CN112410326A

CN112410326A - In vitro screening method of aptamer, aptamer and kit for detecting target molecule

Info

Publication number: CN112410326A
Application number: CN202011335595.8A
Authority: CN
Inventors: 向宇; 顾春梅; 徐潇
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-11-25
Filing date: 2020-11-25
Publication date: 2021-02-26
Anticipated expiration: 2040-11-25
Also published as: CN112410326B

Abstract

The present invention provides a method for in vitro screening of aptamers, the method comprising: providing a random library having phosphorothioate-based modifications and binding group modifications thereon, said binding group being adapted to bind to a target molecule, said phosphorothioate-based modifications being bound to a binding group; contacting the random library with a target, and separating the random library combined with the target molecule to be used as a target aptamer obtained by screening. The random library adopted by the invention is modified with phosphorothioate group and binding group, and the binding group generates affinity action with the target to shorten the distance between the random library and the target, so that the initial activity of the random library combined with the target is endowed, the random library and the target are more easily combined, and the aptamer is more easily screened successfully.

Description

In vitro screening method of aptamer, aptamer and kit for detecting target molecule

Technical Field

The present invention relates to the field of biology. In particular, the invention relates to methods for in vitro screening of aptamers, and kits for detecting target molecules.

Background

Aptamers (aptamers) refer to RNA or single-stranded DNA that can form a spatial structure and specifically bind to a target substance, which is a wide range of target substances, including proteins, small molecules, metal ions, and even whole cells. The acting force of the aptamer combined with the target substance is mainly various weak acting forces including hydrogen bonds, intermolecular acting force, electrostatic adsorption or base stacking force and the like. Aptamers have several advantages over antibodies (a protein): a. can be artificially synthesized, and has low production cost and stable performance; b. can endure the transformation from high temperature to low temperature without harsh storage conditions; c. in vitro screening is carried out, so that whether the target object has immunogenicity or not does not need to be considered; d. the structure is flexible, and other groups are easy to modify.

aptamers are typically screened by an in vitro screening technique known as the EXponential enrichment of Ligands by EXponential Evolution (SELEX). Firstly, a compound containing 10 is synthesized by a method of combinatorial chemistry¹⁴-10¹⁵Random DNA libraries of different DNA sequences that will form countless different spatial structures provide the opportunity for several DNA sequences to be present in the random DNA library that specifically bind to the target. In the first round of screening, the constructed random library is incubated with a target, and after incubation, DNA sequences capable of being combined with the target and DNA sequences incapable of being combined with the target are separated by a certain method. The sequence capable of binding to the target is then amplified by PCR, allowing this portion of the DNA sequence to be exponentially enriched. Subsequently, single-stranded PCR amplification products are isolated and the isolated single-stranded DNA library is continued to be incubated with the target for the next round of screening. After several rounds of screening, DNA sequences that bind specifically to the target and do not bind to the target analog are isolated from the DNA library.

Research shows that certain chemical modification on the aptamer can make the aptamer have stronger binding capacity with a target. However, there is some blindness and randomness to the chemical modification on existing aptamers. The choice of modification site is one of the key considerations, for some of which chemical modification may result in poor aptamer binding to the target. This presents challenges to rational design of existing aptamers for chemical modification to increase their affinity to the target. On the other hand, the traditional SELEX random library only consists of A, T, G, C four bases, and the formed structure has strong randomness and no initial activity of binding with a target, so that aptamer screening is difficult. Therefore, successful screening of aptamers is aggressive through the introduction of functional groups in random libraries that can bind to the target. Traditional chemical modification SELEX is usually achieved by introducing non-natural base modification, which first requires synthesis of new non-natural base monomer molecules and then introduction of new base molecules into a random library by PCR, making the sequence and structure that can be formed by the random library more abundant. However, this method is difficult to synthesize monomers, and when PCR is performed using DNA containing unnatural bases as a template and unnatural base monomers, the polymerase that can be selected for PCR amplification is limited, and thus the fidelity of PCR products is challenging.

Therefore, the in vitro screening method of the aptamer has yet to be studied.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.

In one aspect of the invention, the invention features a method for in vitro screening of aptamers. According to an embodiment of the invention, the method comprises: providing a random library having phosphorothioate-based modifications and binding group modifications thereon, said binding group being adapted to bind to a target molecule, said phosphorothioate-based modifications being bound to a binding group; contacting the random library with a target, and separating the random library combined with the target molecule to be used as a target aptamer obtained by screening.

The random library adopted in the method provided by the embodiment of the invention has phosphorothioate-based modification and binding group modification, and the affinity between the binding group and the target is utilized to shorten the distance between the random library and the target, so that the screening is easy. Compared with the traditional non-natural base synthesis, the invention synthesizes and modifies random libraries of various functional groups based on the reaction of the PS-random library and the binding groups. The method can modify different molecules or functional groups on a random library according to different target molecules, and has the advantages of simple synthetic method and wide applicability.

In addition, after the target aptamer is screened, the binding group can be easily removed. Compared with the PCR with DNA carrying non-natural bases as a template and a target molecule, the PCR is carried out by taking the aptamer modified by the phosphorothioate group as the PCR template, so that the polymerase is more friendly and the PCR is easier.

According to an embodiment of the present invention, the method for in vitro screening of aptamers as described above may further have the following additional technical features:

according to an embodiment of the invention, the phosphorothioate group is modified on a random library by: and carrying out PCR reaction by using a thio monomer by using the random library as a template to obtain the random library with modification of a thiophosphate group, wherein the thio monomer is selected from thio dATP, thio dTTP, thio dGTP and/or thio dCTP. Compared with the traditional chemical modification SELEX which directly utilizes the non-natural base to carry out GPCR amplification, the invention adopts the thiomonomer to carry out PCR amplification, can use high-assurance polymerase to carry out PCR reaction, and has higher fidelity of PCR products.

According to the embodiment of the invention, thio dATP is used as a thio monomer, and the fixed base A is 2-10.

According to an embodiment of the present invention, the DNA polymerase used in the PCR reaction is selected from phusion enzyme, Q5 enzyme or taq enzyme. This can improve the fidelity of the PCR product. According to a preferred embodiment of the invention, the polymerase is a Phusion enzyme.

According to an embodiment of the invention, the binding group is modified onto the random library by alkylation of the molecule to be bound with the phosphorothioate group.

According to an embodiment of the present invention, the molecule to be bound is selected from the group consisting of bromophenylboronic acid (formula 1), 4-aldehyde benzyl bromide (formula 2), 4-bromomethylphenylthiol (formula 3).

According to an embodiment of the present invention, when the molecule to be bound is phenylboronic acid, the target molecule is a polyhydroxy compound, such as adenosine, a polysaccharide; when the molecule to be combined is 4-aldehyde benzyl bromide, the target molecule is various amino-containing compounds or protein molecules, such as streptavidin; when the molecule to be bound is 4-bromomethylthiophenol, the target molecule is a compound that can interact with a thiol group, such as arsenic acid.

According to the embodiment of the invention, after the random library combined with the target sequence is separated, the obtained library is contacted with alkali liquor, so that the combined groups are removed, and the target library is obtained. By contacting the library with an alkaline solution, the binding groups can be effectively removed, and a target library which can be used for PCR amplification can be obtained. Compared with the traditional method of performing PCR by using DNA with non-natural bases as a template in chemical modification SELEX, the method provided by the invention has the advantages that the target random library with binding groups removed by alkali liquor is used as a PCR template, the polymerase is more friendly, and the PCR fidelity is higher.

According to the embodiment of the invention, the pH value of the alkali liquor is 10-13. Thereby, the binding groups, such as phenylboronic acid, are removed quickly and efficiently.

In another aspect of the invention, the invention features an aptamer. According to the embodiment of the invention, the aptamer has a phosphorothioate group and a binding group modification thereon; the aptamer is obtained by the in vitro screening method for aptamers as described above. Thus, aptamers according to embodiments of the invention can specifically bind to a target molecule.

In yet another aspect of the invention, the invention provides a kit for detecting a target molecule. According to an embodiment of the invention, the kit comprises: an aptamer having a phosphorothioate-based modification and a binding group modification, the binding group being adapted to bind to a target molecule. The target molecules can be specifically detected by using the kit according to the embodiment of the invention.

According to an embodiment of the present invention, the aptamer is obtained by the in vitro screening method for aptamers as described above.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic flow chart of a method for modifying functional groups with affinity for a target in a random library (R-random library);

FIG. 2 shows the structural formula of a PS modified monomer;

FIG. 3 shows a schematic flow diagram of a method for removing R groups from an R-random library;

FIG. 4 shows a scheme of a chemical modification SELEX based on a post-PS modification reaction;

FIG. 5 is a graph showing the results of PCR using thiodATP, dTTP, dCTP, dGTP as monomers;

FIG. 6 shows a schematic representation of denaturing PAGE characterization of the reaction of PS-random libraries with Br-BO;

FIG. 7 shows a schematic representation of denaturing PAGE characterization of lye on BO-random libraries for BO removal;

figure 8 shows a schematic of the extent of advancement of s.a. with the number of screening rounds.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

The present invention provides a novel method for in vitro screening of aptamers, see fig. 4, which introduces molecules or functional groups capable of specifically binding to target molecules on a random library based on the reactivity of Phosphorothioate (PS), thereby conferring initial affinity to the target to the random library, and bringing the distance between the random library and the target closer, so that the screening of aptamers becomes easy.

The invention firstly establishes a random library, and the random library is provided with N basic groups of 5nt-60nt in the middle except primers at two ends. The initial random library was obtained by solid phase synthesis, using it as a template, and PCR was performed with thiomonomers to obtain a PS-modified random library (see fig. 1). Taking adenosine as the screening target as an example, adenosine is an o-hydroxy compound, so the random library was chosen to be modified with phenylboronic acid. Boronic acids and ortho-hydroxy compounds readily form dynamic chemical bond borates, and thus, theoretically, upon introduction of a phenylboronic acid into a random library, the phenylboronic acid-random library will draw the distance between the random library and adenosine through the force of the phenylboronic acid and adenosine, giving nucleic acid sequences that readily form structures that bind adenosine an opportunity to approach adenosine and successfully bind to adenosine.

Specifically, the structure-switching SELEX is adopted for nucleic acid aptamer screening, and the random library sequence is as follows: ATACCAGCTTATTCAATT-N20-GGTCTTTCTGTTCT-N30-AGATAGTAAGTGCAATCT, wherein the sequence of the B-DNA is as follows: biotin-AAAAAAAAAAAAAGAACAGAAAGACC, random library, could be hybridized to B-DNA through the intermediate GGTCTTTCTGTTCT sequence and linked to avidin modified magnetic beads through biotin on B-DNA. The forward primer was AP 1: 5'-ATACCAGCTTATTCAATT-3', and the reverse primer is TER-AP 2: 5'-A20-Spacer 18-AGATTGCACTTACTATCT-3', the thiomonomer is thio dATP (2 '-deoxyribose-5' -O- (1-thiophosphite)), and the other monomers are general monomers dTTP, dGTP and dCTP (see figure 2), and the PCR result is shown in figure 5. 100 ntPS-random library bands were excised and purified for recovery.

The PS-random library is then reacted with a brominated binding molecule to obtain a random library modified with a binding molecule. Taking the bromine band molecule Br-R as an example, the PS-random library and Br-R are taken as reactants, and the two reactants are dissolved in a buffer system with the pH value of 4-10, such as phosphate buffer, MES (4-morpholine ethanesulfonic acid) buffer, MOPS (3- (N-morpholine) propanesulfonic acid) buffer, HEPES (hydroxyethyl piperazine ethanesulfonic acid) buffer and the like. Adding organic solvent to dissolve bromo-molecule completely, reaction concentration of PS-random library is 10nM-1mM, reaction concentration of Br-R is 100 μ M-100mM, and reaction is carried out at 10-60 deg.C for 0.5-200 h. After the reaction, the product (random library-R) can be purified by removing the excessive small molecules through an ultrafilter (Amicon), or the reaction mixture can be subjected to denaturing PAGE, and then the product can be recovered and purified by gel cutting.

The reaction product was characterized by denaturing PAGE and the results are shown in FIG. 6. The reaction products of the reaction of the PO-random library with Br-BO showed no shift change relative to the denaturing PAGE of the PO-random library, and the reaction products of the reaction of the PS-random library with Br-BO showed slower migration rate than the reactants. This indicates that BO was successfully modified onto the PS-random library. Referring to FIG. 3, the present inventors subsequently tested whether the BO-random library was successfully stripped by alkali treatment, and as a result, as shown in FIG. 7, the gel-running shift of the product obtained after alkali treatment was comparable to that of the PS-random library, indicating that the alkali can effectively strip small molecules from the random library and restore the small molecules to the PS-random library. PCR using the PS-random library as a template provides higher fidelity of the product than PCR using the BO-random library as a template.

The BO-random library was then hybridized with B-DNA and successfully immobilized on avidin magnetic spheres by biotin-avidin reaction. The magnetic beads are then incubated with adenosine molecules in buffer at room temperature. After a period of incubation, the BO-random library, which binds adenosine, will dissociate from the magnetic sphere. Collecting a BO-random library dissociated from the magnetic spheres, treating with alkaline solution to remove BO, hydrolyzing the BO-random library into a PO-random library (natural non-modified random library), performing PCR amplification by using the obtained PO-random library as a template and thiol-dATP, dTTP, dGTP and dCTP as monomers, performing denaturing PAGE (polyacrylamide gel electrophoresis) characterization and cutting gel for recovery, and obtaining a new round of PS-random library. Then according to the above procedure, synthesis of BO-random library, incubation with target, elution of nucleic acid sequence with affinity, BO removal, PCR amplification and gel cutting recovery of new round of PS-random library are carried out. Repeating the steps, and enriching to obtain a nucleic acid sequence with strong affinity with adenosine after a plurality of rounds of screening.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

1. Construction of a PS-random library

First obtained by solid phase synthesisObtaining a random library with the sequence of ATACCAGCTTATTCAATT-N20-GGTCTTTCTGTTCT-N30-AGATAGTAAGTGCAATCT, and synthesizing B-DNA with the sequence of: biotin-AAAAAAAAAAAAAGAACAGAAAGACC, random library, could be hybridized to B-DNA through the intermediate GGTCTTTCTGTTCT sequence and linked to avidin modified magnetic beads through biotin on B-DNA. Meanwhile, primers for PCR were synthesized, the forward primer being AP 1: 5'-ATACCAGCTTATTCAATT-3', and the reverse primer is TER-AP 2: 5'-A20-Spacer 18-AGATTGCACTTACTATCT-3', the thiomonomer is selected from thio dATP, and the other monomers are selected from natural monomers dTTP, dGTP and dCTP. To a 200. mu.l PCR tube were added 2.5. mu.l AP1, 2.5. mu.l TER-AP2, 1. mu.l 10mM thio dATP, 0.5. mu.l dTTP, 0.5. mu.l dGTP, 0.5. mu.l dCTP, 41.5. mu.l l H₂O, 0.5. mu.l Phusion enzyme and 0.5. mu.l random library template. Placing the PCR system on a PCR instrument, performing denaturation at 98 deg.C for 3min, denaturation at 98 deg.C for 20s, annealing at 56 deg.C for 20s, extending at 72 deg.C for 20s, circulating for 30 times, extending at 72 deg.C for 5min, and keeping at 4 deg.C. The PCR products were identified by running 10% denaturing PAGE, and the results are shown in FIG. 5. Subsequently, the gel strips of the target fragments (100nt PS-random library) were excised into 1.5mL centrifuge tubes, to which 300. mu.L of PBS buffer was added and mixed well with a vortex shaker, and then placed on a rotating rack and shaken at room temperature overnight. The next day, the supernatant was collected and the PS-random library was recovered by ethanol precipitation.

2. Preparation of a random Phenylboronic acid (BO) library

The PS-random library was dissolved in 30 μ l of water, and 30 μ l of 100mM PBS buffer (pH 6), 30 μ l of 50mM 4- (bromomethyl) phenylboronic acid (Br-BO) dissolved in DMF, and 30 μ l of DMF were added thereto, mixed uniformly by a vortex shaker, and then placed on a rotating rack to react at room temperature for 20 hours. The reaction products were identified by running PAGE, and the results are shown in FIG. 6. After completion of the reaction, the reaction mixture was centrifuged and washed 6 times at 12000rpm for 12 minutes using an Amicon-10k ultrafilter tube and water to remove excess unreacted Br-BO.

3. Immobilization of BO-random libraries to magnetic spheres

The BO-random library is hybridized by DNA sequence (B-DNA: biotin-AAAAAAAAAAAAAGAACAGAAAGACC) complementary paired with it, and then reacted with avidin on the magnetosphere by biotin on the B-DNAFixed to the magnetic ball. 1 equivalent of the BO-random library was mixed with 5 equivalents of B-DNA in a screening buffer (50mM MOPS, 1mM MgCl)₂100mM NaCl and 5mM KCl, pH 7.4), heated at 98 ℃ for 5min and then slowly cooled to room temperature. Then, the BO-random library-B-DNA was mixed with avidin magnetic beads at room temperature and shaken for 30 min. After the reaction was completed, the supernatant was removed, and the magnetic beads were washed 10 times with a screening buffer.

4. Incubation of random libraries with targets

Since the random library-BDNA complex is always in dissociation equilibrium, there is always a portion of the DNA library that will dissociate from the resin column without the addition of adenosine. After the DNA was fixed to the magnetic sphere and washed 10 times, background collection was performed. In order to compare the self-dissociation conditions of the DNA libraries in different screening rounds, a blank dissociation time, namely the same incubation time, is set in each screening round, and adenosine is not added to determine the self-dissociation quantity. For example, when the incubation time is 1h, we first leave the DNA complex-magnetic beads which have been washed 10 times at room temperature for 1h, and after 1h, 500. mu.L of the screening buffer is added to collect the first effluent (B1), followed by 500. mu.L of the screening buffer and collecting the second effluent (B2). Then a small volume of saturated adenosine solution was added, gently stirred with a pipette, and the mixture was incubated at room temperature for 1 h. After 1h, 500. mu.L of the selection buffer was added to the magnetic beads, and the first effluent was collected (E1), followed by 500. mu.L of the selection buffer and the second effluent (E2), and in this way, collection of E3, E4, E5, etc. was continued. The affinity of the target to the DNA library was characterized by E1/B1 (structure-switching activity, S.A.). The results are shown in fig. 8, and after 8 rounds of screening, the s.a. was significantly improved.

5. Removal of BO from BO-random libraries

The BO in the BO-random library is removed to make the template simpler in the next PCR, and the template is more beneficial to the recognition and extension of polymerase, so that the fidelity of the PCR is not influenced. Since the amount of BO-random library bound to the target is extremely small and difficult to characterize by denaturing PAGE, the present invention uses freshly prepared BO-random libraries for BO removal experiments. 5pmol-BO was added to a NaOH solution having a pH of 13 and left at room temperature for 20min, and the reaction product was neutralized by adding a certain amount of acetic acid. After completion of the reaction, the product was characterized by 10% denaturing PAGE and the results are shown in FIG. 7.

6. Exponential amplification of random libraries bound to a target

See step 1 for details.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

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Claims

1. A method for in vitro screening of aptamers, comprising:

providing a random library having phosphorothioate-based modifications and binding group modifications thereon, said binding group being adapted to bind to a target molecule, said phosphorothioate-based modifications being bound to a binding group;

contacting the random library with a target, and separating the random library combined with the target molecule to be used as a target aptamer obtained by screening.

2. The method of claim 1, wherein the phosphorothioate group is modified on the random library by:

taking the random library as a template, carrying out PCR reaction by using a sulfo-monomer and dNTP to obtain a random library modified by a thiophosphate group,

wherein the thio monomer is selected from thio dATP, thio dTTP, thio dGTP and/or thio dCTP;

preferably, thio dATP is used as the thio monomer, and the fixed base A is 2-10.

3. The method according to claim 2, wherein the DNA polymerase used in the PCR reaction is selected from phusion enzyme, Q5 enzyme or taq enzyme;

preferably, the polymerase is a Phusion enzyme.

4. The method of claim 1, wherein the binding group is modified to the random library by alkylation of the molecule to be bound with the phosphorothioate group;

optionally, the molecule to be bound is selected from bromobenzeneboronic acid, 4-aldehyde benzyl bromide, 4-bromomethylphenylthiol;

when the molecules to be combined are bromobenzene boric acid, the target molecules are polyhydroxy compounds;

when the molecules to be combined are 4-aldehyde benzyl bromide, the target molecules are various compounds or protein molecules containing amino;

when the molecule to be combined is 4-bromomethylthiophenol, the target molecule is a compound capable of interacting with a sulfhydryl group.

5. The method of claim 1, wherein after isolating the random library bound to the target sequence, the resulting library is contacted with a base solution to remove the binding groups and provide a library of interest that can be used for PCR amplification.

6. The method of claim 5, wherein the pH of the alkaline solution is 10 to 13.

7. An aptamer, wherein the aptamer has a phosphorothioate group and a binding group modification thereon;

the aptamers are obtained by the in vitro screening method for aptamers according to claims 5 and 6.

8. A kit for detecting a target molecule, comprising: an aptamer having a phosphorothioate-based modification and a binding group modification, the binding group being adapted to bind to a target molecule.

9. The kit according to claim 8, wherein the aptamer is obtained by the in vitro screening method for an aptamer according to any one of claims 1 to 6.