Use of graphene oxide in the preparation of single-stranded DNA secondary library for aptamer screening

Abstract

The present application relates to the application of graphene oxide in the preparation of single-stranded DNA secondary library for aptamer screening. Specifically, graphene oxide is incubated with the product after asymmetric PCR amplification, and the single-stranded DNA secondary library on graphene oxide can be obtained after centrifugation and washing. The method of the present application is simple, does not affect the specificity and affinity of aptamer, has the advantages of low cost and high efficiency, overcomes the problems of existing single-stranded DNA secondary library preparation methods, such as many steps, high operation requirements and high cost, and helps to screen high-quality aptamer.

Description

Technical Field

[0001] This invention relates to the application of graphene oxide in the preparation of single-stranded DNA secondary libraries for nucleic acid aptamer screening. Background Technology

[0002] Nucleic acid aptamers offer significant advantages over antibodies, including ease of sequencing, synthesis, and amplification, and hold immense promise for applications in medical, food, and environmental testing. Aptamers are derived from randomly synthesized single-stranded DNA (ssDNA) as an initial library. Through systematic evolution of ligands by exponential enrichment (SELEX) and subsequent rounds of screening of secondary libraries, ssDNA that binds to targets with high affinity and specificity is obtained.

[0003] The preparation of ssDNA secondary libraries is crucial for nucleic acid aptamer screening. The initial ssDNA library consists of a large number of randomly sequenced ssDNA molecules. After screening from the initial library, some ssDNA molecules that bind to the target are collected, amplified by PCR, and then prepared as ssDNA secondary libraries for the next round of screening. Currently, methods for preparing ssDNA secondary libraries include: conventional PCR-exonuclease digestion. 【1】 Conventional PCR-Biotin-Streptavidin Magnetic Bead Separation Method 【2】 Asymmetric PCR-gel separation method 【3】 .

[0004] References

[0005] 【1】.Uemachi,H.,Kasahara,Y.,Tanaka,K.,Okuda,T.,Yoneda,Y.,&Obika,S.(2021).Hybrid-Type SELEX for the Selection of Artificial Nucleic AcidAptamers Exhibiting Cell Internalization Activity.Pharmaceutics,13(6),888.https: / / doi.org / 10.3390 / pharmaceutics13060888;

[0006] 【2】.Sun, S., Liu, H., Hu, Y., Wang, Y., Zhao, M., Yuan, Y., Han, Y., Jing, Y., Cui, J., Ren, X., Chen, X., & Su, J. (2022). Selection and identification of a novel ssDNAaptamer targeting human skeletal muscle. Bioactive materials, 20, 166–178. https: / / doi.org / 10.1016 / j.bioactmat.2022.05.016;

[0007] 【3】.El-Husseini,DM,Sayour,AE,Melzer,F.,Mohamed,MF,Neubauer,H.,&Tammam,RH(2022).Generation and Selection of Specific Aptamers TargetingBrucella Species through an Enhanced Cell-SELEX Methodology.Internationaljournal of molecular sciences, 23(11), 6131. ​​https: / / doi.org / 10.3390 / ijms23116131.

[0008] (1) Preparation of ssDNA secondary libraries by conventional PCR-exonuclease digestion method:

[0009] The conventional PCR-exonuclease digestion method involves adding Lambda exonuclease to the conventional PCR amplification product to digest the double-stranded DNA (dsDNA) produced by PCR amplification, thereby obtaining ssDNA. To remove undigested dsDNA, digestion debris, nucleases, etc., the digested amplification product still requires polyacrylamide gel electrophoresis, gel excision, and purification. The yield of ssDNA obtained by this method is extremely low.

[0010] (2) Preparation of ssDNA secondary libraries by conventional PCR-biotin-streptavidin magnetic bead method:

[0011] The conventional PCR-biotin-streptavidin magnetic bead method involves PCR amplification using one of the primers labeled with biotin. After amplification, streptavidin magnetic beads are added to the product, allowing the biotin-carrying dsDNA to bind to the streptavidin-carrying beads, thus separating the dsDNA from the amplification product. The dsDNA bound to the magnetic beads is then treated with an alkaline method or a heat method to release unlabeled ssDNA. However, alkaline or heat treatment of ssDNA results in low yields and affects the specificity and affinity of nucleic acid aptamers.

[0012] (3) Asymmetric PCR method – gel extraction and purification to prepare ssDNA secondary libraries:

[0013] Asymmetric PCR – gel extraction purification – uses unequal amounts of a primer pair for PCR amplification. In the initial cycles, the amplification product is dsDNA; however, when the low-concentration primers (restriction primers) are depleted, and only the high-concentration primers (unrestriction primers) remain, PCR amplification produces a large amount of ssDNA. Asymmetric PCR – gel extraction purification involves polyacrylamide gel electrophoresis, gel extraction, metal bath heating, column separation or magnetic separation, high-speed centrifugation, washing, and elution, among other experimental steps. This method is time-consuming, involves many steps, and requires highly skilled technicians.

[0014] In summary, current methods for preparing ssDNA secondary libraries used in each round of nucleic acid aptamer screening suffer from numerous steps, high operational requirements, and high costs. To obtain high-quality nucleic acid aptamers, the problems with current ssDNA secondary library preparation methods urgently need to be addressed. Summary of the Invention

[0015] Currently, the preparation of ssDNA secondary libraries in nucleic acid aptamer screening involves numerous technical steps, high operational requirements, and high costs. To address these issues, this invention provides the application of graphene oxide in the preparation of single-stranded DNA secondary libraries, specifically, the preparation of ssDNA secondary libraries using graphene oxide, which can be directly applied to each round of nucleic acid aptamer screening.

[0016] When using graphene oxide to construct ssDNA secondary libraries, it is only necessary to incubate the graphene oxide with the product of asymmetric PCR amplification, followed by centrifugation and washing to obtain the ssDNA secondary library on the graphene oxide. The method is simple, does not affect the specificity and affinity of nucleic acid aptamers, and has the advantages of low cost and high efficiency. It overcomes the problems of multiple steps, high operational requirements and high cost in the preparation of existing single-stranded DNA secondary libraries, and helps to screen for high-quality nucleic acid aptamers.

[0017] To date, in the process of nucleic acid aptamer screening, graphene oxide has only been used to adsorb ssDNA that has not been bound to the target, and the adsorbed graphene oxide is removed by centrifugation; currently, graphene oxide has not been used for the preparation of ssDNA secondary libraries.

[0018] This invention creatively utilizes graphene oxide for the construction of ssDNA secondary libraries, innovatively combining the carboxyl groups on the surface of graphene oxide, which deprotonate at neutral pH, resulting in a negative charge. Negatively charged graphene oxide electrostatically repels similarly negatively charged ssDNA or dsDNA. However, under suitable cation concentration conditions in the buffer solution, ssDNA can strongly adsorb onto graphene oxide through the aromatic and hydrophobic rings on its exposed bases, via π-π stacking and hydrophobic interactions; while dsDNA, with its bases hidden in a double helix structure, can only weakly adsorb onto graphene oxide.

[0019] In some embodiments of the present invention, the above-described incubation system contains 0.125-0.25 mg / ml of graphene oxide (GO).

[0020] In some embodiments of the present invention, the cation is Mg2+. Experiments have shown that Mg2+ in the concentration range of 10-20 mM is beneficial for reducing self-desorption.

[0021] In some embodiments of the present invention, a GO-ssDNA library is constructed by the following steps:

[0022] S1. During the screening of nucleic acid aptamers, asymmetric PCR is used to amplify the ssDNA that has been screened in the previous round and is bound to the target.

[0023] S2. Graphene oxide adsorbs ssDNA from asymmetric PCR products to form ssDNA-GO.

[0024] S3. Centrifuge to precipitate the precipitate, then wash to obtain the ssDNA-GO secondary library from the asymmetric PCR product. This ssDNA-GO secondary library can be used directly for the next round of screening.

[0025] The beneficial effects of this invention are as follows: This invention utilizes an ssDNA-GO secondary library constructed based on graphene oxide, overcoming the shortcomings of existing methods for preparing single-stranded DNA secondary libraries. Compared with current methods for preparing single-stranded DNA secondary libraries, this method is simpler to operate, lower in cost, and more efficient, making it suitable for nucleic acid aptamer screening or other fields requiring ssDNA libraries. Attached Figure Description

[0026] Figure 1The agarose gel electrophoresis image shows that the ssDNA bound to the target in the previous round of nucleic acid aptamer screening was amplified by asymmetric PCR to produce ssDNA, which was then adsorbed by graphene oxide.

[0027] Lane 1. dsDNA band generated by conventional PCR amplification of ssDNA library; Lane 2. ssDNA band generated by asymmetric PCR amplification; Lane M. Standard DNA Marker; Lane 3. Supernatant after incubation of asymmetric PCR product with graphene oxide and centrifugation; Lane 4. Precipitate after incubation of asymmetric PCR product with graphene oxide and centrifugation.

[0028] Figure 2 The quantitative real-time PCR amplification curve shows that the adsorption system in which graphene oxide (GO) adsorbs ssDNA from asymmetric PCR products, after centrifugation, the precipitate (GO-ssDNA), after being washed twice, was incubated with the next round of target (water as a control) and then centrifuged again. The amount of ssDNA in the supernatant obtained was 300 times higher than that in the control, indicating that the target competitively and effectively binds to ssDNA from GO-ssDNA.

[0029] Curve 1: Target, Ct value: 6.95; Curve 2: Water, Ct value: 15.17; Curve P: Positive control of qPCR reaction, Ct value: 18.73; Curve N: Negative control of qPCR reaction, no Ct.

[0030] Figure 3 A is an agarose gel electrophoresis image [lanes 1-3. ssDNA prepared by the method of this invention (repeated 3 times); lanes 4-6. ssDNA prepared by asymmetric PCR-gel purification method (repeated 3 times)]; B is the quantification of the grayscale of the electrophoretic bands, column 1 (black) is the quantification of lanes 1-3; column 2 (white) is the quantification of lanes 4-6, showing that under the same sample and quantity of asymmetric PCR products, the amount of ssDNA prepared by the method of this invention is significantly greater than that prepared by the asymmetric PCR-gel purification method, indicating that the secondary library of ssDNA prepared by the method of this invention has a more efficient library capacity. Detailed Implementation

[0031] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with the accompanying drawings and embodiments, is provided below.

[0032] Example 1:

[0033] This embodiment constructs a single-stranded DNA secondary library in graphene oxide and applies it to nucleic acid aptamer screening, including the following steps:

[0034] Step 1: PCR amplification

[0035] During the nucleic acid aptamer screening process, asymmetric PCR amplifies the ssDNA that has bound to the target from the previous round of screening.

[0036] The asymmetric PCR reaction system is as follows:

[0037]

[0038]

[0039] The standard PCR reaction system is as follows:

[0040]

[0041] The 2×Taq PCR enzyme premix was purchased from Beijing Tiangen Biotech Co., Ltd., product number KT201.

[0042] The primer amounts and primer ratios for the asymmetric PCR reaction are as follows:

[0043] Upstream primer: 5 μl 10 μM upstream primer; Downstream primer: 1 μl 1 μM downstream primer; Upstream primer / downstream primer = 50

[0044] The initial ssDNA library consists of 76 bp of nucleotides, with a 40 bp random sequence in the middle and 18 bp primer sequences at both ends.

[0045] All primers and ssDNA libraries were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0046] Amplification procedures for asymmetric PCR and conventional PCR:

[0047]

[0048] The asymmetric PCR product was used as sample 1, and the regular PCR product was used as sample 2.

[0049] Step 2: Graphene oxide (GO) adsorbs ssDNA from the asymmetric PCR product to form ssDNA-GO.

[0050] The reaction system for the adsorption of ssDNA by graphene oxide is as follows:

[0051]

[0052] The 0.5 mg / ml graphene oxide was purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd., item number 100675.

[0053] The reaction conditions for graphene oxide to adsorb ssDNA were 350 rpm and constant temperature incubation at 25℃ for 40 min.

[0054] The reaction conditions for separating ssDNA from the asymmetric PCR product were centrifugation at 12000 rpm for 15 min, followed by aspirating the supernatant as sample 3 and retaining the ssDNA-GO precipitate as sample 4. (Note: At this point, a large amount of ssDNA has adsorbed onto GO. GO is separated from the supernatant by centrifugation. The separated supernatant and GO precipitate are added to the electrophoresis loading buffer for gel electrophoresis detection, which can reveal...) Figure 1 The gel image of the supernatant showed no ssDNA bands, while the gel image of the GO precipitate showed clear ssDNA bands, which verifies that most of the ssDNA was adsorbed onto the GO precipitate.

[0055] Samples 1-4 were analyzed by agarose gel electrophoresis. The agarose gel concentration was 2.5%. The marker used was a 50bp DNA ladder, purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number M1800. Figure 1 The actual nucleic acid loading volume for all samples was 2 μl, and the DNA marker loading volume was 2.5 μl. Figure 1 As can be seen, gel electrophoresis of asymmetric PCR products only shows the ssDNA band. Figure 1 Lane 2 shows that ssDNA was effectively amplified; further, the supernatant in lane 3 shows no ssDNA band, while the precipitate of this adsorption system, after washing, showed an ssDNA band on gel electrophoresis (lane 4), indicating that GO effectively adsorbed ssDNA.

[0056] Step 3: Centrifuge to precipitate, then wash to obtain the ssDNA-GO secondary library.

[0057] Slowly add 50 μl of 1×PBS buffer (containing 5 mM Mg) to the ssDNA-GO precipitate obtained in step two. 2+ Then centrifuge at 12000 rpm for 5 min, discard the supernatant and retain the precipitate to obtain the ssDNA-GO secondary library.

[0058] Additionally, the kit used for preparing ssDNA secondary libraries using the asymmetric PCR-gel purification method was purchased from Shanghai Sangon Biotech Co., Ltd., catalog number B518745. Specific experimental procedures were performed according to the kit instructions (reference webpage: www.sangon.com / productDetail?productInfo.code=B518745).

[0059] The experiments of preparing ssDNA secondary libraries using the ssDNA-GO secondary library and the asymmetric PCR-gel purification method were each repeated three times.

[0060] ssDNA samples prepared by GO separation and gel excision purification were detected by agarose gel electrophoresis. Because the experiment was repeated 3 times, a total of 6 samples were obtained, which were labeled as sample 1 to sample 6. The actual nucleic acid loading volume of all samples was 1 μl, and the DNA marker loading volume was 2.5 μl.

[0061] The amount of ssDNA secondary libraries prepared from asymmetric PCR products was compared between the method of this invention and the asymmetric PCR-gel extraction purification method using relative quantification. The analysis software used was the Tanon-1600 gel imaging processing system from Shanghai Tianneng Technology Co., Ltd., and the data were statistically analyzed using GraphPad Prism 8.0 software from GraphPad Software. The agarose gel electrophoresis results and relative quantification results are shown below. Figure 3 As shown in the figure, under the same sample size and quantity of asymmetric PCR products, the method of this invention produces a significantly larger amount of ssDNA than the method prepared by asymmetric PCR-gel purification. This indicates that the method of this invention produces a more efficient ssDNA secondary library.

[0062] Step 4: Use the ssDNA-GO secondary library for the next round of screening to test the binding ability of the target to the ssDNA-GO secondary library.

[0063] The reaction system for target adsorption of ssDNA is as follows:

[0064]

[0065] The reaction system of the control group (without the target molecule, but with an equal volume of ddH2O added) is as follows:

[0066]

[0067]

[0068] The GO-ssDNA secondary libraries in the target experimental group and the control group had the same source and quantity.

[0069] The incubation conditions for the target experimental group / control group were 350 rpm and constant temperature incubation at 25℃ for 30 min.

[0070] After incubation, the samples from the target experimental group / control group were centrifuged at 12,000 rpm for 15 min, and then the supernatant was carefully aspirated as sample 4-1 and sample 4-2.

[0071] In step four, qPCR is used to detect samples 4-1 and 4-2, and a positive control P (2 μl 10 pMssDNA library) and a negative control N (2 μl ddH2O) are set up.

[0072] The qPCR reaction system is as follows:

[0073]

[0074] The 2×qPCR enzyme premix was purchased from Beijing Lanjieke Technology Co., Ltd., product number BL705A.

[0075] The amplification procedure for qPCR is as follows:

[0076]

[0077] The amplification curves of sample 4-1, sample 4-2, positive control P, and negative control N were analyzed using the built-in software of the MA-6000 qPCR instrument from Suzhou Yarui Biotechnology Co., Ltd. The results are as follows: Figure 2 As shown. From Figure 2 It can be seen that after incubating GO-ssDNA with the target of the next round of screening (using water as a control) and then centrifuging, the target in the supernatant effectively bound the ssDNA from GO-ssDNA. The amount of ssDNA in the obtained supernatant was about 300 times higher than that of the control (sample 4-2) (Note: According to the relative quantification method -- 2-ΔCt method, the fold difference in ssDNA amount between curve 1 and curve 2 = 2^-(6.95-15.17) = 2^8.22 = 298.17. Reference: Pryor, RJ, & Wittwer, CT. (2006). Real-time polymerase chain reaction and melting curve analysis. Methods in Molecular Biology, 336, 19-32.). This indicates that the GO-ssDNA secondary library constructed by GO has almost no effect on the specificity and affinity of nucleic acid aptamers.

[0078] The above results show that, through this invention, after obtaining the ssDNA-GO secondary library, it can be applied to subsequent screening of nucleic acid aptamers. After adding the target solution—generally a protein or small molecule—to the ssDNA-GO precipitate, the affinity ssDNA will desorb from the GO and adsorb onto the target molecule in the supernatant. PCR amplification of the target-ssDNA in the supernatant can amplify the affinity ssDNA. Then, based on the ssDNA enrichment, it can proceed to the next round of nucleic acid aptamer screening. In the experiment of step four, ssDNA was bound using both the target and water. It was found that in the presence of the target, a large amount of affinity ssDNA desorbed from the GO and then bound to the target in the supernatant. However, in the absence of the target, only a small portion of the ssDNA self-desorbed and entered the supernatant.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Use of graphene oxide for the preparation of a single-stranded DNA secondary library for aptamer selection, characterized in that, The single-stranded DNA is adsorbed on the graphene oxide; The asymmetric PCR product is incubated with the graphene oxide, centrifuged and washed to obtain a ssDNA secondary library on the graphene oxide; The incubation system comprises 0.125-0.25 mg / ml of graphene oxide and 10-20 mM of Mg 2+ ; The graphene oxide is used for the construction of the ssDNA secondary library, and the carboxyl groups contained on the surface of the graphene oxide are deprotonated at neutral pH, and the graphene oxide is negatively charged, and the negatively charged graphene oxide and the ssDNA or dsDNA with the same negative charge are electrostatically repelled, but under the condition of a suitable cation concentration in the buffer, the ssDNA is strongly adsorbed on the graphene oxide through the aromatic ring and the hydrophobic ring on the exposed base by π-π stacking and hydrophobic interaction force; and the bases of the dsDNA are hidden in the double helix structure, and the dsDNA can only be weakly adsorbed on the graphene oxide; The GO-ssDNA library is constructed by the following steps: S1. In the process of aptamer screening, the ssDNA combined with the target in the asymmetric PCR amplification of the last round of screening; S2. The ssDNA in the asymmetric PCR product is adsorbed on the graphene oxide to form ssDNA-GO; S3. After centrifugal precipitation, the ssDNA-GO secondary library is obtained from the asymmetric PCR product by washing, and the ssDNA-GO secondary library is directly used for the next round of screening.

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