, respectively, as capture and detection aptamers (Table 1). Using streptavidin as a linker, the biotinylated detection aptamer was conjugated with sfGFP or NLuc. Biotinylated sfGFP and NLuc were individually prepared by in vitro protein expression of plasmids pK7Avi-sfGFP and pK7NLuc-Avi encoding sfGFP and NLuc with an Avi- tag at the C-terminus.

In situ biotinylation of the synthesized protein was achieved by performing in vitro expression (ALIVE) using a reporter gene conjugated with an aptamer as a template in the presence of 1 mM biotin (FIG. 1). Expression of the biotinylated protein was confirmed by a gel-shift assay after adding 250 µg/ml streptavidin to the reaction mixture.

4. ALIVE (aptamer-linked in vitro expression) analysis

Streptavidin was dissolved in PBS (phosphate-buffered saline) at a concentration of 10 μg/ml, loaded at 100 μl into a 96-well polystyrene plate, and incubated overnight at 4° C. to coat. After washing with PBS-T [0.1% (v/v) Tween-20 in PBS], each well of the plate was incubated with 100 μl of biotinylated capture aptamer at room temperature for 1 hour. Then, the plate was blocked with 300 μl of 1% bovine serum albumin solution at room temperature for 2 hours. After washing the plate with PBST, 10 nM of human α-thrombin solution was added to each well of various volumes. After incubation at room temperature for 2 hours, the plate was washed twice with PBST buffer, and 50 µl of a 66.5 nM reporter-coating detection aptamer solution was added. After incubation for 1 hour, the plate was washed with PBST, and 100 μl of the reaction mixture for in vitro expression of the reporter protein was added. Expression in vitro was performed in a water bath at 30° C. for 3 hours.

In the assay in which sfGFP is expressed as a reporter, 20 μl of the reaction mixture was diluted with 190 μl of PBS and its fluorescence was measured using a Victor X2 microplate reader (Perkin Elmer, Waltham, MA, USA).

The ALIVE assay using NLuc as a reporter is similar except that the reporter gene fused to the conjugated aptamer is replaced with NLuc and the bioluminescence signal is measured after adding the same volume of NLuc substrate solution after the in vitro expression step. Followed the procedure. Bioluminescence was measured at 450 nm in a CLARIO Star microplate reader (BMG Labtech, Ortenberg, Baden-Wurttemberg, Germany).

Example 1. readable Aptamer Expression in vitro

Since the aptamer is composed of a nucleic acid, it can additionally be easily fused to a DNA sequence. Based on this, a DNA molecule having a dual function was designed, consisting of two parts: a single-stranded module containing a target-binding DNA aptamer; and a double-stranded expression module encoding a reporter protein. As a result, the structure was created in situ of the reporter protein. It was predicted that it could be used as a'readable' aptamer that reports the presence or absence of a target.

As an exemplary model, a DNA structure including the sequence of an anti-thrombin DNA aptamer and sfGFP gene in one molecule was prepared. Two unique thrombin aptamers have been reported that bind to different positions on the thrombin molecule. These aptamers, designated Apt15 and Apt29, were designed to be adjacent to the 5'-end of the forward primer used to PCR amplify the sfGFP gene. A C ₁₂ spacer was inserted between the aptamer and primer sequences so that the aptamer site remained as a single strand after PCR. The resulting readable aptamers were named Apt15-sfGFP and Apt29-sfGFP (FIG. 2).

It was confirmed that both Apt15-sfGFP and Apt29-sfGFP aptamers could induce in vitro expression of sfGFP as efficiently as the control sfGFP gene lacking the aptamer sequence. As a result of synthesizing a protein in a reaction mixture for protein expression in vitro using two readable aptamers and a control DNA, all three types of DNA constructs produced about 13 μM of sfGFP (FIG. 3 ).

Then, when the two readable aptamers and the control DNA were physically immobilized on the surface, it was investigated whether they affect the expression efficiency in vitro. The readable aptamer was prepared using a 5'-biotinylated reverse primer during PCR, and 10 ng of biotinylated readable aptamer was immobilized on the streptavidin-coated surface.

As a result, as disclosed in FIG. 4, the synthesized sfGFP fluorescence levels were similar regardless of whether two readable aptamer DNAs were immobilized on the streptavidin-coated surface by biotin. In addition, it was confirmed that the expression level of sfGFP is proportional to the DNA concentration in the range of 0.8-23 pM (FIG. 5). From these results, it was determined that the synthesis (translation) of the reporter protein could be amplified in the aptamer capable of target binding.

Example 2. By ALIVE method Thrombin detection

In Example 2, an aptamer pair-based sandwich assay for thrombin was established using readable aptamers. One of the pair of thrombin-binding aptamers (Apt15 and Apt29) was synthesized as 3'-biotin and used as a'capture aptamer', and the other fused with an expression module for a reporter protein used as a readable'detection aptamer'. Became.

Thrombin was analyzed using Apt29 as a capture aptamer and Apt15-sfGFP as a readable detection aptamer. To a 96-well microplate coated with streptavidin, 10 μM of biotinylated Apt29, various concentrations of thrombin and Apt15-sfGFP were sequentially supplied. In vitro expression of Apt15-sfGFP increased fluorescence in proportion to the supplied amount of thrombin (FIG. 6A ). However, the fluorescence change was less sensitive to the concentration of thrombin when the capture and detection aptamers were exchanged (FIG. 6B), and when the capture and detection aptamers were exchanged with each other as disclosed in FIG. 7, a very high background signal was shown. . It was determined that the increase in background signal was due to non-specific binding of Apt29-sfGFP to the surface. Based on these results, later experiments decided to use biotinylated Apt29 as a'capture aptamer' and Apt15 as a'detection aptamer'.

Example 3. NLuc ALIVE assay using as a signal generating enzyme

Luminescence analysis is much more sensitive than colorimetric and fluorescence analysis and has a wide dynamic range. In order to further improve the sensitivity and robustness of the ALIVE assay, the sfGFP gene of the assay configuration was replaced with a gene encoding Nano-Glo luciferase (NLuc).

Because NLuc can produce 100 times more photons than commonly used luciferases such as firefly luciferase and Renilla luciferase, we chose a processed version of the deep-sea shrimp luciferase called NLuc. More importantly, compared to other types of luciferase, NLuc is better compatible with the conditions of the present invention for protein expression in vitro.As disclosed in FIG. 8, NLuc has a much higher level than firefly luciferase. Was expressed.

The solubility of firefly luciferase synthesized in vitro was as low as 10% at a standard reaction temperature of 30° C., but NLuc was expressed in a soluble form and almost dissolved at a wide range of reaction temperatures. Therefore, at standard temperature, the NLuc gene can act as an efficient and powerful expression module for ALIVE analysis.

When the ALIVE analysis was performed using an aptamer for detection with an NLuc gene (Apt15-NLuc), the limit of detection (LOD) of antithrombin was 2.7aM (Fig. 9).

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Analysis methods using the constructs of aptamers and reporter <130> PN20159 <150> KR 2019-0074003 <151> 2019-06-21 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> T7-15up-Forward <400> 1 tcgatcccgc gaaattaata cgactcacta tagg 34 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GTB-Reverse <400> 2 caaaaaaccc ctcaagaccc gtttaga 27 <210> 3 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> GRB-C12-apt15-reverse <400> 3 ggttggtgtg gttggccccc ccccccccaa aaaacccctc aagacccgtt taga 54 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> apt15 <400> 4 tttttttttt tttttggttg gtgtggttgg 30 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> apt29 <400> 5 tttttttttt tttttagtcc gtggtagggc aggttggggt gact 44

Claims (11)

(1) fixing a capture aptamer capable of capturing a target to a support;
(2) after step (1), adding a sample containing a target to react so that the capture aptamer can capture the target, and washing to remove unreacted residue;
(3) coupling the detection aptamer to which the reporter gene is conjugated to the target captured by the capture aptamer in step (2);
(4) after the step (3), synthesizing a reporter protein through in vitro expression (ALIVE) using the reporter gene conjugated to the detection aptamer as a template; And
(5) Analyzing the reporter protein synthesized by the in vitro expression (ALIVE) to detect the content of the target; an analysis method using an aptamer-reporter gene fusion. The method of claim 1, wherein when the reporter protein is an enzyme that catalyzes the color development or luminescence reaction of a substrate, between steps (4) and (5), a reaction product is obtained by adding a substrate to obtain a signal to be detected. Amplifying step; analysis method using an aptamer-reporter gene fusion further comprising. The method of claim 2, wherein the enzyme catalyzing the color development reaction of the substrate is horseradish peroxidase, alkaline phosphatase, glucosidase, tyrosinase, and Analysis method using an aptamer-reporter gene fusion, characterized in that any one selected from GUS (β-glucuronidase). The method of claim 2, wherein the enzyme catalyzing the luminescence reaction of the substrate is luciferase. The method of claim 1, wherein the reporter gene is a gene encoding a fluorescent protein. The aptamer-reporter gene of claim 5, wherein the fluorescent protein is any one selected from GFP (Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), RFP (Red Fluorescent Protein), and Equorin. Analysis method using fusion. The method of claim 1, wherein in the step (2), the sample is any one selected from blood, plasma, serum, urine, or lymph fluid. The method of claim 1, wherein the capture aptamer in step (1) is bound to a support using biotin as a linker. The method of claim 1, further comprising a spacer between the detection aptamer and the reporter gene. The method of claim 1, wherein the in vitro expression (ALIVE) using the reporter gene conjugated to the detection aptamer as a template is carried out under conditions of a reaction temperature of 20 to 40°C and a reaction time of 2 to 6 hours. Analysis method using the aptamer-reporter gene fusion. The method of claim 1, wherein the capture aptamer or detection aptamer is single-stranded or double-stranded DNA, or single-stranded or double-stranded RNA.

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