CN112410326B

CN112410326B - In vitro screening method of nucleic acid aptamer, nucleic acid aptamer and kit for detecting target molecule

Info

Publication number: CN112410326B
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: 2023-05-12
Anticipated expiration: 2040-11-25
Also published as: CN112410326A

Abstract

The invention provides an in vitro screening method of a nucleic acid aptamer, which comprises the following steps: providing a random library having phosphorothioate group modifications and binding group modifications thereon, the binding group being adapted to bind to a target molecule, the phosphorothioate group being bound to a binding group; contacting the random library with a target, and separating the random library combined with the target molecule as a target nucleic acid aptamer obtained by screening. According to the invention, phosphorothioate groups and binding groups are modified in the random library, and the binding groups have an affinity effect with the target, so that the distance between the random library and the target is shortened, the initial binding activity of the random library and the target is endowed, the random library and the target are easier to bind, and the nucleic acid aptamer is easier to be screened successfully.

Description

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

Technical Field

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

Background

Aptamer refers to RNA or single-stranded DNA capable of forming a certain spatial structure and specifically binding to a target substance, and the target substance is wide in range and includes proteins, small molecules, metal ions and even whole cells. The binding forces of the aptamer to the target substance are mainly various weak forces including hydrogen bonding, intermolecular forces, electrostatic adsorption or base stacking forces, etc. The aptamer has several advantages over the antibody (a protein): a. can be synthesized artificially, and has low production cost and stable performance; b. can withstand the transition from high temperature to low temperature without requiring harsh storage conditions; c. the in vitro screening results, thus, without regard to whether the target is immunogenic; d. flexible structure and easy modification of other groups.

The aptamers are typically screened by an in vitro screening technique known as the exponential enrichment system evolution technique (Systematic Evolution of Ligands by EXponential enrichment, SELEX). First, a composition containing 10 is synthesized by combinatorial chemistry ¹⁴ -10 ¹⁵ Random DNA libraries of different DNA sequences, which will form a myriad of different spatial structures, thus providing the possibility that several DNA sequences exist in the random DNA library that specifically bind to the target. First wheelIn screening, the constructed random library is incubated with a target, and after incubation, DNA sequences capable of binding to the target and DNA sequences incapable of binding to the target are separated by a certain method. The sequence capable of binding to the target is then amplified by PCR, resulting in exponential enrichment of this portion of the DNA sequence. Subsequently, the single strand is used to isolate the PCR amplified product, and the isolated single strand DNA library is further incubated with the target for the next round of screening. After several rounds of screening, DNA sequences that specifically bind to the target and do not bind to the target analog are isolated from the DNA library.

Studies have shown that certain chemical modifications on aptamers can result in greater binding to targets. However, chemical modifications on existing aptamers are somewhat blind and random. The choice of modification site is one of the key factors to consider, and for some key sites, chemical modification thereon may result in difficulties in aptamer binding to the target. This presents challenges for rational design of existing aptamers to improve their affinity for targets. On the other hand, the random library of conventional SELEX consists of only A, T, G, C bases, and the formed structure has strong randomness and no initial activity of binding to the target, so that aptamer screening is difficult. Thus, successful screening of aptamers is positive by introducing functional groups in the randomized library that can bind to the target. Traditional chemical modification SELEX is usually realized by introducing non-natural base modification, and the method firstly needs to synthesize new non-natural base monomer molecules, and then introduces the new base molecules into a random library through PCR, so that the sequence and structure which can be formed by the random library are more abundant. However, this method has difficulty in synthesizing monomers, and has a problem in that, when PCR is performed using a DNA containing an unnatural base as a template and an unnatural base, polymerase enzyme to be selected for PCR amplification is limited, and fidelity of a PCR product is challenging.

Thus, current methods for in vitro screening of nucleic acid aptamers remain to be investigated.

Disclosure of Invention

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

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

The random library used in the method according to the embodiment of the invention has phosphorothioate group 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 random library is easy to screen. Compared with the traditional unnatural base synthesis, the invention synthesizes the random library for modifying various functional groups based on the reaction of the PS-random library and the binding group. The method can modify different molecules or functional groups on a random library according to different target molecules, and has simple synthesis method and wide applicability.

In addition, after screening the target nucleic acid aptamer, the binding group can be easily removed. Compared with the PCR with DNA carrying unnatural base as the template and target molecule, the PCR template prepared by using the aptamer modified by phosphorothioate group is more friendly to polymerase and easier to PCR.

According to an embodiment of the present invention, the in vitro screening method for a nucleic acid aptamer 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 with a thio-phosphate group modified random library by using the random library as a template, 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 non-natural bases to carry out GPCR amplification, the invention adopts the thio monomer to carry out PCR amplification, can use high-assurance polymerase to carry out PCR reaction, and has higher fidelity of PCR products.

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

According to an embodiment of the invention, the DNA polymerase used for the PCR reaction is selected from the group consisting of a phusion enzyme, a Q5 enzyme and a taq enzyme. Thus, the fidelity of the PCR product can be improved. 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 a random library by alkylation of the molecule to be bound with the phosphorothioate group.

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

According to an embodiment of the invention, when the molecule to be bound is bromophenylboronic acid, the target molecule is a polyhydroxy compound, such as adenosine, a polysaccharide; when the molecule to be bound is 4-aldehyde benzyl bromide, the target molecule is a variety of amino-containing compounds or protein molecules, such as streptavidin; when the molecule to be bound is 4-bromomethyl thiophenol, the target molecule is a compound that can interact with a thiol group, such as arsinic acid.

According to an embodiment of the invention, after isolation of the random library binding to the target sequence, the resulting library is contacted with an alkaline solution such that the binding groups are removed, resulting in a library of interest. By contacting the library with an alkaline solution, the binding groups can be effectively removed, resulting in a library of interest that can be used for PCR amplification. Compared with the traditional chemical modification of SELEX which uses DNA with unnatural base as a template for PCR, the invention uses the target random library with the binding group removed by alkali liquor as a PCR template, which is more friendly to polymerase and has higher PCR fidelity.

According to an embodiment of the invention, the pH value of the alkaline solution is 10-13. Thus, the binding group, such as phenylboronic acid, is rapidly and efficiently removed.

In another aspect of the invention, the invention provides a nucleic acid aptamer. According to the embodiment of the invention, the aptamer is provided with phosphorothioate groups and binding group modifications; the nucleic acid aptamer is obtained by the in vitro screening method of the nucleic acid aptamer. Thus, nucleic acid aptamers according to embodiments of the invention may specifically bind to target molecules.

In yet another aspect of the invention, a kit for detecting a target molecule is provided. According to an embodiment of the invention, the kit comprises: a nucleic acid aptamer having a phosphorothioate modification and a binding group modification, the binding group being adapted to bind to a target molecule. Target molecules can be specifically detected using the kit according to embodiments of the present invention.

According to an embodiment of the invention, the nucleic acid aptamer is obtained by the in vitro screening method of the nucleic acid aptamer described previously.

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 foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 shows a schematic flow diagram of a method for modifying functional groups with affinity for a target at random libraries (R-random libraries);

FIG. 2 shows the structural formula of PS-modified monomers;

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

FIG. 4 shows a chemical modification SELEX flow diagram based on a PS post-modification reaction;

FIG. 5 shows a graph of the results of PCR using thio dATP, dTTP, dCTP, dGTP as a monomer;

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

FIG. 7 shows a schematic representation of denaturing PAGE of lye versus BO removal from a BO-random library;

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

Detailed Description

Embodiments of the present invention are described in detail below. 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 nucleic acid aptamers, see fig. 4, based on Phosphorothioate (PS) reactivity, by introducing molecules or functional groups on a random library that specifically bind to target molecules, thereby conferring initial affinity to the random library for the target, and by approaching the distance between the random library and the target, allowing easy aptamer screening.

The invention firstly establishes a random library, wherein the random library is N bases with the middle of 5nt-60nt except the primers at the two ends. The initial random library was obtained by solid phase synthesis, and PCR was performed using the template and a thio monomer to obtain a PS modified random library (see FIG. 1). Taking adenosine as a screening target as an example, adenosine is an o-hydroxy compound, and thus modifying phenylboronic acid on a random library was chosen. Boric acid and ortho-hydroxy compounds tend to form dynamic chemical bond borates, and thus, in theory, after introducing phenylboronic acid into a random library, the phenylboronic acid-random library will pull the distance between the random library and adenosine by the force of phenylboronic acid and adenosine, allowing the nucleic acid sequence that tends to form a structure capable of binding to adenosine to have an opportunity to access and successfully bind to adenosine.

Specifically, the nucleic acid aptamer screening is performed by using a struct-switching SELEX, and the random library sequences are as follows: ATACCAGCTTATTCAATT-N20-GGTCTTTCTGTTCT-N30-AGATAGTAAGTGCAATCT, B-DNA sequence: a random library of biotin-AAAAAAAAAAAAAGAACAGAAAGACC can be hybridized to B-DNA via an intermediate GGTCTTTCTGTTCT sequence and then attached to an avidin modified magnetic sphere via a biotin on the B-DNA. The forward primer is AP1:5'-ATACCAGCTTATTCAATT-3' reverse primer is TER-AP2: the 5'-A20-Spacer18-AGATTGCACTTACTATCT-3' was thiodATP (2 '-deoxydenosine-5' -O- (1-thiotriphosphite)) as the thio monomer, the common monomer dTTP, dGTP, dCTP (see FIG. 2) as the other monomers, and the PCR result was shown in FIG. 5. 100 ntPS-random library bands were excised and purified for recovery.

The PS-random library is then reacted with bromobinding molecules to obtain a random library modified with binding molecules. Taking bromine-band molecule Br-R as an example, a PS-random library and Br-R are taken as reactants, and the two reactants are dissolved in a buffer system with the pH value between 4 and 10, such as phosphate buffer, MES (4-morpholinoethanesulfonic acid) buffer, MOPS (3- (N-morpholino) propanesulfonic acid) buffer, HEPES (hydroxyethyl piperazine ethanamic acid) buffer and the like. Adding organic solvent to dissolve brominated molecules thoroughly, reacting PS-random library at 10nM-1mM, br-R at 100 μM-100mM, and reacting at 10-60 deg.C for 0.5-200 h. After the reaction, the excessive small molecules can be removed through an ultrafilter (Amicon), the product (random library-R) can be purified, and the reaction mixture can be subjected to denaturing PAGE, and then the product is subjected to gel cutting recovery and purification.

The reaction products were characterized by denaturing PAGE and the results are shown in FIG. 6. After the PO-random library is reacted with Br-BO, the reaction product has no displacement change relative to the denaturation PAGE of the PO-random library, and after the PS-random library is reacted with Br-BO, the migration speed of the reaction product on the denaturation PAGE is slower than that of the reactant. This suggests that BO was successfully modified to PS-random libraries. Referring to FIG. 3, the present invention then tested whether the alkali-treated BO-random library could successfully remove BO from the random library, and the results are shown in FIG. 7, the run-out displacement of the product obtained after alkali treatment is comparable to that of the PS-random library, indicating that alkali can effectively remove small molecules from the random library and recover the PS-random library. PCR with PS-random library as template is more fidelity than PCR with BO-random library as template.

The BO-random library was then hybridized to B-DNA and successfully immobilized to an avidin magnetic sphere by a biotin-avidin reaction. Then the magnetic ball and the adenosine molecule are placed in buffer solution and incubated at room temperature. After a period of incubation, the random library of BO-capable of binding to adenosine will dissociate from the magnetic beads. Collecting the BO-random library dissociated from the magnetic ball, treating with alkali liquor to remove BO, hydrolyzing the BO-random library into PO-random library (natural non-modified random library), and performing PCR amplification with the obtained PO-random library as template and thio-dATP, dTTP, dGTP, dCTP as monomer, performing denaturing PAGE characterization and gel cutting recovery to obtain new PS-random library. Then, according to the above procedure, BO-random library synthesis, target incubation, elution of affinity nucleic acid sequence, BO removal, PCR amplification and new round of PS-random library gel cutting recovery are carried out. And (3) repeatedly carrying out a plurality of rounds of screening, and enriching to obtain the nucleic acid sequence with stronger affinity with the adenosine.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

1. Construction of PS-random libraries

Firstly, a random library is obtained through solid phase synthesis, the sequence is ATACCAGCTTATTCAATT-N20-GGTCTTTCTGTTCT-N30-AGATAGTAAGTGCAATCT, and in addition, B-DNA is synthesized, and the sequence is: a random library of biotin-AAAAAAAAAAAAAGAACAGAAAGACC can be hybridized to B-DNA via an intermediate GGTCTTTCTGTTCT sequence and then attached to an avidin modified magnetic sphere via a biotin on the B-DNA. Meanwhile, a primer for PCR is synthesized, and the forward primer is AP1:5'-ATACCAGCTTATTCAATT-3' reverse primer is TER-AP2: the 5'-A20-Spacer18-AGATTGCACTTACTATCT-3' is formed by using thiodATP as a thio monomer, and using a natural monomer dTTP, dGTP, dCTP as other monomers. To a 200. Mu.l PCR tube was added 2.5. Mu.l AP1, 2.5. Mu.l TER-AP2, 1. Mu.l 10mM thiodATP, 0.5. Mu.l dTTP, 0.5. Mu.l dGTP, 0.5. Mu.l dCTP, 41.5. Mu. l H ₂ O, 0.5. Mu.l Phusion enzyme and 0.5. Mu.l random library template. Placing the above PCR system on a PCR instrument, denaturing at 98deg.C for 3min, denaturing at 98deg.C for 20s, annealing at 75deg.C for 20s, extending at 72deg.C for 20s, and extending at 72deg.C for 5min after 30 times of circulationAnd then maintained at 4 ℃. PCR products were identified by 10% denaturing PAGE and the results are shown in FIG. 5. Subsequently, the strips of the target fragments (100 nt PS-random library) were cut into 1.5mL centrifuge tubes, 300. Mu.L of PBS buffer was added thereto, and mixed well with a vortex shaker, and then placed on a spin stand and shaken overnight at room temperature. The next day, the supernatant was taken and the PS-random library therein was recovered by ethanol precipitation.

2. Preparation of phenylboronic acid (BO) -random library

The PS-random library was dissolved in 30. Mu.l of water, and 30. Mu.l of 100mM PBS buffer (pH=6), 30. Mu.l of 50mM 4- (bromomethyl) phenylboronic acid (Br-BO) dissolved in DMF and 30. Mu.l of DMF were added thereto, and after mixing uniformly with a vortex oscillator, they were allowed to react at room temperature for 20 hours on a rotating frame. The reaction products were identified by running denaturing PAGE and the results are shown in FIG. 6. After completion of the reaction, the reaction mixture was washed by centrifugation with Amicon-10k filter tubes and water 6 times at 12000rpm for 12 minutes each to remove the excess unreacted Br-BO.

3. Fixation of BO-random library to magnetic spheres

The BO-random library is immobilized on the magnetic beads by hybridization with its complementary pair of DNA sequences (B-DNA: biotin-AAAAAAAAAAAAAGAACAGAAAGACC) and then by reaction of biotin on the B-DNA with avidin on the magnetic beads. 1 equivalent of BO-random library was mixed with 5 equivalents of B-DNA in screening buffer (50 mM MOPS, 1mM MgCl) ₂ 100mM NaCl and 5mM KCl, pH 7.4), heated at 98℃for 5min, and then cooled slowly to room temperature. Then, the BO-random library-B-DNA was mixed with avidin beads and shaken at room temperature for 30min. After the reaction was completed, the supernatant was removed, and the magnetic beads were washed 10 times with the screening buffer.

4. Incubation of random library with target

Since the random library-BDNA complex is always in dissociation equilibrium, there is always a fraction of the DNA library that will dissociate from the resin column without the addition of adenosine. After DNA was immobilized on the magnetic ball and washed 10 times, background collection was performed. To facilitate comparison of the dissociation of the DNA library itself in different rounds of screening, we set a blank dissociation time-the same incubation time-for each round of screening, without adenosine, to determine the amount of dissociation itself. For example, when the incubation time is 1h, we first left the DNA complex-magnetic beads washed 10 times at room temperature for 1h, then add 500. Mu.L of screening buffer after 1h, collect the first effluent (B1), then add 500. Mu.L of screening buffer, collect the second effluent (B2). Then, a small volume of saturated adenosine solution was added, and the mixture was incubated at room temperature for 1 hour with gentle stirring using a pipette. After 1h, 500. Mu.L of screening buffer was added to the magnetic beads, and the first effluent (E1) was collected, followed by 500. Mu.L of screening buffer, and the second effluent (E2) was collected, and thus E3, E4, E5, etc. were collected continuously. E1/B1 was used to characterize the affinity of the target to the DNA library (structure-switching activity, S.A.). The results are shown in fig. 8, and after 8 rounds of screening, the s.a. is significantly improved.

5. Removal of BO from BO-random libraries

The BO on the BO-random library is removed to make the template simpler in the next PCR, and is more beneficial to the recognition and extension of polymerase so as to prevent the fidelity of the PCR from being affected. Since the amount of the BO-random library bound to the target is very small and is difficult to characterize by denaturing PAGE, the BO removal experiment is performed by using a freshly prepared BO-random library. 5pmol-BO was added to NaOH solution of pH 13 and allowed to react at room temperature for 20 minutes, and a certain amount of acetic acid was added to the reaction product to neutralize it. After the reaction was completed, the products were characterized by 10% denaturing PAGE, and the results are shown in FIG. 7.

6. Exponential amplification of random libraries binding to targets

See step 1 in particular.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means 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 present invention. In this specification, schematic representations of the above terms are not necessarily directed 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, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

SEQUENCE LISTING

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Claims

1. An in vitro screening method for a nucleic acid aptamer, comprising:

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

contacting the random library with a target, and separating the random library combined with the target molecule as a target nucleic acid aptamer obtained by screening; the phosphorothioate groups were modified on the random library by:

taking the random library as a template, carrying out PCR reaction by using a thio monomer and dNTPs to obtain a random library with phosphorothioate group modification,

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

the binding group is modified onto a random library by alkylating the molecule to be bound with the phosphorothioate group;

the molecule to be bound is selected from 4- (bromomethyl) phenylboronic acid.

2. The method according to claim 1, characterized in that thiodATP is used as the thio monomer and the immobilized base A is 2-10.

3. The method of claim 2, wherein the DNA polymerase used in the PCR reaction is selected from the group consisting of a phusion enzyme, a Q5 enzyme, and a taq enzyme.

4. The method of claim 2, wherein the DNA polymerase used in the PCR reaction is a Phusion enzyme.

5. The method of claim 1, wherein after isolating the random library that binds to the target sequence, the resulting library is contacted with an alkaline solution such that the binding groups are removed, resulting in a library of interest that can be used for PCR amplification.

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

7. A nucleic acid aptamer, characterized in that the nucleic acid aptamer has phosphorothioate groups and binding group modifications thereon;

the nucleic acid aptamer obtained by the in vitro screening method of the nucleic acid aptamer of claims 5 and 6;

the molecule to be bound is selected from 4- (bromomethyl) phenylboronic acid.

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

the nucleic acid aptamer is obtained by the in vitro screening method of the nucleic acid aptamer according to any one of claims 1 to 4.