阅读说明：本技术 一种基于交联反应的核酸适配体的快速筛选方法及筛选获得的结构开关型核酸适配体 (rapid screening method of aptamer based on crosslinking reaction and structure switch type aptamer obtained through screening ) 是由 娄新徽 乔娜 吴晓 刁冬林 于 2018-06-08 设计创作，主要内容包括：本发明涉及一种基于交联反应的核酸适配体的快速筛选新方法及其通过该方法筛选获得的人血清白蛋白的结构开关型核酸适配体。本方法中靶标与核酸适配体文库进行均相孵育,然后通过交联反应将靶标捕获在磁珠上,这样与靶标结合的核酸序列被捕获在磁珠上；将磁性分离出来的与靶标结合的核酸序列通过聚合酶链反应(PCR)进行扩增；将双链PCR产物进行单链制备得到次级文库,次级文库进入下一轮的筛选。将上述过程循环2-3轮可以实现对文库的快速富集。该方法还可以与其它筛选技术联用,大幅提高筛选效率。按照本方法经3轮富集后的文库仅经过1轮基于文库固定的筛选方法就得到了具有高亲和力和选择性的HSA的SSA。(The invention relates to a novel method for rapidly screening aptamer based on cross-linking reaction and a structural switch type aptamer of human serum albumin obtained by screening through the method. In the method, a target and a nucleic acid aptamer library are subjected to homogeneous incubation, and then the target is captured on magnetic beads through a cross-linking reaction, so that a nucleic acid sequence combined with the target is captured on the magnetic beads; amplifying the magnetically isolated target-bound nucleic acid sequence by Polymerase Chain Reaction (PCR); and (3) carrying out single-chain preparation on the double-chain PCR product to obtain a secondary library, and screening the secondary library in the next round. The process is circulated for 2-3 rounds to realize the rapid enrichment of the library. The method can be used together with other screening technologies, so that the screening efficiency is greatly improved. According to the method, the library after 3 rounds of enrichment is subjected to only 1 round of screening method based on library fixation to obtain SSA of HSA with high affinity and selectivity.)

1. A method for rapidly screening aptamers based on a crosslinking reaction, which is characterized by comprising the following steps: step 1, library heat treatment; step 2, activating magnetic beads; step 3, magnetic bead negative screening; step 4, incubating the library and the target; step 5, capturing the nucleic acid sequence combined with the target through a cross-linking reaction; step 6, eluting the bound nucleic acid on the magnetic beads; step 7, PCR; and 8, preparing single-stranded DNA.

2. The method of claim 1, wherein the above steps 1-8 are cycled for 2-3 rounds to achieve rapid enrichment of the library.

3. The method according to claim 1 or 2,

step 1, library heat treatment as follows: taking a proper amount of library to carry out heat treatment in a buffer solution: heating in water bath at 95 ℃, quenching in ice bath, and standing at room temperature;

step 2, activating the magnetic beads as follows: activating the magnetic beads modified by carboxyl with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to generate magnetic beads modified by NHS active ester;

and 3, magnetic bead negative screening as follows: taking part of the NHS activated magnetic beads prepared in the step 2 to incubate with the library in the step 1, carrying out magnetic separation, and using a supernatant in the step 4;

step 4, library and target incubation are as follows: mixing and incubating the library collected in the step 3 and a target according to a certain molar ratio in a screening buffer solution;

step 5, capturing the nucleic acid sequence bound to the target by a cross-linking reaction as follows: incubating the NHS activated magnetic beads prepared in the step 2 with the library and target mixed solution in the step 4, carrying out magnetic separation, and cleaning the magnetic beads;

Step 6, eluting the bound nucleic acid on the magnetic beads as follows: incubating a certain amount of proteinase K with the magnetic beads obtained in the step 5, or adding a buffer solution into the magnetic beads obtained in the step 5, heating, incubating, and collecting supernate;

Step 7, PCR is as follows: carrying out PCR on the eluent obtained in the step 6, and purifying a PCR product by an ethanol precipitation method;

step 8, single-stranded DNA preparation as follows: preparing single strands by using lambda phage exonuclease, purifying the products of the lambda phage exonuclease digestion by an ethanol precipitation method, quantifying the products in each round by ultraviolet, wherein the obtained single strand DNA library is a secondary library, and the secondary library is subjected to the next round of screening or clone sequencing.

4. The method of claim 3, further comprising the steps of:

(1) 1-8, circulating for 2-3 rounds to realize the rapid enrichment of the library to form an enrichment library, and hybridizing and incubating the enrichment library with a biotin-modified probe which is complementary to a middle fixed sequence of the library;

(2) fixing the double-stranded library formed in the step (1) on streptavidin-coated magnetic beads;

(3) incubating library immobilized magnetic beads and a target, carrying out magnetic separation after incubation is finished, and collecting supernatant;

(4) and (3) PCR: carrying out PCR on the supernatant obtained in the step (3), and purifying a PCR product by an ethanol precipitation method;

(5) Cloning and sequencing;

(6) selecting a representative enrichment sequence for chemical synthesis;

(7) Chemically synthesized sequences were tested for affinity, selectivity and functionality.

5. A method for rapidly screening aptamers based on a crosslinking reaction, the method comprising: a first round of screening of homogeneous Tb aptamers based on a crosslinking reaction, a second round of screening of homogeneous Tb aptamers based on a crosslinking reaction, a third round of screening of Tb aptamers based on a target immobilized magnetic bead method and a fourth round of screening of Tb aptamers based on a magnetic bead method,

Wherein the first round of screening comprises the steps of:

(1) Heat treatment of the starting DNA library;

(2) The library was incubated with Tb;

(3) activating the magnetic beads;

(4) Incubating the magnetic beads with the DNA-Tb complex;

(5) DNA elution;

(6) Performing small-scale PCR to obtain a gel electrophoresis result picture;

(7) Selecting the optimal number of amplification rounds according to the gel electrophoresis result diagram in the step (6), and carrying out PCR amplification on all samples obtained in the step (5);

(8) after the PCR reaction is finished, mixing the obtained samples in pairs to ensure that the volume of each group of samples is 200 mu L, and carrying out ethanol precipitation;

(9) carrying out small-scale lambda Exo enzyme digestion;

(10) Carrying out large-scale lambda Exo enzyme digestion;

(11) Precipitating lambda Exo enzyme digestion products by using ethanol, wherein the operation process is the same as that of (8);

(12) The quantitative determination of the ultraviolet light is carried out,

wherein the second round of screening comprises the steps of:

(1) Heat treatment of the enriched DNA library;

(2) Library and Tb incubation, activated magnetic bead, magnetic bead and DNA-Tb compound incubation, DNA elution, PCR, lambda Exo enzyme digestion, ethanol precipitation and ultraviolet quantification are all screened according to the first round;

Wherein the third round of screening comprises the steps of:

(1) Activating the magnetic beads;

(2) coupling Tb to the surface of the magnetic beads;

(3) Incubating the protein-coated magnetic beads with the enriched library;

(4) DNA elution;

(5) PCR, lambda Exo enzyme digestion, ethanol precipitation and ultraviolet quantification refer to the first round of screening,

Wherein the fourth screening comprises the steps of:

(1) activating the magnetic beads and coupling Tb to the surfaces of the magnetic beads refer to the third screening (1) and (2);

(2) Incubating the magnetic beads coated with the protein with DNA;

(3) DNA elution, PCR, lambda Exo digestion, ethanol precipitation and UV quantification were all referred to the third round of screening.

6. The method of claim 5, further comprising the steps of: clone sequencing and selecting representative sequences for chemical synthesis and affinity determination.

7. a method for rapidly screening aptamers based on a crosslinking reaction, the method comprising: a first round of screening for a homogeneous HSA aptamer based crosslinking reaction, a second round of screening for a homogeneous HSA aptamer based crosslinking reaction, a third round of screening for a homogeneous HSA aptamer based crosslinking reaction, and a fourth round of screening for a library-immobilized SSA,

Wherein the first round of screening comprises the steps of:

(1) Heat treatment of the starting DNA library;

(2) Activating the magnetic beads;

(3) magnetic bead negative screening;

(4) Incubation of the library with HSA;

(5) activating the magnetic beads during incubation of the library with HSA;

(6) DNA elution;

(7)PCR；

(8) performing gel cutting purification on the first round screening product by using a UNIQ-10 column type PAGE gel DNA recovery kit;

Wherein the second round of screening comprises the steps of:

(1) heat treatment of the starting DNA library;

(2) activating the magnetic beads;

(3) magnetic bead negative screening;

(4) Incubation of the library with HSA;

(5) Activating the magnetic beads during incubation of the library with HSA;

(6) DNA elution;

(7)PCR；

(8) cutting and purifying;

(9) lambda Exo enzyme digestion, ethanol precipitation and ultraviolet quantification,

Wherein the third round of screening comprises the steps of:

The operation steps of the second round of enrichment library are the same as those of the second round of screening except that 0.048nmol of the second round of enrichment library is input,

Wherein the fourth screening comprises the steps of:

(1) Hybridizing the third round of products;

(2) cleaning the magnetic beads;

(3) fixing magnetic beads on the library;

(4) incubating library immobilized magnetic beads with HSA;

(5) PCR, purifying PCR products by an ethanol precipitation method, purifying PCR products by a UNIQ-10 column PAGE gel DNA recovery kit, generating single chains by using lambda Exo, purifying lambda Exo enzyme digestion products by the ethanol precipitation method, and quantifying by ultraviolet.

8. the method of claim 7, further comprising the steps of: cloning and sequencing; representative sequences were selected for chemical synthesis and affinity determination as well as selectivity and functionality testing.

9. A structurally switched aptamer screened according to the method of any one of claims 1 to 8.

10. the structural switch aptamer of claim 9, wherein said structural switch aptamer is Tb-1, Tb-2, Tb-3, Tb-4, HSA-1, HSA-2, HSA-3, HSA-4, HSA-5 or HSA-6.

Technical Field

the invention relates to a rapid screening method of aptamer based on crosslinking reaction and a structure switch type aptamer of human serum albumin obtained by screening, belonging to the technical field of biology.

Background

The aptamer screening technique, namely Enrichment ligand Evolution (SELEX) (Nature,1990,346(6287), 818-. The basic principle of SELEX technology is to incubate an oligonucleotide (DNA or RNA) library with a specific target molecule, separate the oligonucleotide bound to the target molecule by a separation technique, perform exponential amplification by a PCR technique, prepare a secondary library by single-stranded the obtained product, and restart a new round of screening using the secondary library as the initial library of the next round. Through multiple rounds of screening processes, the aptamer with high affinity and high specificity is finally obtained.

the SELEX technique has been widely used in various fields since its introduction. However, the traditional SELEX technology is cumbersome to operate and has a long screening period. In order to accelerate the process of screening for aptamers, various improved SELEX techniques have been developed in recent years, shortening the screening cycle. These improved techniques have mostly replaced traditional affinity column or cellulose acetate membrane based separation techniques with more efficient or convenient separation methods by improving the separation steps in the SELEX process. These improved methods include: magnetic bead separation (Nucleic Acids Research 2003,31(18), e110-e118.), capillary electrophoresis (Analytical Chemistry 2004,76(18), 5387. times. 5392.), gel electrophoresis (Nucleic Acids Research 2005,33(17), e141-e147.), microfluidic chip method (Proc. Natl. acid. Sci. U.S.A.2009,106,2989-2994.), surface plasmon resonance (Analytical Biochemistry 2005,342, 312. times. 317). Although various aptamer screening techniques have been developed in recent years, screening of aptamers is still challenging, often facing the problem of screening failure, and new screening techniques are urgently needed.

Among the current various aptamer screening technologies, the method based on target immobilization is most widely applied due to its convenient operation. However, in the method for screening the aptamer for immobilizing the target molecule, the solid phase matrix has serious non-specific adsorption, and the target is immobilized on the solid phase surface to mask the binding site of the target, so that the conformation of the target is influenced, and great uncertainty is caused. These factors all contribute to inefficient and even failure of aptamer screening. The method of screening for homogeneous aptamers avoids such problems without the need to immobilize the target molecule to a solid phase medium, but rather incubate the library directly with the target molecule in solution, followed by isolation. Because the target molecules are in a free state in the screening process, which is the same as the state in an actual sample, the aptamer obtained by the homogeneous screening technology is theoretically more suitable for later application. Current homogeneous aptamer screening techniques include capillary electrophoresis (Journal of the American Chemical society 2005,127(9), 3165-.

Although homogeneous aptamer screening methods overcome the problems of traditional methods of aptamer screening for immobilized target molecules, they still have certain drawbacks. For example, capillary electrophoresis requires special equipment and specialized personnel, and high voltage also affects the properties of the target protein; low separation efficiency of membrane separation, centrifugal separation and electrophoretic separation can result in low screening efficiency; the separation efficiency of graphene separation is greatly affected by the physical properties of targets and is difficult to control. There is an urgent need to develop homogeneous aptamer screening techniques that are simpler to operate, more efficient to separate, and more easily controlled.

in addition, while the structural switch type aptamer (SSA) is widely applied in the field of biosensing, the current methods for screening SSA are inefficient (proc.natl.acad.sci.u.s.a.2010,107,14053-14058.) and require 15-20 screening cycles, and the obtained aptamers have poor selectivity and low affinity, and the development of an efficient SSA screening technology is urgently needed.

disclosure of Invention

the invention aims to provide a novel method for rapidly screening aptamer based on cross-linking reaction, and the SSA of the human serum albumin obtained by screening through the method.

the invention provides a rapid screening method of a nucleic acid aptamer based on a crosslinking reaction, which comprises the following steps: step 1, library heat treatment; step 2, activating magnetic beads; step 3, magnetic bead negative screening; step 4, incubating the library and the target; step 5, capturing the nucleic acid sequence combined with the target through a cross-linking reaction; step 6, eluting the bound nucleic acid on the magnetic beads; step 7, PCR; and 8, preparing single-stranded DNA.

Wherein the above steps 1-8 are cycled for 2-3 rounds to achieve rapid enrichment of the library.

in the above-mentioned method, the first step of the method,

Step 1, library heat treatment as follows: taking a proper amount of library to carry out heat treatment in a buffer solution: heating in water bath at 95 ℃, quenching in ice bath, and standing at room temperature;

Step 2, activating the magnetic beads as follows: activating the magnetic beads modified by carboxyl with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to generate magnetic beads modified by NHS active ester;

and 3, magnetic bead negative screening as follows: taking part of the NHS activated magnetic beads prepared in the step 2 to incubate with the library in the step 1, carrying out magnetic separation, and using a supernatant in the step 4;

Step 4, library and target incubation are as follows: mixing and incubating the library collected in the step 3 and a target according to a certain molar ratio in a screening buffer solution;

Step 5, capturing the nucleic acid sequence bound to the target by a cross-linking reaction as follows: incubating the NHS activated magnetic beads prepared in the step 2 with the library and target mixed solution in the step 4, carrying out magnetic separation, and cleaning the magnetic beads;

Step 6, eluting the bound nucleic acid on the magnetic beads as follows: incubating a certain amount of proteinase K with the magnetic beads obtained in the step 5, or adding a buffer solution into the magnetic beads obtained in the step 5, heating, incubating, and collecting supernate;

step 7, PCR is as follows: carrying out PCR on the eluent obtained in the step 6, and purifying a PCR product by an ethanol precipitation method;

Step 8, single-stranded DNA preparation as follows: preparing single strands by using lambda phage exonuclease, purifying the products of the lambda phage exonuclease digestion by an ethanol precipitation method, quantifying the products in each round by ultraviolet, wherein the obtained single strand DNA library is a secondary library, and the secondary library is subjected to the next round of screening or clone sequencing.

the method also comprises the following steps:

(1) 1-8, circulating for 2-3 rounds to realize the rapid enrichment of the library to form an enrichment library, and hybridizing and incubating the enrichment library with a biotin-modified probe which is complementary to a middle fixed sequence of the library;

(2) Fixing the double-stranded library formed in the step (1) on streptavidin-coated magnetic beads;

(3) Incubating library immobilized magnetic beads and a target, carrying out magnetic separation after incubation is finished, and collecting supernatant;

(4) and (3) PCR: carrying out PCR on the supernatant obtained in the step (3), and purifying a PCR product by an ethanol precipitation method;

(5) cloning and sequencing;

(6) selecting a representative enrichment sequence for chemical synthesis;

(7) chemically synthesized sequences were tested for affinity, selectivity and functionality.

In another aspect, the present invention provides a method for rapid screening of aptamers based on a crosslinking reaction, the method comprising: a first round of screening of homogeneous Tb aptamers based on a crosslinking reaction, a second round of screening of homogeneous Tb aptamers based on a crosslinking reaction, a third round of screening of Tb aptamers based on a target immobilized magnetic bead method and a fourth round of screening of Tb aptamers based on a magnetic bead method,

Wherein the first round of screening comprises the steps of:

(1) heat treatment of the starting DNA library;

(2) the library was incubated with Tb;

(3) Activating the magnetic beads;

(4) Incubating the magnetic beads with the DNA-Tb complex;

(5) DNA elution;

(6) Performing small-scale PCR to obtain a gel electrophoresis result picture;

(7) Selecting the optimal number of amplification rounds according to the gel electrophoresis result diagram in the step (6), and carrying out PCR amplification on all samples obtained in the step (5);

(8) after the PCR reaction is finished, mixing the obtained samples in pairs to ensure that the volume of each group of samples is 200 mu L, and carrying out ethanol precipitation;

(9) carrying out small-scale lambda Exo enzyme digestion;

(10) Carrying out large-scale lambda Exo enzyme digestion;

(11) precipitating lambda Exo enzyme digestion products by using ethanol, wherein the operation process is the same as that of (8);

(12) the quantitative determination of the ultraviolet light is carried out,

Wherein the second round of screening comprises the steps of:

(1) Heat treatment of the enriched DNA library;

(2) Library and Tb incubation, activated magnetic bead, magnetic bead and DNA-Tb compound incubation, DNA elution, PCR, lambda Exo enzyme digestion, ethanol precipitation and ultraviolet quantification are all screened according to the first round;

wherein the third round of screening comprises the steps of:

(1) Activating the magnetic beads;

(2) coupling Tb to the surface of the magnetic beads;

(3) incubating the protein-coated magnetic beads with the enriched library;

(4) DNA elution;

(5) PCR, lambda Exo enzyme digestion, ethanol precipitation and ultraviolet quantification refer to the first round of screening,

wherein the fourth screening comprises the steps of:

(1) activating the magnetic beads and coupling Tb to the surfaces of the magnetic beads refer to the third screening (1) and (2);

(2) Incubating the magnetic beads coated with the protein with DNA;

(3) DNA elution, PCR, lambda Exo digestion, ethanol precipitation and UV quantification were all referred to the third round of screening.

the method also comprises the following steps: clone sequencing and selecting representative sequences for chemical synthesis and affinity determination.

In still another aspect, the present invention provides a method for rapid screening of aptamers based on a crosslinking reaction, the method comprising: a first round of screening for a homogeneous HSA aptamer based crosslinking reaction, a second round of screening for a homogeneous HSA aptamer based crosslinking reaction, a third round of screening for a homogeneous HSA aptamer based crosslinking reaction, and a fourth round of screening for a library-immobilized SSA,

Wherein the first round of screening comprises the steps of:

(1) Heat treatment of the starting DNA library;

(2) activating the magnetic beads;

(3) magnetic bead negative screening;

(4) Incubation of the library with HSA;

(5) Activating the magnetic beads during incubation of the library with HSA;

(6) DNA elution;

(7)PCR；

(8) Performing gel cutting purification on the first round screening product by using a UNIQ-10 column type PAGE gel DNA recovery kit;

wherein the second round of screening comprises the steps of:

(1) Heat treatment of the starting DNA library;

(2) activating the magnetic beads;

(3) magnetic bead negative screening;

(4) Incubation of the library with HSA;

(5) Activating the magnetic beads during incubation of the library with HSA;

(6) DNA elution;

(7)PCR；

(8) Cutting and purifying;

(9) lambda Exo enzyme digestion, ethanol precipitation and ultraviolet quantification,

Wherein the third round of screening comprises the steps of:

The operation steps of the second round of enrichment library are the same as those of the second round of screening except that 0.048nmol of the second round of enrichment library is input,

Wherein the fourth screening comprises the steps of:

(1) hybridizing the third round of products;

(2) Cleaning the magnetic beads;

(3) fixing magnetic beads on the library;

(4) incubating library immobilized magnetic beads with HSA;

(5) PCR, purifying PCR products by an ethanol precipitation method, purifying PCR products by a UNIQ-10 column PAGE gel DNA recovery kit, generating single chains by using lambda Exo, purifying lambda Exo enzyme digestion products by the ethanol precipitation method, and quantifying by ultraviolet.

The method also comprises the following steps: cloning and sequencing; representative sequences were selected for chemical synthesis and affinity determination as well as selectivity and functionality testing.

In yet another aspect, the present invention provides a structurally switched aptamer screened according to any of the methods described above. The structural switch type aptamer is Tb-1, Tb-2, Tb-3, Tb-4, HSA-1, HSA-2, HSA-3, HSA-4, HSA-5 or HSA-6.

specific Experimental procedures of the invention

one screening process of the method of the invention comprises the following steps:

1. Library heat treatment: taking a proper amount of library to carry out heat treatment in a buffer solution: heating in water bath at 95 deg.c, quenching in ice bath, and setting at room temperature.

2. Activating magnetic beads: and activating the magnetic beads modified by carboxyl groups by using N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride, EDC) to generate magnetic beads modified by NHS active ester.

3. and (3) magnetic bead negative screening: and (3) taking part of the NHS activated magnetic beads prepared in the step (2) to incubate with the library in the step (1), carrying out magnetic separation, and using the supernatant in the step (4).

4. Library and target incubation: and (3) mixing and incubating the library collected in the step 3 and the target according to a certain molar ratio in a screening buffer.

5. Capturing nucleic acid sequences bound to the target by a cross-linking reaction: and (4) incubating the NHS activated magnetic beads prepared in the step (2) with the library and target mixed solution in the step (4), carrying out magnetic separation, and washing the magnetic beads.

6. Elution of bound nucleic acids on magnetic beads: and (3) incubating a certain amount of proteinase K with the magnetic beads obtained in the step (5), or adding a buffer solution into the magnetic beads obtained in the step (5), heating, incubating, and collecting supernatant.

7. And (3) PCR: and (4) carrying out PCR on the eluent obtained in the step (6), and purifying a PCR product by an ethanol precipitation method.

8. Preparation of single-stranded DNA: single-strand preparation is carried out by using lambda phage exonuclease (lambda Exo), lambda Exo enzyme digestion products are purified by an ethanol precipitation method, and products in each round are quantified by ultraviolet. The single-stranded DNA library obtained is a secondary library which is subjected to the next round of screening or clonal sequencing.

and (3) circulating the steps 1-8 for 2-3 rounds to realize the rapid enrichment of the library. The enriched library is subjected to the next stage of screening for aptamers, such as screening for SSA.

screening for SSA included the following steps:

(1) hybridizing and incubating the enrichment library with a biotin-modified probe complementary to an intermediate immobilization sequence of the library;

(2) fixing the double-stranded library formed in the step (1) on streptavidin-coated magnetic beads;

(3) Incubating library immobilized magnetic beads and a target, carrying out magnetic separation after incubation is finished, and collecting supernatant;

(4) And (3) PCR: carrying out PCR on the supernatant obtained in the step (3), and purifying a PCR product by an ethanol precipitation method;

(5) Cloning and sequencing;

(6) selecting a representative enrichment sequence for chemical synthesis;

(7) Chemically synthesized sequences were tested for affinity, selectivity and functionality.

the method of the invention has the following advantages:

1. the method does not need solid phase fixation of the target, overcomes the defect that the target is fixed on the solid phase in the prior screening technology, avoids the problems of surface fixation and shielding of target binding sites and influence on target conformation, greatly reduces non-specific adsorption of a solid phase medium to a library, and is very favorable for accelerating the screening efficiency of the aptamer;

2. according to the method, the nucleic acid sequence combined with the target is separated by a magnetic separation technology, expensive or special equipment is not needed, the operation is simple and rapid, and any condition optimization is not needed;

3. the method can be used for the pre-enrichment of the library, and the efficiency and the success rate of the screening technology of the existing aptamer are improved;

4. the method can be used for rapidly screening the SSA, and the obtained SSA has high selectivity and strong affinity.

drawings

FIG. 1 is a schematic diagram of the method of the present invention.

FIG. 2 is a flowchart of a prior art target-immobilization-based aptamer magnetic bead screening method used in example 1 of the present invention.

FIG. 3 is a graph showing an affinity test of the thrombin aptamer Tb-2 obtained by screening in example 1 of the present invention.

FIG. 4 is a flow chart of SSA screening based on library immobilization as employed in example 2 of the present invention.

FIG. 5 is the percent elution of the first three homogeneous cross-linking reaction-based screens of example 2 determined using the real-time quantitative PCR technique (RT-PCR) of the present invention; the percent elution is the proportion of nucleic acid sequences in the library that bind to the target in the total input library.

FIGS. 6A and 6B are graphs of the ratio of nucleic acids in the supernatant of washed and incubated target proteins of each magnetic bead to the total input library in SSA screening example 2 using RT-PCR assay of the present invention (FIG. 6A) and the melting temperature of nucleic acids in the background and target incubators (FIG. 6B).

FIG. 7 is a graph showing an affinity assay of HSA aptamer HSA-6 screened in example 2 of the present invention.

FIGS. 8A and 8B are graphs showing the structural switching performance and specificity of HSA-6 tested by the present invention, i.e., fluorescence method (FIG. 8A) and gel electrophoresis method (FIG. 8B); the fluorescence intensity of the supernatant after magnetic separation was measured in FIG. 8A; figure 8B gel electrophoresis of magnetically separated supernatants after incubation with different targets.

Detailed Description

FIG. 1 is a schematic diagram of the method of the present invention. As shown in fig. 1, each screening cycle includes 1. library heat treatment; 2. activating the magnetic beads to generate magnetic beads modified by NHS active ester; 3. and (3) magnetic bead negative screening: NHS activated magnetic beads and the library in 1 are incubated, magnetically separated, and the nucleic acid sequence combined with the activated magnetic beads is removed; 4. incubating the library with a target; 5. capturing nucleic acid sequences bound to the target by a cross-linking reaction; 6. elution of bound nucleic acids on magnetic beads: carrying out thermal elution or hydrolysis of the target by proteinase K to realize nucleic acid elution; 7, PCR; 8. preparation of single-stranded DNA.

FIG. 2 is a flowchart of a prior art target-immobilization-based aptamer magnetic bead screening method used in example 1 of the present invention. As shown in fig. 2, each screening cycle includes 1. activated magnetic beads; 2. fixing the target on the magnetic bead; 3. incubating the library with protein-coated magnetic beads; 4. eluting the nucleic acid combined on the magnetic beads by proteinase K; 5, PCR; 6. lambda Exo single stranded DNA preparation. FIG. 3 is a graph showing an affinity test of the thrombin aptamer Tb-2 obtained by screening in example 1 of the present invention.

FIG. 4 is a flow chart of SSA screening based on library immobilization as employed in example 2 of the present invention. As shown in FIG. 4, each screening cycle includes 1. library hybridization to the intermediate complementary sequence; 2. the hybrid chain is fixed on the magnetic bead; 3. incubating the magnetic beads with the fixed hybrid chains with the target; 4, PCR; 5. lambda Exo single strand preparation. FIG. 5 is the percent elution of the first three homogeneous cross-linking reaction-based screens of example 2 determined using the real-time quantitative PCR technique (RT-PCR) of the present invention; the percent elution is the proportion of nucleic acid sequences in the library that bind to the target in the total input library.

FIGS. 6A and 6B are graphs of the ratio of nucleic acids in the supernatant of washed and incubated target proteins of each magnetic bead to the total input library in SSA screening example 2 using RT-PCR assay of the present invention (FIG. 6A) and the melting temperature of nucleic acids in the background and target incubators (FIG. 6B). FIG. 7 is a graph showing an affinity assay of HSA aptamer HSA-6 screened in example 2 of the present invention. FIGS. 8A and 8B are graphs showing the structural switching performance and specificity of HSA-6 tested by the present invention, i.e., fluorescence method (FIG. 8A) and gel electrophoresis method (FIG. 8B); the fluorescence intensity of the supernatant after magnetic separation was measured in FIG. 8A; figure 8B gel electrophoresis of magnetically separated supernatants after incubation with different targets.