Combined element for screening aptamer and use thereof, and high-throughput screening method for aptamer

Abstract

A high-throughput screening method for an aptamer, and a use. An implementation route is divided into three parts, a first part is construction of a library of a pre-enriched aptamer and sequencing same, a second part is generation of an aptamer cluster on a slide, and a third part is combination of a target protein and the aptamer cluster. Provided is a method for high-throughput rapid detection of an aptamer. The method has the beneficial effects: sequencing time, sequence synthesis and affinity re-verification time are saved, the affinity verification costs are greatly saved, rapid screening of aptamers can be promoted, and the actual use of aptamers is promoted along with the increase in the types and numbers of aptamers. The existing platform BGISEQ 500 of Beijing Genomics Institute can be used to grow an aptamer cluster on a chip by using DNB as a template, and the use of sequencers is expanded by integrating sequencing, sequence synthesis and affinity identification.

Description

Combination elements for screening nucleic acid aptamers and their applications, as well as high-throughput screening methods for nucleic acid aptamers Technical Field

[0001] The present invention relates to the field of nucleic acid aptamers, in particular to a combination element for screening nucleic acid aptamers and an application thereof, as well as a high-throughput screening method for nucleic acid aptamers. Background Art

[0002] Aptamers are typically single-stranded DNA or RNA molecules that can fold into a specific structure, allowing them to bind specifically and with high affinity to target molecules. Aptamers have a wide range of target molecules, including proteins, enzymes, growth factors, antibodies, and other biomacromolecules, as well as small molecules such as metal ions, nucleotides, and amino acids. Aptamers are easy to modify and label, and can be synthesized in vitro through chemical synthesis. They can be modified at specific sites, such as with enzymes, biotin, radioactive isotopes, and biotin. Aptamers also exhibit good stability. Aptamers are essentially nucleic acids with a certain tolerance to temperature, pH, and ionic strength, making them easier to store, transport, and use over a long period of time. Compared to antibodies, they possess greater stability.

[0003] Systematic evolution by ligand exponential enrichment (SELEX) is a traditional method for screening nucleic acid aptamers. The basic principle of SELEX technology is to chemically synthesize a single-stranded oligonucleotide library in vitro, mix it with the target substance, and through repeated screening and amplification, wash away nucleic acid molecules that do not bind to the target substance or have high affinity for the target substance, and separate and purify nucleic acid aptamers with high affinity for the target substance. The traditional SELEX method is cumbersome to operate and has a long screening cycle. After screening, whether selecting single clones for affinity verification by sequencing or selecting sequences for affinity verification after high-throughput sequencing, it takes a lot of time and cost, and the throughput is very low. Generally, only a few or dozens of sequences can be selected for verification. Since sequences with high abundance are not necessarily sequences with affinity, selecting only a few sequences for affinity verification is likely to miss sequences that truly have affinity.

[0004] Summary of the Invention

[0005] In view of this, the present invention provides a high-throughput screening method for nucleic acid aptamers.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a combination element for screening nucleic acid aptamers, characterized by comprising any of the following items or a combination thereof:

[0008] (I) Aptamer library:

[0009] The nucleic acid aptamer library includes a first fixed sequence, a random sequence and a second fixed sequence from the 5' end to the 3' end;

[0010] (II), Primer set:

[0011] The primer set includes an upstream primer and a downstream primer;

[0012] The upstream primer includes a first linker sequence and a first complementary sequence;

[0013] The first complementary sequence includes: a sequence that is partially or fully complementary to the first fixed sequence;

[0014] The downstream primer includes a second linker sequence and a second complementary sequence;

[0015] The second complementary sequence includes: a sequence that is partially or fully complementary to the second fixed sequence;

[0016] (III), nucleic acid circularization reagents and / or digestion reagents;

[0017] (IV) a carrier coupled with a linker primer: the linker primer can hybridize with the DNB obtained after cyclization of the aptamer;

[0018] Furthermore, it also includes (V) a fluorescent primer; the fluorescent primer can hybridize with the linker primer.

[0019] In some specific embodiments of the present invention, the nucleic acid aptamer library has:

[0020] 1. the nucleotide sequence shown in SEQ ID No. 1; or

[0021] II. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in I and having the same or similar function as the nucleotide sequence shown in I; or

[0022] III. A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in I, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in I.

[0023] In some specific embodiments of the present invention, the upstream primer has:

[0024] IV, the nucleotide sequence shown in SEQ ID No. 2; or

[0025] V. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in IV and having the same or similar function as the nucleotide sequence shown in IV; or

[0026] VI. a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in IV, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in IV;

[0027] The downstream primer has:

[0028] VII. the nucleotide sequence shown in SEQ ID No. 3; or

[0029] VIII. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in VII and having the same or similar function as the nucleotide sequence shown in VII; or

[0030] IX. A nucleotide sequence having at least 80% homology to the nucleotide sequence shown in VII, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in VII.

[0031] In some specific embodiments of the present invention, the aptamer library is used as a template, and the upstream and downstream primers are used to perform overhang PCR amplification and purification to obtain a template DNA; the template DNA has the nucleotide sequence shown in SEQ ID No. 4. The template DNA described in the present invention is merely an example to demonstrate the specific effects of the present invention. The sequence of the template DNA can be selected or replaced according to the specific detection purpose. Any template DNA can be screened using the combination elements for screening nucleic acid aptamers described in the present invention. Any template DNA is within the scope of protection of the present invention and is not limited herein.

[0032] In some specific embodiments of the present invention, the fluorescent primers include, but are not limited to, ROX fluorescent primers, such as AF532, or any fluorescent protein that can be recognized by a sequencer's fluorescence channel. The fluorescent proteins include, but are not limited to, AF647 fluorescent protein. The ROX fluorescent primers have the nucleotide sequence shown in SEQ ID No. 5. Any fluorescent primer or fluorescent protein known in the art is within the scope of protection of the present invention and is not limited thereto.

[0033] The present invention provides the use of the combination element in any of the following items:

[0034] (I) high-throughput screening of nucleic acid aptamers;

[0035] (II) preparing reagents, kits or devices for high-throughput screening of nucleic acid aptamers;

[0036] (III) preparing a reagent, a kit or a device for detecting the target molecule of the nucleic acid aptamer.

[0037] In some specific embodiments of the present invention, the target molecules include but are not limited to proteins, polypeptides, ions, small molecules, macromolecular polymers, nucleic acids and living cells;

[0038] Furthermore, the target molecule includes syphilis antigen;

[0039] Furthermore, the target molecule is modified with a fluorescent protein or a fluorescent small molecule. The viral antigens described in the present invention are merely examples to demonstrate the specific effects of the present invention. The target molecule can be selected or replaced according to the specific detection purpose. Any target molecule can be screened using the combination elements for screening nucleic acid aptamers described in the present invention. Any target molecule is within the scope of protection of the present invention and is not limited herein.

[0040] The present invention provides a method for screening nucleic acid aptamers, which is based on the combination elements, and obtains nucleic acid aptamers that meet the requirements according to sequence information and protein binding results.

[0041] In some specific embodiments of the present invention, the screening method comprises the following steps:

[0042] Obtaining a nucleic acid aptamer library with sequencing adapters connected at both ends, circularizing the nucleic acid aptamer library with adapters using a DNB preparation technology to obtain DNBs and sequence them;

[0043] Dissociating the sequencing chain generated by the sequencing, hybridizing the DNB with the adapter primer on the carrier, extending the nucleic acid aptamer sequence on the adapter primer, and then digesting the DNB to obtain a carrier loaded with the nucleic acid aptamer cluster;

[0044] The fluorescent primer is hybridized with the linker primer on the carrier loaded with the nucleic acid aptamer cluster, and then the fluorescent target protein is bound to the nucleic acid aptamer to obtain a protein binding result;

[0045] According to the sequence information of the nucleic acid aptamer at each position and the binding result with the protein, the nucleic acid aptamer that meets the requirements is obtained.

[0046] In some specific embodiments of the present invention, the step of obtaining a nucleic acid aptamer library with sequencing adapters connected to both ends comprises:

[0047] constructing nucleic acid aptamers according to the first fixed sequence, the random sequence, and the second fixed sequence in sequence to form a nucleic acid aptamer library;

[0048] Using an upstream primer with the first adapter sequence and a downstream primer with the second adapter sequence, the first adapter sequence and the second adapter sequence are connected to both ends of the nucleic acid aptamer by Overhang PCR to obtain a nucleic acid aptamer library with sequencing adapters connected to both ends.

[0049] The present invention provides a reagent or a kit comprising the combination element.

[0050] The present invention provides a device comprising the combined element.

[0051] The present invention provides a syphilis antigen nucleic acid aptamer, wherein the syphilis antigen nucleic acid aptamer has:

[0052] I. a nucleotide sequence as shown in any of SEQ ID Nos. 6 to 8; or

[0053] II. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in I and having the same or similar function as the nucleotide sequence shown in I; or

[0054] III. A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in I, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in I.

[0055] The syphilis antigen nucleic acid aptamer can be obtained by screening using the nucleic acid aptamer screening method with syphilis antigen as the target.

[0056] The present invention provides the use of the syphilis antigen nucleic acid aptamer in preparing a reagent, a kit or a device for detecting syphilis.

[0057] The present invention provides application of the syphilis antigen nucleic acid aptamer in detecting syphilis.

[0058] The beneficial effects brought about by the technical solution provided by the present invention are as follows:

[0059] 1. The present invention provides a high-throughput, rapid detection method for nucleic acid aptamers, integrating sequencing, sequence synthesis, and affinity identification. This method saves sequencing time, sequence synthesis time, and affinity revalidation time, while significantly reducing affinity validation costs. It can promote the rapid screening of nucleic acid aptamers, and as the types and number of nucleic acid aptamers increase, it will promote the practical application of nucleic acid aptamers.

[0060] 2. The present invention utilizes the BGISEQ 500 platform of BGI to extend nucleic acid aptamer clusters on the chip using DNB as template, which has high throughput and can screen more nucleic acid aptamers simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 shows DNB, aptamer cluster, and aptamer cluster bound to fluorescently labeled target protein;

[0062] FIG2 is a schematic diagram showing the nucleic acid aptamer library plus sequencing adapter sequence in the present invention; wherein,

[0063] Figure 3 shows the circularization of the linker-added library using BGI's DNB preparation technology;

[0064] Figure 4 shows the use of the DBCO click method to couple hybridization primers to a chip after sequencing, and to extend new nucleic acid aptamers after binding to the template DNA;

[0065] FIG5 shows a sequencing chip after template DNB digestion and the nucleic acid aptamer clusters on the chip;

[0066] Figure 6 shows the hybridization of ROX fluorescent primer and adapter primer and the binding of AF647 fluorescent protein to the nucleic acid aptamer on the chip;

[0067] FIG7 shows the binding results of AF647 fluorescent protein and nucleic acid aptamer;

[0068] FIG8 shows the binding of fluorescent target (TP) protein to nucleic acid aptamer;

[0069] FIG9 shows the statistical results of the binding ratio of fluorescent proteins with more than 1000 aptamers;

[0070] Figure 10 shows the TP-11 SPR verification results;

[0071] Figure 11 shows the TP-65 SPR verification results;

[0072] FIG12 shows the TP-93 SPR verification results. DETAILED DESCRIPTION

[0073] The present invention discloses a high-throughput screening method for nucleic acid aptamers. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0074] The screened aptamer library was subjected to high-throughput sequencing using the BGISEQ 500 sequencing platform. After sequencing, the aptamers were grown in situ and bound to the fluorescently labeled target protein, as shown in Figure 1. By combining sequence information with protein binding information, affinity aptamers can be quickly obtained.

[0075] The experimental principle of the present invention is as follows:

[0076] Part 1: Library Construction and Sequencing

[0077] The aptamer library consists of fixed sequences at both ends and a random sequence in the middle. The fixed sequences at both ends are used for PCR. Overhang primers contain adapter sequences, and sequencing adapter sequences are added to both ends of the library through PCR, as shown in Figure 2. After library construction, the adapter-added library is circularized using BGI's preparative DNB technology, as shown in Figure 3, and then sequenced.

[0078] Overhang PCR is primer extension PCR. Overhang primers carry complementary sequences and adapter sequences to the fixed sequences at both ends of the library. After PCR amplification, adapter sequences can be added to both ends of the library to facilitate subsequent sequencing.

[0079] The splint oligo used for library circularization has a sequence that is complementary to both ends of the nucleic acid aptamer library with the addition of a linker. It is mixed with the single strand of the nucleic acid aptamer library with the addition of a linker and ligated to circularize the library.

[0080] DNB, or DNA nanoball, is based on rolling circle amplification, which performs linear rolling circle amplification on the circularized library to form a single DNA strand containing a large number of identical libraries. The DNA single strand folds freely to form a DNA nanoball.

[0081] Part II: Preparation of Aptamer Cluster Slides

[0082] After sequencing, the sequencing strands are dissociated on the chip. New adapter primers (capture oligo) are then coupled to the chip surface using the DBCO click method. These adapter primers can then hybridize with the DNBs, allowing them to grow into aptamers, as shown in Figure 4. The template DNBs are then digested, leaving only the aptamer clusters required for the experiment on the sequencing chip, as shown in Figure 5.

[0083] The reason for dissociating the sequencing chain is that the sequencing chain will affect the hybridization of the adapter primer and DNB in ​​the next step.

[0084] Part III: Fluorescence localization primer hybridization and protein binding

[0085] Using the BGISEQ 500loader, the ROX fluorescent primer was first hybridized with the adapter primer, as shown in Figure 6. The fluorescent target protein was then allowed to bind to the aptamer on the chip, and a photo was taken. The chip binding image is shown in Figure 7.

[0086] If the fluorescent target protein binds to an aptamer, the location of the aptamer will emit fluorescence, indicating that the aptamer can bind to the target protein.

[0087] Part 4: Algorithm Analysis

[0088] After sequencing, the fastq file is processed to find the sequence information corresponding to each position on the chip. Based on the protein binding image, the fluorescent location on the chip is found, thereby extracting the information of the aptamer bound to the protein. Finally, the aptamer sequence-protein binding ratio can be calculated. By comparing the differences in protein binding ratios between different sequences, the sequence with the highest binding ratio is identified as the one with affinity.

[0089] The raw materials and reagents used in the nucleic acid aptamer high-throughput screening method provided by the present invention can all be purchased from the market.

[0090] The present invention will be further described below in conjunction with the embodiments:

[0091] Example 1 Construction and sequencing of pre-enriched nucleic acid aptamer library screened by SELEX

[0092] 1. Construct the library using the overhang PCR method.

[0093] Prepare the following PCR system: 0.5 μL Q5 High-Fidelity DNA Polymerase, 0.25 μmol / LF primer, 0.25 μmol / LR primer, 5 ng single-stranded DNA template, and make up to 50 μL with molecular-grade water.

[0094] Place the PCR mixture on a PCR instrument and run the following program: 98°C for 30 s, 12 cycles of 98°C for 10 s, 67°C for 15 s, and 72°C for 15 s, and finally incubate at 72°C for 2 min, then cool to 4°C for storage.

[0095] After the reaction, the PCR product was purified using 1.5X AMPure magnetic beads (Beckman). The purification steps were described in the AMPure magnetic beads manual.

[0096] Library sequence: 5'-CTTCTGCACGCCTCCTTCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGAGACGAGATCGGCGGACACT-3' (SEQ ID No. 1)

[0097] PCR F primer sequence: 5'p-GAACGACATGGCTACGATCCGACTTCTTCTGCACGCCTCCTTCC-3' (SEQ ID No. 2)

[0098] PCR primer sequence: 5'-TGTGAGCCAAGGAGTTGATCGGACCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTAGTGTCCGCCGATCTCGTCTCC-3' (SEQ ID No. 3)

[0099] The template DNA sequence obtained after amplification and purification is as follows: 5'p-GAACGACATGGCTACGATCCGACTTCTTCTGCACGCCTCCTTCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGAGACGAGATCGGCGGACACTAGTCGGAGGCCAAGCGGTCTTAGGAAGACAATAGGTCCGATCAACTCCTTGGCTCACA-3' (SEQ ID No. 4)

[0100] 2. Cyclize the template obtained in step 1. Prepare the following reaction system:

[0101] 20 ng of library, 20 μL of DNB preparation buffer (BGI), and molecular grade water to make up to 40 μL.

[0102] After thorough mixing, place the mixture on a PCR instrument, incubate at 95°C for 3 minutes, then react at 40°C for 3 minutes, and store at 4°C. Add 40 μL of DNB polymerase mix III (BGI) and 4 μL of DNB polymerase mix IV (BGI) to the above system, mix thoroughly, and immediately place the mixture on a PCR instrument. React at 35°C for 25 minutes. After the reaction is complete, immediately add 20 μL of stop buffer (BGI), mix slowly by pipetting up and down 8-10 times with a widened pipette tip, and quantify using the Qubit ssDNA Kit.

[0103] 3. Sequencing. Prepare DNBs from the cyclized product obtained in step 2 and mix the resulting DNBs with a certain proportion of the balanced library (the balanced library proportion is sufficient to be greater than 40%) in a total volume of 100 μL. Load the DNBs onto the BGISEQ 500 sequencing slide according to the instructions in the BGISEQ 500SE100 sequencing kit. Sequence the sequence according to the BGISEQ 500SE100 sequencing kit instructions, setting the sequencing length to 76 bp. The resulting Fq file is ready for use.

[0104] Example 2 Preparation of nucleic acid aptamer cluster slide

[0105] 1. Formamide removal of sequencing strands

[0106] Formamide was passed into the BGISEQ 500 sequencing slide after sequencing in Example 1 to remove the sequencing strands hybridized on the DNB.

[0107] 2. Chip surface modification

[0108] The BGISEQ-500 platform chip surface was connected to NHS-PEG4-Azide with the following structure: The chip surface was modified according to the following method: NHS-PEG4-Azide (564.58 g / mol) was added at a concentration of 45 μmol / L and 100 mL was prepared. Method:

[0109] Store at -20°C to avoid repeated freezing and thawing. The concentration of DECO-primer is 1 μmol / L and diluted with PBS.

[0110] 3. Primer-probe coupling

[0111] BGI Liuhe custom synthesized the following primer and probe sequences:

[0112] DBCO (linker) -ACAGAACGACATGGCTACGATCCGACTT (SEQ ID No. 121, which contains the first fixed sequence complementary sequence, the PCR amplification site sequence, and the intermediate sequence). 1 μmol / L of the above primer probe sequence was diluted with PBS and passed onto the azide-modified chip for reaction at room temperature for 1 hour or overnight.

[0113] 4. Aptamer Synthesis

[0114] Prepare a mixture of dNTP and Hifi polymerase, use DNB as template, Capture oligo as primer, and dNTP as substrate to extend the nucleic acid aptamer sequence.

[0115] 5. Digestion of DNB

[0116] A single-strand specific endonuclease mixture is prepared to remove the single strand, leaving behind the aptamer cluster.

[0117] Example 3 Fluorescent primer hybridization and binding to fluorescent target protein

[0118] 1. Hybridization fluorescent primer: BGI Liuhe customized ROX modified primer sequence: 5'ROX-GTCGGATCGTAGCCATGTCGTTC-3' (SEQ ID No. 5)

[0119] The primers were diluted to 1 μmol / L with 5×SSC, loaded onto the slide prepared in Example 2, and hybridized at room temperature for 30 minutes.

[0120] 2. Target protein binding: Dilute the AF647-modified fluorescent protein TP with DPBS and load it onto the prepared sequencing slide. Incubate at room temperature for 30 minutes. Rinse off excess protein with DPBS.

[0121] 3. Photographing: The photographing range here is consistent with the position and area photographed during sequencing in Example 1.

[0122] 4. Data Analysis:

[0123] (1) The sequence position and sequence information measured in Example 1 were matched with the protein binding information obtained in Example 3. The matching information showed that the positions of the sequencing sequence and the protein binding sequence could be matched twice.

[0124] (2) Based on the matching results, the protein binding results of the nucleic acid aptamer are further output, and sequences with different binding ratios are selected for further SPR affinity verification. The data are as follows:

[0125] Table 1

[0126] Example 4 Aptamer SPR Verification

[0127] 1. Sequence synthesis: BGI Liuhe customized the nucleic acid aptamers obtained by data analysis in Example 3:

[0128] 2.SPR verification:

[0129] (1) Preparation of capture surface and binding assay

[0130] The CM5 sensor chip module was embedded in a BIAcore instrument, and a filtered, degassed DPBS buffer solution was used as the flow system. A pre-enrichment procedure was run to determine the optimal buffer conditions and concentrations for coupling the ligand and tagged proteins to the chip. Dextran carboxylates on the chip were activated by injecting 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). A low-pH sodium acetate solution containing the ligand or tagged protein was then introduced. The proteins were immobilized on the chip surface via amino groups forming bonds with activated carboxyl groups on the chip. Finally, excess activated carboxylates were blocked with ethanolamine treatment, and unbound ligands were rinsed away with the buffer, completing the capture surface preparation.

[0131] Through chip surface testing and regeneration condition selection, the binding experimental conditions were finally determined: aptamer analysis concentration of 500 nM, association time of 180 seconds, dissociation time of 180 seconds, regeneration reagent of 2 M NaCl, regeneration time of 30 seconds, instrument flow rate of 30 μl / min, and instrument temperature of 25°C. The response value of the reference channel (coupled to the tag protein GST) was subtracted from the response value of the analytical channel (coupled to the ligand protein TP) to determine the actual response value of the aptamer to the TP syphilis antigen. The affinity of the aptamer was further screened based on the actual response value.

[0132] (2) Kinetic analysis of protein-aptamer interactions

[0133] After confirming the affinity of the aptamer for TP based on the binding experiment in the previous step, the aptamer was diluted with DPBS to 0, 31.25, 62.5, 125, 250, 500, and 1000 nM concentrations. The solution was centrifuged to remove bubbles. After the baseline stabilized, the samples were injected sequentially from low to high concentrations. After the program was completed, the experimental data was kinetically fitted using Biacore Evaluation software to obtain a more accurate fitting curve and dissociation equilibrium constant (KD).

[0134] High-throughput identification of syphilis (TP) antigen aptamers

[0135] Syphilis (TP) antigen nucleic acid aptamers were screened and identified according to the experimental protocols described in Examples 1 to 4. The results are as follows:

[0136] 1. The binding of fluorescent target protein and nucleic acid aptamer is shown in Figure 8;

[0137] 2. Protein binding ratio results: The protein binding ratios of more than 1000 sequences are shown in Figure 9;

[0138] We selected 112 sequences with varying binding ratios for SPR validation. The average binding ratio for the affinity sequences was 28.15%, while the average binding ratio for all sequences was 24.09%. Subsequent SPR validation results revealed that the selected sequences exhibited a fast dissociation pattern with the protein, resulting in a slightly higher binding ratio for positive sequences than for standard sequences. For slow-dissociating sequences, the difference in binding ratios between positive and negative sequences was even greater.

[0139] 3. List some of the nucleic acid aptamers for TP antigens and the SPR results. The three nucleic acid aptamers for TP antigens are shown in the following table:

[0140] Table 2

[0141] The TP-11 SPR results are shown in FIG10 , the TP-65 SPR results are shown in FIG11 , and the TP-93 SPR results are shown in FIG12 .

[0142] The above is a detailed introduction to a method for high-throughput identification of syphilis (TP) antigen nucleic acid aptamers provided by the present invention. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above examples is only used to help understand the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, the present invention can also be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for screening a combination of nucleic acid aptamers, characterized in that: Includes any or any combination of the following: (I) Nucleic acid aptamer library: The nucleic acid aptamer library includes a first fixed sequence, a random sequence, and a second fixed sequence in sequence from the 5' end to the 3' end; (II), Primer set: The primer set includes an upstream primer and a downstream primer; The upstream primer includes a first adapter sequence and a first complementary sequence; The first complementary sequence includes: a sequence that is partially or completely complementary to the first fixed sequence; The downstream primer includes a second adapter sequence and a second complementary sequence; The second complementary sequence includes: a sequence that is partially or completely complementary to the second fixed sequence; (III), nucleic acid circularization reagents and / or digestion reagents; (IV), a carrier coupled to a linker primer: the linker primer can hybridize with the DNB obtained after cyclization of the nucleic acid aptamer; Furthermore, it also includes (V) a fluorescent primer; the fluorescent primer can hybridize with the linker primer.

2. The combination element according to claim 1, characterized in that The nucleic acid aptamer library has: I. The nucleotide sequence shown in SEQ ID No.1; or II. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in I and having the same or similar function as the nucleotide sequence shown in I; or III. A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in I, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in I.

3. The composite element according to claim 1 or 2, characterized in that: The upstream primer has: IV, the nucleotide sequence shown in SEQ ID No.2; or V. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in IV and having the same or similar function as the nucleotide sequence shown in IV; or VI, a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in IV, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in IV; The downstream primer has: VII. the nucleotide sequence shown in SEQ ID No. 3; or VIII. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in VII and having the same or similar function as the nucleotide sequence shown in VII; or IX. A nucleotide sequence having at least 80% homology to the nucleotide sequence shown in VII, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in VII.

4. The composite element according to any one of claims 1 to 3, characterized in that The fluorescent primers include but are not limited to ROX fluorescent primers, such as AF532 and any other fluorescent primers that can be recognized by the fluorescent channel of the sequencer.

5. Use of the combination element according to any one of claims 1 to 4 in any of the following items: (I) high-throughput screening of nucleic acid aptamers; (II) preparing reagents, kits or devices for high-throughput screening of nucleic acid aptamers; (III) preparing a reagent, a kit or a device for detecting the target molecule of the nucleic acid aptamer.

6. The use according to claim 5, characterized in that The target molecules include, but are not limited to, proteins, polypeptides, ions, small molecules, macromolecular polymers, nucleic acids and living cells; Further, the target molecule includes syphilis antigen; Furthermore, the target molecule is modified with fluorescent protein or fluorescent small molecule.

7. A method for screening nucleic acid aptamers, characterized in that: Based on the combination element as described in any one of claims 1 to 4, a nucleic acid aptamer that meets the requirements is obtained according to the sequence information and the protein binding result.

8. The screening method according to claim 7, characterized in that The steps include: Obtaining a nucleic acid aptamer library with sequencing adapters connected at both ends, circularizing the nucleic acid aptamer library with adapters by using a DNB preparation technology to obtain DNBs and sequence them; Dissociating the sequencing chain generated by the sequencing, hybridizing the DNB with the adapter primer on the carrier, extending the nucleic acid aptamer sequence on the adapter primer, and then digesting the DNB to obtain a carrier loaded with the nucleic acid aptamer cluster; The fluorescent primer is hybridized with the linker primer on the carrier loaded with the nucleic acid aptamer cluster, and then the fluorescent target protein is combined with the nucleic acid aptamer to obtain the protein binding result; According to the sequence information of the nucleic acid aptamer at each position and the binding result with the protein, the nucleic acid aptamer that meets the requirements is obtained.

9. The method according to claim 8, characterized in that The step of obtaining a nucleic acid aptamer library with sequencing adapters connected to both ends comprises: constructing nucleic acid aptamers according to the first fixed sequence, the random sequence, and the second fixed sequence in sequence to form a nucleic acid aptamer library; Using an upstream primer with the first adapter sequence and a downstream primer with the second adapter sequence, the first adapter sequence and the second adapter sequence are connected to both ends of the nucleic acid adapter by Overhang PCR to obtain a nucleic acid adapter library with sequencing adapters connected to both ends.

10. A reagent or a kit, characterized in that The invention comprises a combination element as claimed in any one of claims 1 to 4.

11. The device, characterized in that The invention comprises a combination element as claimed in any one of claims 1 to 4.

12. Syphilis antigen nucleic acid aptamer, characterized in that have: I. A nucleotide sequence as shown in any of SEQ ID No. 6 to 8; or II. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in I and having the same or similar function as the nucleotide sequence shown in I; or III. A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in I, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in I.

13. Use of the syphilis antigen nucleic acid aptamer according to claim 12 in any of the following items: (I) preparing reagents, kits or devices for detecting syphilis; (II) Detection of syphilis.

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