CN111690655B - Chemically modified base-containing single-stranded DNA aptamer capable of specifically recognizing anthrax protective antigen PA83 and application thereof - Google Patents

Abstract

The invention discloses a single-stranded DNA aptamer containing a chemically modified base and capable of specifically recognizing anthrax protective antigen PA83 and application thereof. The invention takes anthrax protective antigen (PA83) as a target, utilizes a nucleic acid library containing chemically modified nucleotide, and screens out a high-affinity aptamer AP5 by a paramagnetic particle method SELEX, wherein the aptamer can specifically recognize PA83 protein in the environment of various interference proteins; the aptamer can be applied to an aptamer biosensor based on a surface plasmon resonance technology and a fluorescence polarization technology and is used for detecting PA83 protein so as to detect the bacillus anthracis, and the aptamer has the potential of being applied to the construction of a novel detection technology of the protective antigen of the bacillus anthracis.

Description

Chemically modified base-containing single-stranded DNA aptamer capable of specifically recognizing anthrax protective antigen PA83 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a single-stranded DNA aptamer containing a chemically modified base and capable of specifically recognizing anthrax protective antigen PA83 and application thereof.

Background

The form of infection with Bacillus anthracis is spore, and can enter the body from skin wounds, alimentary canal or lungs, wherein aerosol transmission is the main transmission path of bioterrorism attacks. The protective antigen PA is an important component of the anthrax toxin complex. The anthrax protective antigens commonly referred to are PA83 and PA63, and the American military institute for infectious diseases and the American center for disease control research show that large amounts of PA83 are present in the culture supernatant of anthrax spore germination and in the body fluids of patients at the pre-infection stage. Therefore, the research and development of the identification element for identifying the PA83 protein have important significance for the bacillus anthracis detection technology.

Detection techniques using aptamers as recognition elements, which have been developed in recent years, exhibit excellent performance and are attracting attention. Compared with an antibody, the aptamer has the advantages of low preparation cost, strong thermal stability, easy modification, artificial directional optimization and the like. However, the current initial library based on the natural base system only has four variables ACT (U) G which are inferior to the combination of twenty amino acids of the antibody, and in order to ensure the specificity of the aptamer, the random base sequence is not suitable to be overlong, the suitable length is generally 25-42 base numbers, and the information content of the initial library is further limited.

A plurality of studies at home and abroad show that the application of the chemically modified nucleotide to library construction can greatly improve the information content of the library, so that the aptamer with more excellent performance is screened out, but different modification modes have great influence on the function of the aptamer.

Disclosure of Invention

An object of the present invention is to provide an aptamer or a derivative thereof.

The aptamer or the derivative thereof provided by the invention is any one of the following 1) -7):

1) the single-stranded DNA molecule shown in the sequence 1, wherein W at the 12 th position, 29 th position, 31 th position and 33 th position of the sequence 1 is Trp-dU, and Y at the 13 th position and the 27 th position is Tyr-dU;

the Trp-dU is a modified indole group at the C5 position of dU;

the Tyr-dU is a phenol group modified on the C5 position of dU;

2) deleting or adding one or more nucleotides to the aptamer shown in 1) to obtain a derivative of the aptamer with the same function as the aptamer;

3) carrying out nucleotide substitution or modification on the aptamer shown in 1) to obtain a derivative of the aptamer with the same function as the aptamer;

4) transforming the skeleton of the aptamer shown in 1) into a phosphorothioate skeleton to obtain a derivative of the aptamer with the same function as the aptamer;

5) an RNA molecule coded by the aptamer shown in 1) to obtain an aptamer derivative with the same function as the aptamer;

6) peptide nucleic acid encoded by the aptamer shown in 1), and obtaining a derivative of the aptamer with the same function as the aptamer;

7) adding a signal molecule and/or an active molecule and/or a functional group and/or a radionuclide to one or more ends of the aptamer shown in any one of 1) to 6) to obtain a derivative of the aptamer having the same function as the aptamer.

The aptamer derivative represented by 7) is characterized in that a fluorescent group, a biotin group or a radionuclide is labeled at the 5 'end or the 3' end of any one of the aptamers represented by 1) to 6).

The aptamer derivative shown in the 7) is 10A at the 5' end of the aptamer shown in the 1), and the aptamer with 10A added is obtained; and then biotin is labeled at the 5' end of the aptamer added with 10A.

The application of the aptamer in preparing a product with at least one of the following functions 1) to 7) is also within the protection scope of the invention:

1) detecting PA83 protein;

2) recognizing PA83 protein;

3) capturing the PA83 protein;

4) specifically binds to PA83 protein;

5) detecting the bacillus anthracis;

6) detecting whether a sample to be detected contains PA83 protein;

7) and detecting whether the sample to be detected contains the bacillus anthracis or not.

In the application, the product is a kit or an aptamer biosensor based on a surface plasmon resonance technology or a detection product based on a fluorescence polarization technology.

It is another object of the present invention to provide a product comprising the aptamer as described above.

The product has at least one function of 1) to 7) as follows:

1) detecting PA83 protein;

2) recognizing PA83 protein;

3) capturing the PA83 protein;

4) specifically binds to PA83 protein;

5) detecting the bacillus anthracis;

6) detecting whether a sample to be detected contains PA83 protein;

7) and detecting whether the sample to be detected contains the bacillus anthracis or not.

The invention takes anthrax protective antigen (PA83) as a target, utilizes a nucleic acid library containing chemically modified nucleotide, and utilizes a paramagnetic particle method SELEX to screen out a high-affinity aptamer AP5, the aptamer can specifically recognize PA83 protein under the environment of various interference proteins, and simultaneously has no affinity with other proteins such as calf serum protein hemagglutinin protein and the like, so that the aptamer can be applied to an aptamer biosensor based on a surface plasmon resonance technology, and the detection limit of PA83 protein is 10ng/mL (about 6.06 pM); the method is applied to a fluorescence polarization technology for detecting PA83 protein, and has the function of quantitatively detecting PA83 protein with the concentration range of 15.6nM-1 MuM, thereby detecting the bacillus anthracis; the aptamer has wide application prospect, is expected to become a novel nucleic acid molecule for detecting the bacillus anthracis, has the potential of being applied to constructing a novel detection technology for the protective antigen of the bacillus anthracis, and has huge potential in detection application.

Drawings

FIG. 1 is a deoxyuracil with a modified amino acid side chain group at position C5.

FIG. 2 shows the affinity of AP1-AP5 for the target protein PA.

FIG. 3 shows AP-5 characterization of affinity using SPR instrumentation.

FIG. 4 shows the structural prediction of modified AP 5.

FIG. 5 is the dynamic detection of PA protein by aptamer biosensing based on SPR technique.

FIG. 6 is a PA aptamer specificity assay.

FIG. 7 shows detection of PA83 protein by FITC-labeled streptavidin-conjugated PA83 aptamer.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of chemically modified base-containing Single-stranded DNA aptamer that specifically recognizes anthrax protective antigen PA83

One, design and customization of nucleic acid libraries for chemically modified nucleic acids

Because the RNA aptamer is easy to degrade by nuclease, the invention selects and screens the single-stranded DNA aptamer with stable structure in the detection environment. Deoxyuracils (shown in FIG. 1) modified with tryptophan, tyrosine and lysine side chain groups (indole, phenol and amine groups) at C5 were selected for introduction into nucleic acid library preparations, denoted Trp-dU, Tyr-dU, Lys-dU, respectively. The indole group, the phenol group and the amine group have the characteristics of non-polarity, polarity without charge and polarity with positive charge respectively. The total length of the nucleic acid library is 80bp, CAGGGGACGCACCAAGG-N40-CCATGACCCGCGTGCTGACATCG, the front end and the rear end are fixed sequences for amplification, and only comprise natural nucleotide ATCG; the random sequences in the middle section are respectively designed to be 40, and are synthesized by a solid-phase phosphoramidite triester method, wherein the adding ratio of Trp-dU to Tyr-dU to Lys-dU to dT to dA to dG to dC is 1:1:1:1:4:4: 4.

Second, magnetic bead method SELEX

1. Coupling biotin-labeled PA83 protein to streptavidin-modified magnetic beads

1.1 spin-up M-280 streptavidin magnetic particles (10mg/mL), 50. mu.L (0.5mg) was pipetted into a 1.5mL tube using a 100. mu.L pipette. Standing on a magnetic frame for 5min, and removing the supernatant.

1.2 Add 250. mu.L buffer and spin wash the magnetic particles. The supernatant was discarded with a precision pipettor. The washing was repeated 2 more times, and the magnetic particles were washed 3 times in total (labeled: magnetic particle X tube);

1.3 at room temperature, add 100 μ L buffer containing 15 μ g target protein to the magnetic particle X tube, incubate slowly shaking at room temperature for 30 minutes;

1.4 place the X-tube containing the coupling reaction on a magnetic frame, discard the buffer with a precision pipettor, wash with 200. mu.L of buffer, repeat 2 times. Finally, 100. mu.L of buffer was added to the X-tube (named: T1).

2. Pre-processing nucleic acid libraries

2.1 dissolving the library containing the chemically modified nucleic acid in 2OD with 590 mu LPBS buffer solution, wherein the final concentration is 100 mu M, and uniformly mixing by shaking;

2.2 standing in a water bath kettle at 95 ℃ for 5min, and naturally cooling to room temperature so as to form a stable tertiary structure, thereby obtaining the pretreated nucleic acid library.

Third, negative selection

3.1 adding the nucleic acid library pretreated by the second intermediate 2 into an EP tube sealed overnight by BSA, uniformly mixing the nucleic acid library at room temperature for 2 hours in a vortex manner, taking the supernatant, and removing an aptamer combined with the tube wall;

3.2 adding the nucleic acid library after 3.1 treatment into an M-280 magnetic bead tube which is not coupled with PA83 protein, and removing the aptamer combined with the M-280 magnetic bead to obtain a nucleic acid library A.

Fourth, Positive screening

4.1 spin incubation of "nucleic acid library A" with T1 from 1 of the second above for 2h at room temperature with mixing;

4.2 placing the magnetic beads in a magnetic frame for 10min, washing the magnetic beads for 3 times by PBST containing 0.1% Tween 20, wherein the volume of washing liquid is 200 mu L each time;

4.3 discard the supernatant, resuspend the beads in 50. mu.L of PBS at pH 7.2, label "library Y".

Fifthly, aptamer dissociation and purification

5.1 to 50. mu.L of the above "library Y", 50. mu.L of 1N NaOH was added. Incubate at 65 ℃ for 30 min. Then 40. mu.L of 2M Tris-Cl (pH 5.5) was added to neutralize the NaOH;

5.2 adsorbing the magnetic particles by using a magnetic frame, and transferring the supernatant into a 1.5mLEP tube;

5.3 purification of "library Y" (as per instructions) using EZ-10 column PCR product purification kit.

Six, two positive screening

6.1 Add 15. mu.L of "library Y" to 30. mu.L of 500nM PA83 labeled protein (uncoupled beads) and vortex incubate for 45min at room temperature;

6.2 taking 10 mu L M-280 streptavidin magnetic particles, washing for 3 times by PBST, and discarding the supernatant;

6.3 adding the mixed liquid in the first step into the EP tube in the step 2, and performing vortex incubation for 2h at room temperature;

6.4 Place on magnetic frame for 10min, discard the supernatant, PBST wash 5 times, 50 u L pure water heavy suspension, 4 degrees C preservation, labeled "aptamer AP".

3. The aptamer sequence was determined by sequencing and comparing the databases.

20 μ L of the "aptamer AP" was taken and sent to the technology company for sequencing and database determination of the modified nucleotide sites.

5 candidate aptamers AP1-AP5 targeting PA protein were screened by using a magnetic bead method SELEX. The sequence abundant sequence (top five of the repetition rate) in the sequencing synthesis result is used as a candidate aptamer, and the specific sequence is shown in table 1.

Table 1 aptamer sequence targeting PA83 protein

In Table 1 above, column 1 is the name of candidate aptamer, column 2 is the sequence of candidate aptamer targeting PA protein, wherein W, X, Y is Trp-dU, Tyr-dU and Lys-dU, respectively, wherein Trp-dU is the modification of tryptophan side chain group (indole group) at C5 position of dU, Tyr-dU is the modification of tyrosine Tyr side chain group (phenol group) at C5 position of dU, Lys-dU is the modification of lysine Lys side chain group (amine group) at C5 position of dU (as shown in FIG. 1); column 3 is the respective aptamer repetition rate.

Fifthly, comparing and evaluating the affinity of the aptamer AP1-AP5 by an ELISA method

Adding 10 sequences of dA to the 5' end of each sequence of the candidate aptamers AP1-AP5 obtained in the fourth step, and labeling biotin: 5 '-Biotin-AAAAAAAA-AP-3', respectively obtaining an aptamer AP1 labeled with Biotin, an aptamer AP2 labeled with Biotin, an aptamer AP3 labeled with Biotin, an aptamer AP4 labeled with Biotin and an aptamer AP5 labeled with Biotin; will each beThe aptamers labeled biotin were dissolved in PBS (pH 7.4, available from Gibco)^TMThe product number is 10010001), the concentration is 100pM, and an aptamer AP1 solution marked with biotin, an aptamer AP2 solution marked with biotin, an aptamer AP3 solution marked with biotin, an aptamer AP4 solution marked with biotin and an aptamer AP5 solution marked with biotin are respectively obtained; wherein the solute is an aptamer of the respective labeled biotin.

PBS without any aptamer was used as a blank.

The affinity of each aptamer sequence to the target is determined by an ELISA method by taking the aptamer labeled with biotin as a primary antibody and taking the streptavidin labeled with HRP as a secondary antibody. The method comprises the following specific steps:

1. coating antigen: PA83 protein (purchased from List Biological Labs, cat # 171E) was diluted to 10mg/ml with coating solution, coated in microwell plates, 0.5. mu.g/well, and an equal amount of coating solution (purchased from Biotechnology (Shanghai) Co., Ltd., cat # E661004) was added to the blank wells overnight at 4 ℃;

2. and (3) sealing: the wells of the plate were discarded and rinsed five times with PBST (0.5% w/v Tween-20 (from Sigma, cat. No. 44112) in PBS buffer pH 7, 5min each time, 2% BSA blocking solution (from Shanghai Biotech, cat. No. 520035) was added at 200. mu.L/well and blocked in a 37 ℃ incubator for 2 h;

3. addition of biotinylated aptamer: the plate was discarded and rinsed five times with PBST for 5min each. Respectively putting the aptamer AP1-AP5 solution with the concentration of 100pM labeled biotin into a microporous plate, wherein the input amount of each hole is 100 mu L, and each group is parallel to two multiple holes to be used as a group to be detected;

wells were blanked with PBS alone.

Incubating for 2h in a thermostat at 37 ℃;

4. adding a secondary antibody: the plate was discarded and rinsed five times with PBST for 5min each. A1: 10000 dilution of HRP-labeled streptavidin (available from Shanghai Biotech, cat # D111054) (PBS (pH 7.4, available from Gibco)^TMGood No. 10010001)) as "Adding the secondary antibody into a microplate, incubating for 1h in a thermostat at 37 ℃ at 100 mu L/hole;

5. color reading value: the plate was discarded and rinsed five times with PBST for 5min each. Adding 100 μ L/hole TMB color developing solution (purchased from Biotechnology engineering (Shanghai) Ltd., product No. E661007), keeping away from light for 15min, adding equal amount of stop solution (2M H)₂SO₄) And reading the OD450 value.

And comparing the change values of the OD450 absorbance of each group to be detected with that of the blank group, wherein the greater the change of the absorbance, the stronger the capacity of the aptamer for combining with the PA 83.

As shown in FIG. 2, AP1-AP5 can bind to PA immobilized on the bottom of the microplate, and AP5 has the highest affinity with the target protein PA 83.

Sixth, AP5 affinity characterization

AP5 was diluted at various concentrations of 10, 5, 2.5, 1.25, 0.625nM and aptamer affinity was characterized using an SPR (surface plasmon resonance) detector to balance the dissociation constant Kd as determined by Bai, Chenjun, et al, aptamer Selection and Application in multiple Binding-Based electric impedance Detection of activated H1N1Virus, biosensors & Bioelectronics (2018): S5609566318302203.

The SPR molecular dynamics results are shown in FIG. 3, the binding rate constant K of AP5_onIs 1.29X10^6M-1S-1Dissociation rate constant K_offIs 1.36X10^-3S^-1Equilibrium dissociation constant K_d＝K_off/K_on1.068nM, higher affinity than the currently reported aptamers, meeting the requirement of project development.

Example 2 application of aptamer AP5 in detection of PA83 protein

First, biotin-labeled aptamer AP5

The Biotin-labeled aptamer AP5 was synthesized by adding a sequence of 10 dA sequences to the 5' end, 5' -Biotin-AAAAAAAAAA-AP5-3 ' (5 ' -Biotin-AAAAAAAAAAGCCCACGGCGGWYCGCCGGCCACAGTYAWCWGWGGTGGGC-3 '), and the secondary structure of the aptamer was predicted using RNAstructure software as shown in FIG. 4.

Second, aptamer biosensor based on surface plasmon resonance technology is constructed by aptamer AP5

Methods for constructing aptamer Biosensors based on surface plasmon resonance technology can be found in Wang S, Dong Y, Liang X.development of a SPR aptamer associated with oriented aptamers for direct capture and detection of tetracycline in multiple holes samples [ J ]. Biosensors & Bioelectronics,2018,109:1, as follows:

1. fixing the dextran nano gold chip on a fluid chamber of an SPR instrument, and adjusting the flow rate to 25 mu L/min;

2. 306.72mg EDC and 46.08mg NHS were weighed respectively, and added into 4m ultrapure water, 100. mu.L each was mixed well and added into the sample introduction tube A. Another 0.5mg of streptavidin was added to 1mL of 10mM sodium acetate solution, and 100. mu.L was added to the sample tube B. Adding 100 mu L of ethanolamine aqueous solution with the pH value of 8.5 and the concentration of 1M serving as confining liquid into the sample injection tube C to obtain EDC/NHS mixed solution;

3. running a streptavidin fixing program, activating EDC/NHS mixed solution for 7min, fixing streptavidin for 10min, and blocking ethanolamine for 10 min;

4. preparing 10nmol/L of the prepared biotin-labeled aptamer AP5 by taking PBST as a buffer solution, taking 100 ul for fixation, setting the fixation time to be 10min and the fixation flow rate to be 10 muL/min, and sealing for 10min by using ethanolamine after the fixation is finished; a biosensor was constructed.

5. And (3) testing the sensitivity:

PA83 solution with concentration of 0ng/ml,1ng/ml,10ng/ml,50ng/ml and 100ng/ml was prepared as the sample to be tested (PBS (pH 7.4, available from Gibco)^TMItem number 10010001), solute PA83 protein was purchased from List Biological Labs, item number 171E), binding time was set to 90s, dissociation time was set to 120s, flow rate was set to 10 μ L/min, 20 mhhcl was selected as regeneration liquid, regeneration time was set to 30 s;

the sensitivity test result is shown in figure 5, the biosensor can complete sample detection within 5min, and the PA protein with the concentration of more than 10ng/ml can lead out obvious fluctuation of a detection signal.

6. And (3) specific detection:

samples containing the interfering protein were prepared, 100. mu.L each,

sample 1: solution containing 100ng/ml Hemagglutinin (HA) protein: consists of a solute and a solvent, PBS (pH 7.4, from Gibco)^TM100,10001), the solute is HA (available from Beijing Shenzhou Yinqiao science and technology Co., Ltd.: cat # 11055-V08B), and the concentration of the solute in the solution is 100 ng/ml;

sample 2: bovine Serum Albumin (BSA) solution containing 100 ng/ml: consists of a solute and a solvent, PBS (pH 7.4, from Gibco)^TMItem number 10010001), solute BSA (available from bio-engineering (shanghai) gmbh, item number C102301), solute concentration in solution 100 ng/ml;

sample 3: solution containing 100ng/ml Hemagglutinin (HA) protein +100ng/ml BSA protein: consists of a solute and a solvent, PBS (pH 7.4, from Gibco)^TMStock number 10010001), the solutes are HA and BSA, and the concentrations of the solutes in the solution are both 100 ng/ml;

sample 4: solution containing 100ng/ml Hemagglutinin (HA) protein +100ng/ml BSA protein +100ng/ml PA83 protein: consists of a solute and a solvent, PBS (pH 7.4, from Gibco)^TM100,10010001) with solutes of HA, BSA and PA83, wherein the concentrations of the solutes in the solution are all 100 ng/ml;

sample 5: PBST.

The binding time was set to 90s, the dissociation time to 120s, the flow rate to 10. mu.L/min, 20mM HCl was selected as the regeneration liquid, and the regeneration time to 30 s.

The results of the specificity test are shown in fig. 6, and it can be seen that the aptamer AP5 can specifically bind to PA protein under the environment of various interfering proteins.

Aptamer AP5 establishment of aptamer-Based PA83 protein fluorescence Polarization technology the aptamer-Based PA83 protein fluorescence Polarization technology can be found in Hirotaka Minagawa, et al.fluorescence Polarization-Based Rapid Detection System for Salivary Biomarkers Using modified DNA Aptamers contacting Base-applied bases, anal. chem.2020,92, 1780-1787:

in the biotin-labeled aptamer AP5 solution, the solute was the biotin-labeled aptamer AP5 as described above, and the solvent was PBS (pH 7.4, available from Gibco)^TMGoods number 10010001)

A solution of biotinylated aptamer AP5 (20 nM concentration) was mixed with a solution of FITC-labeled streptavidin (5nM) purchased from Shanghai Producer under the code D110512 in PBS (pH 7.4 from Gibco)^TMStock number 10010001)) were mixed in equal volumes and reacted at a temperature of 25 ℃ for 30min to obtain a mixture. The mixture was mixed with a PA83 protein solution (PBS (pH 7.4, from Gibco, Inc.) at various concentrations (15.6nM, 31.2nM, 62.4nM, 312nM, 500nM, 1000nM) in PBS^TM10010001, and PA83 protein as solute), reacting for 10min, and detecting with fluorescence polarization technique (excitation wavelength 485 and emission wavelength 535) by a SpectraMax M5/M5e multifunctional microplate reader.

As shown in fig. 7, it can be seen that the aptamer AP5 has the ability to detect PA83 protein at a concentration ranging from 15.6nM to 1 μ M, and the fitting curve formula is y 1.8945ln (x) +2.1256, and the pearson correlation coefficient is calculated to be 0.9602, and P0.0023 < 0.05.

The results show that the aptamer AP5 has high affinity for a target PA83 protein, and can specifically recognize the PA83 protein in the environment of various interference proteins; the aptamer can be applied to an aptamer biosensor based on a surface plasmon resonance technology and a fluorescence polarization technology and is used for detecting PA83 protein so as to detect the bacillus anthracis, and the aptamer has the potential of being applied to the construction of a novel detection technology of the protective antigen of the bacillus anthracis.

SEQUENCE LISTING

<110> China people liberation force disease prevention control center

<120> single-stranded DNA aptamer containing chemically modified base and capable of specifically recognizing anthrax protective antigen PA83 and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 40

<212> DNA

<213> Artificial sequence

<400> 1

gcccacggcg gwycgccggc cacagtyawc wgwggtgggc 40

Claims (5)

1. An aptamer which is any one of the following 1) to 2):

1) the single-stranded DNA molecule shown in the sequence 1, wherein W at the 12 th position, 29 th position, 31 th position and 33 th position of the sequence 1 is Trp-dU, and Y at the 13 th position and the 27 th position is Tyr-dU;

the Trp-dU is a modified indole group at the C5 position of dU;

the Tyr-dU is a phenol group modified on the C5 position of dU;

2) adding 10A to the 5' end of the aptamer shown in 1) to obtain the aptamer added with 10A; and then biotin is labeled at the 5' end of the aptamer added with 10A.

2. Use of the nucleic acid aptamer of claim 1 for preparing a product having at least one of the following functions 1) to 2):

1) detecting PA83 protein;

2) capturing the PA83 protein.

3. The use of the nucleic acid aptamer of claim 1 in the preparation of a product for detecting bacillus anthracis.

4. Use according to claim 2 or 3, characterized in that: the product is a kit or an aptamer biosensor based on a surface plasmon resonance technology or a detection product based on a fluorescence polarization technology.

5. A product for detecting or capturing PA83 protein, comprising the aptamer of claim 1.

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