High-speed DNA aptamer selection method using continuous addition and aggregation of gold nanoparticles

Abstract

The present invention relates to a high-speed method for selecting a DNA aptamer through a continuous process of adding gold nanoparticles to a reaction solution containing a target material and a single-stranded nucleic acid library and identifying aggregation. By using the method, an aptamer specifically binding to the target material can be effectively selected within a short time without a nucleic acid amplification process.

Description

Ultra-fast DNA aptamer screening using sequential addition and aggregation of gold nanoparticles

[0001] The present invention relates to a method for ultra-high-speed screening of DNA aptamers that specifically bind to a target substance by continuously adding gold nanoparticles to a reaction solution containing a target substance and a single-stranded nucleic acid library and confirming aggregation.

[0002] An aptamer is a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. Its etymology comes from the Latin word “aptus,” which means “fitting.”

[0003] Since the aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was first developed by Larry Gold's research team at the University of Colorado in 1990, many aptamers capable of binding to various target molecules, including small-molecule organic compounds, peptides, and proteins, have been continuously discovered.

[0004] The gold nanoparticle-based SELEX technique (GNP-SELEX) has the advantage of not requiring separate target immobilization and easily observing the binding of the target to a single-stranded nucleic acid library in real time between rounds by utilizing the colorimetric method of gold nanoparticles after treatment with a high concentration of salt (KR No. 10-2442161). However, some target substances can affect the gold nanoparticle surface regardless of the binding of the single-stranded nucleic acid library, making the GNP-SELEX technique inapplicable in these cases. Furthermore, the GNP-SELEX technique can be time-consuming because it involves amplification of the single-stranded nucleic acid library selected in each round. Therefore, a time-saving aptamer selection method that is independent of the characteristics of the target substance is needed.

[0005] In the above circumstances, the present inventors have devised a novel method to solve the conventional problems. This method comprises the steps of i) preparing solutions with and without target material added to a high-concentration single-stranded nucleic acid library, ii) serially diluting each solution, adding gold nanoparticles at each round and centrifuging, and iii) using the supernatant after centrifugation for the next step and analyzing the degree of aggregation of gold nanoparticles in the supernatant.

[0006] This method is a technology that can easily determine the binding strength of single-stranded nucleic acids and target substances within 4-5 hours by observing the color change of the gold solution remaining in the lower layer after centrifugation, as gold nanoparticles are continuously added at each dilution step.

[0007] This method allows for faster aptamer selection by reducing the number of rounds when using the GNP-SELEX technique by preferentially selecting a single-stranded nucleic acid library with strong binding affinity to the target substance.

[0008] Accordingly, the present invention aims to provide a method for selecting single-stranded nucleic acids having strong binding affinity to a target substance from a single-stranded nucleic acid library by using continuous addition and aggregation of gold nanoparticles.

[0009] To achieve the above purpose, the present invention provides a method for screening a single-stranded nucleic acid that specifically binds to a target substance, comprising the following steps:

[0010] a) A step of reacting a target material or a non-target material with a single-stranded nucleic acid library, respectively;

[0011] b) a step of adding gold nanoparticles to the above a) and causing a reaction;

[0012] c) a step of centrifuging the result of b);

[0013] d) The supernatant is transferred to a new tube, and the precipitated gold nanoparticles are subjected to aggregation;

[0014] e) a step of checking the absorbance of the aggregated gold nanoparticles to determine whether to proceed with additional reactions; and

[0015] f) If additional reactions are required, repeat steps b) to e) by adding target or non-target substances to the supernatant transferred to a new tube.

[0016]

[0017] As used herein, the terms "react," "reacting," and "to react" refer to a phenomenon caused by bringing reactants into proximity through mixing, addition, etc., so that they can interact.

[0018] The terms "nucleic acid" and "single-stranded nucleic acid" used herein follow their commonly known definitions. In particular, since the method for selecting nucleic acids according to the present application and the nucleic acids selected thereby are concepts that can be expanded regardless of the type of nucleic acid, the concept of nucleic acid in the present application is intended to encompass not only DNA and RNA, but also all nucleic acids that could be understood by the level of conventional technology at the time.

[0019] As used herein, the term "nucleic acid library" refers to a collection of at least two different types of nucleic acids. The term "single-stranded nucleic acid library" refers to a collection of at least two different types of single-stranded nucleic acids. The nucleic acid library according to the present application may be in any form, including a form in which the nucleic acids are contained within a microorganism, a form contained within a special formulation (e.g., micelles, liposomes, etc.), or a form dispersed without any specific formulation.

[0020] As used herein, the term "target substance" refers to a specific substance that binds to a single-stranded nucleic acid, which is the purpose of the present application. Furthermore, the term "non-target substance" refers to a substance to which the single-stranded nucleic acid is not intended to bind, when selecting the single-stranded nucleic acid. The target substance and non-target substance may be in any form, including low-molecular-weight compounds, nucleic acids, peptides, proteins, protein variants, lipids, lipid variants, carbohydrates, and carbohydrate variants.

[0021] In the present invention, a single-stranded nucleic acid that specifically binds to a target substance may be referred to as an aptamer.

[0022] In the present invention, the gold nanoparticles may be prepared using a commonly known method for preparing gold nanoparticles. In one specific embodiment of the present invention, the gold nanoparticles may be stabilized with citric acid to have an affinity for single-stranded DNA (ssDNA), and may have an average diameter of 15 nm to 50 nm. In addition, the gold nanoparticles may be prepared by reduction and stabilization with citric acid.

[0023] In one specific embodiment of the present invention, steps a) and b) are performed at room temperature, and the reaction time may be 5 to 40 minutes, preferably 10 to 40 minutes. In addition, the reaction time may vary depending on the concentration of the target substance, single-stranded nucleic acid library, and gold nanoparticles.

[0024] In the present invention, the centrifugation of c) is performed under conditions sufficient to precipitate gold nanoparticles, and in the example, centrifugation was performed at 4°C and 6500 rcf for 30 minutes.

[0025] According to one specific example of the present invention, in step d), the agglomeration of the gold nanoparticle precipitate can be achieved by adding salt, and a salt commonly used in the technical field to which the present invention belongs is used for the agglomeration.

[0026] As shown in Figure 1, the present invention utilizes the color difference that occurs when a target substance or non-target substance is reacted with a single-stranded nucleic acid library, then gold nanoparticles are added and aggregated. Specifically, in the presence of a target substance, the color of the gold nanoparticle precipitate rapidly transitions from red to purple, and the color transition can be quantified by measuring the absorbance of the precipitate.

[0027] In one specific embodiment of the present invention, the absorbance of step e) is determined by measuring the absorbance of the aggregated gold nanoparticles. 520 / E 700 It may be to check the ratio. More specifically, the step e) is to check the E of the aggregated gold nanoparticles by reacting with a non-target substance. 520 / E 700 E of the gold nanoparticles aggregated by reacting with the target substance 520 / E 700 If it is 1.5 to 3 times higher than the ratio, it may be a step to determine whether to perform step f).

[0028] Additionally, the step e) may further include visually confirming the color change of the aggregated gold nanoparticles.

[0029] If it is determined that an additional reaction is to be performed in step e), steps b) to e) are repeated by adding a target substance or a non-target substance to the supernatant transferred to a new tube. According to one specific example of the present invention, steps b) to e) may be repeated 2 to 10 times, and preferably 4 to 10 times.

[0030] In the present invention, the aptamer screening method described above is named Gold Nanoparticle-Facilitated Assembly of Supernatant Transfer (GNP-FAST). GNP-FAST is characterized by its ability to screen aptamers that specifically bind to a target substance in a relatively short period of time without a nucleic acid amplification process. Aptamers selected through GNP-FAST can undergo a step of confirming their sequence through base sequence analysis.

[0031] Using the screening method for a single-stranded nucleic acid that specifically binds to a target substance according to the present invention, DNA aptamers that specifically bind to a target substance can be effectively screened in a short period of time without a separate amplification process.

[0032] Figure 1 schematically shows an aptamer screening method according to the present invention.

[0033] Figure 2 shows the results of confirming the color change of gold nanoparticle precipitate and salt reaction solution (A), the absorbance ratio of gold nanoparticle precipitate and salt reaction solution at 520 nm and 720 nm (B), and the structure of the target substance (C) while conducting aptamer screening using diethylhexyl phthalate (DEHP) as a target substance.

[0034] Figure 3 shows the results of confirming the color change of gold nanoparticle precipitate and salt reaction solution (A), the absorbance ratio of gold nanoparticle precipitate and salt reaction solution at 520 nm and 720 nm (B), and the structure of the target substance (C) while conducting aptamer screening using cortisol as a target substance.

[0035] Figure 4 shows the results of confirming the color change of gold nanoparticle precipitate and salt reaction solution (A), the 520 nm and 720 nm absorbance ratio of gold nanoparticle precipitate and salt reaction solution (B), and the structure of the target substance (C) while conducting aptamer screening using aldosterone as a target substance.

[0036] Figure 5 shows the results of confirming the color change of gold nanoparticle precipitate and salt reaction solution (A), the absorbance ratio of gold nanoparticle precipitate and salt reaction solution at 520 nm and 720 nm (B), and the structure of the target substance (C) while conducting aptamer screening using diethyl phthalate (DEP) as a target substance.

[0037] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0038]

[0039] Experimental method

[0040] 1. Preparation of gold nanoparticles

[0041] A 250 mL round bottom flask was filled with 50 mL of distilled water and a magnetic bar, and heated while stirring on a heating stirrer. When the distilled water boiled, 100 μL of 300 mM gold tetrachloride (HAuCl4) was added, followed by 1 mL of 3% sodium citrate.

[0042] When the solution changed from black to red, the boiling was stopped and the solution was stirred at room temperature for 1 hour to cool. The solution was centrifuged at 6,500 rcf at 4°C for 30 minutes, concentrating the resulting gold nanoparticles fivefold. The diameter of the produced gold nanoparticles was confirmed to have an average diameter of 15 nm to 50 nm.

[0043]

[0044] 2. Single-stranded nucleic acid library

[0045] A single-stranded nucleic acid library with a total length of 60 mer was prepared, which had a random base sequence of 30 mer (N30) with an A:T:C:G ratio of 25:25:25:25 and a 15 mer primer initiation sequence capable of PCR amplification attached to each end.

[0046]

[0047] 5'- ATGCGGATCCGCGC(N30)CGGCGCGAAGCTTGCG -3'

[0048]

[0049] The single-stranded nucleic acid library was incubated at 95°C for 5 minutes to release the nucleic acids, and then incubated at 4°C for 15 minutes to stabilize them. Hereinafter, the single-stranded nucleic acid library is referred to as an "ssDNA library."

[0050]

[0051] 3. Screening for aptamers that react with target substances

[0052] 3-1. Target material preparation

[0053] Diethylhexyl phthalate (DEHP), cortisol, aldosterone, and diethyl phthalate (DEP) were prepared as target substances and dissolved in ethanol.

[0054]

[0055] 3-2. Aptamer screening

[0056] Based on the total reaction volume of 500 μL, 1 μM of the ssDNA library and 2 μM of the target material (all final concentrations) were reacted for 30 minutes at room temperature to ensure sufficient binding. The binding between the target material and the ssDNA library is generally reacted for about 15 minutes at room temperature, but since the present invention uses a low concentration of μM, the reaction was performed for 30 minutes. The reaction solution was adjusted to 450 μL, excluding 50 μL of the gold nanoparticle solution. The binding buffer for the ssDNA library and the target material was 0.2X PBS (0.027 M NaCl, 0.54 mM KCl, 0.86 mM Na2HPO4, 0.28 mM KH2PO4).

[0057]

[0058] The aptamer screening method was performed repeatedly, with steps 1) to 4) being performed once as one round. As a control, a reaction solution without the target substance was used, and gold nanoparticles were dispersed in the binding buffer. The concentration of gold nanoparticles was determined using a dynamic light scattering analyzer (DLS) and an ultraviolet-visible spectrophotometer, and then gold nanoparticles manufactured to be suitable for screening were prepared by concentrating them 5-fold. 1) 50 μL of gold nanoparticles were added to the reaction solution of the ssDNA library and the target substance, and the reaction was performed at room temperature for 15 minutes.

[0059] - The ssDNA library combined with the target substance does not bind to the surface of the gold nanoparticle because it forms a structure, but the ssDNA library not combined with the target substance binds to the surface of the gold nanoparticle through the base of the nucleic acid.

[0060] 2) Centrifuge the reaction solution from Step 1) at 6,500 rcf at 4°C for 30 minutes, and transfer the supernatant to a new tube. Transfer the precipitated gold nanoparticles to a 96-well plate, add 0.2X PBS to sufficiently disperse them, and allow to stabilize at room temperature for 5 minutes.

[0061] - When centrifuged, the ssDNA library bound to the target substance remains in the supernatant, and the ssDNA library that is relatively not bound to the target substance exists together with the gold nanoparticles in the sediment.

[0062] 3) Add 0.8 M NaCl solution to the gold nanoparticles in a 96-well plate and incubate at room temperature for 15 minutes. Afterwards, observe the color change of the gold nanoparticles and measure the absorbance at 520 nm and 720 nm.

[0063] 4) Add the target substance to the supernatant transferred to a new tube and incubate at room temperature for 30 minutes. Since the supernatant contains the ssDNA library that reacted with the target substance, a reaction occurs between the added target substance and the ssDNA library.

[0064]

[0065] The rounds were stopped when the color change between the precipitated gold nanoparticles in the reaction solution containing the target substance and the control solution containing no target substance reached its maximum. The final supernatant was then recovered from the reaction solution containing the target substance, and aptamers that specifically bind to the target substance were selected from the recovered supernatant.

[0066]

[0067] The present applicant named the above-described aptamer screening method Gold Nanoparticle-Facilitated Assembly of Supernatant Transfer (GNP-FAST).

[0068]

[0069]

[0070] Experimental results

[0071] When the target material is present in the reaction solution, the single-stranded nucleic acid library is present in greater quantities in the supernatant compared to the control, and conversely, the single-stranded nucleic acid library is present in less quantity in the precipitated gold nanoparticles.

[0072] When salt is added to the precipitated gold nanoparticles, the aggregation of gold nanoparticles in the sample containing the target substance progresses faster than in the control group, resulting in a rapid color transition from red to purple in the reaction solution. In addition, the absorbance at 700 nm increases due to the aggregation of gold nanoparticles, so E 520 / E 700 The ratio is lower than that of the control group. That is, E 520 / E 700If the ratio is high, it can be seen that the gold nanoparticles are not agglomerated, and if it is high, it can be seen that the gold nanoparticles are agglomerated.

[0073] Both the color change of the gold nanoparticle precipitate visible to the naked eye and the absorbance measurement of the precipitate can be used for aptamer screening, especially for the E between the control and experimental groups. 520 / E 700 If there is a round in which the difference in ratio is the greatest, it can be seen that the aptamer that binds to the target substance was screened in that round. Based on the results of Figures 2 to 4, E of the experimental and control groups 520 / E 700 If the ratio differs by about 2 times or more, it can be considered the most suitable round for screening.

[0074]

[0075] 1. Use of diethylhexyl phthalate (DEHP) as a target substance

[0076] GNP-FAST was performed using diethylhexyl phthalate (DEHP) as the target substance. The color change of the gold nanoparticle precipitate, the absorbance of the gold nanoparticle precipitate at 520 nm and 720 nm, and the concentration of the single-stranded library in the supernatant were determined for each round.

[0077] As a result of the verification, it was found that the color of the reaction changed to purple more quickly when the target substance DEHP was present (Fig. 1A). In addition, when the target substance DEHP was present, E 520 / E 700 This was lower, especially in rounds 2 and 3, compared to the control group and E 520 / E 700 The difference in ratio was significant (Fig. 1B).

[0078]

[0079] 2. Use of cortisol as a target substance

[0080] Similar to the results in Figure 1, the color change of the reaction solution was faster when cortisol was present, and the color change of the reaction solution was particularly noticeable from the second round, E 520 / E 700 It was found that the ratio was significantly reduced (Figures 2A and 2B).

[0081]

[0082] 3. Use of aldosterone as a target substance

[0083] When aldosterone was present, the color change of the reaction solution was faster, and the color change of the reaction solution was particularly noticeable from the second round, E 520 / E 700 It was found that the ratio was significantly reduced (Figures 3A and 3B).

[0084]

[0085] 4. Use of diethyl phthalate (DEP) as a target substance

[0086] When DEHP, cortisol, and aldosterone were used as target substances, the presence of random single-stranded nucleic acids that bind to the target substances in the ssDNA library resulted in a difference in the color of the reaction solution depending on the presence or absence of the target substances added.

[0087] However, when DEP was used as the target substance, there was no significant difference in the color of the reaction solution regardless of the addition of the target substance, and E 520 / E 700 The ratio also did not differ significantly between the control and experimental groups (Figures 4A and 4B). Therefore, it was found that the GNP-FAST method could not be applied in these cases.

Claims

1. A method for screening a single-stranded nucleic acid that specifically binds to a target substance, comprising the following steps: a) A step of reacting a target material or a non-target material with a single-stranded nucleic acid library, respectively; b) a step of adding gold nanoparticles to the above a) and causing a reaction; c) a step of centrifuging the result of b); d) The supernatant is transferred to a new tube, and the precipitated gold nanoparticles are subjected to aggregation; e) a step of checking the absorbance of the aggregated gold nanoparticles to determine whether to proceed with additional reactions; and f) If additional reactions are required, repeat steps b) to e) by adding target or non-target substances to the supernatant transferred to a new tube.

2. In paragraph 1, A method for screening a single-stranded nucleic acid that specifically binds to a target substance, wherein steps a) and b) above are performed at room temperature.

3. In paragraph 1, The absorbance of the above step e) is confirmed by measuring the absorbance of the aggregated gold nanoparticles E 520 / E 700 A screening method for single-stranded nucleic acids that specifically bind to a target substance, the method comprising: confirming the ratio.

4. In paragraph 3, The above step e) is to react with a non-target substance to form E of the aggregated gold nanoparticles. 520 / E 700 E of the gold nanoparticles aggregated by reacting with the target substance 520 / E 700 A method for screening single-stranded nucleic acids that specifically bind to a target substance, wherein step f) is performed when the ratio is 1.5 to 3 times higher.

5. In paragraph 1, A method for screening a single-stranded nucleic acid that specifically binds to a target substance, wherein the step e) further includes visually confirming a color change of the aggregated gold nanoparticles.

6. In paragraph 1, A method for screening a single-stranded nucleic acid that specifically binds to a target substance, wherein the target substance is selected from the group consisting of low-molecular-weight compounds, nucleic acids, peptides, proteins, protein variants, lipids, lipid variants, carbohydrates, and carbohydrate variants.

7. In paragraph 1, A method for screening single-stranded nucleic acids that specifically bind to a target substance, wherein the gold nanoparticles have an average diameter of 15 nm to 50 nm.

8. In paragraph 1, A method for screening a single-stranded nucleic acid that specifically binds to a target substance, wherein the aggregation of the gold nanoparticle precipitate in step d) is achieved by adding salt.

9. In paragraph 1, A method for screening a single-stranded nucleic acid that specifically binds to a target substance, wherein steps b) to e) are repeated 2 to 10 times in step f).

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