CN114807148A - Aptamer for detecting new coronavirus SARS-CoV-2 and application thereof - Google Patents

Aptamer for detecting new coronavirus SARS-CoV-2 and application thereof Download PDF

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CN114807148A
CN114807148A CN202210247345.1A CN202210247345A CN114807148A CN 114807148 A CN114807148 A CN 114807148A CN 202210247345 A CN202210247345 A CN 202210247345A CN 114807148 A CN114807148 A CN 114807148A
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胡小晓
欧海龙
刘匀毅
谭蔚弘
曾洁
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Abstract

The invention discloses a nucleic acid aptamer capable of being used for detecting SARS-CoV-2 Spike RBD (N501Y) protein and application thereof. The invention screens out two nucleic acid aptamers which can be specifically combined with SARS-CoV-2 Spike RBD (N501Y) protein by an in vitro screening technology: NY 36: 5'-AACACGACACTAGTTCGGCTCGCG AACCCATCGCACGTCGTG-3' and NY27: 5'-AAGCCCACATGCCTGTGACCATCGAACAGCAGCAGCGTCGTG-3'; the two aptamers have high specificity, are weakly combined with common RBD protein, and have stronger combining ability with SARS-CoV-2 Spike RBD (N501Y) protein.

Description

Aptamer for detecting novel coronavirus SARS-CoV-2 and application thereof
Technical Field
The invention belongs to the technical field of biology, relates to a nucleic acid aptamer and application thereof, and particularly relates to a nucleic acid aptamer for detecting a new coronavirus and an application method thereof.
Background
SARS-CoV-2 acts as an enveloped single-stranded positive RNA virus. The genome is about 30000 nucleotides in length. The co-encoded 27 proteins include: an RNA polymerase (RdRP), a non-structural protein, a structural protein. The four major structural proteins are: spike sugar (S), membrane (M), envelope (E) and nucleocapsid (N) proteins. Wherein the S protein has a molecular mass of about 175 kDa and is a type I membrane protein comprising two subunits, S1 and S2. S1 mainly contains a Receptor Binding Domain (RBD) responsible for recognizing cell surface receptors. S2 contains essential elements required for membrane fusion. The S protein binds to receptors on the cell surface and plays an important role in the tropism of the virus. It also mediates fusion of the viral membrane with the host cell membrane, ensuring translocation of the viral genome into the cytoplasm. In addition, it is involved in virion assembly. The S protein epitope is the primary antigen that stimulates the formation of neutralizing antibodies and serves as a target for cytotoxic lymphocytes.
The rapid spread of SARS-CoV-2 worldwide is closely related to adaptive mutation of the virus. All 4 structural proteins encoded by the viral genome have a mutation record, including small envelope glycoprotein (E), membrane glycoprotein (M), nucleocapsid (N) protein, and spike (S) protein. The most prominent mutation is the spike protein, which mediates viral entry into cells by binding to the angiotensin converting enzyme 2 (ACE2) receptor. Mutations that occur in the spike protein Receptor Binding Domain (RBD) have the high potential to alter the kinetics and strength of the virus' interaction with target cells. RBD mutation sites exist in the currently popular variant strains Alpha, Beta, Gamma, Delta, Kappa, Iota and Lambda.
Since 11 months 2020, several variants with mutations in RBD have spread rapidly in the United kingdom (B.1.1.7; alpha variant), south Africa (B.1.351; beta variant) and Brazil (P.1; gamma variant). Early epidemiological and clinical studies have shown that these variations have a high transmission capacity in the population. Although phylogenetically different, a common feature of these several varieties is that residue 501 in the RBD is mutated from Asn to Tyr (N501Y). x-ray crystallography and cryoelectron microscopy (cryo-EM) structural studies have established that N501 is a key residue of the spike protein at the RBD and ACE2 interface, involved in critical contacts with several ACE2 residues. In combination with the recent report that the stronger infectivity of the N501Y mutant resulted from better binding to ACE 2. It has been demonstrated that the N501Y mutation significantly increases the ACE2-spike protein binding affinity (Kd) from 22 to 0.44 nM, which may explain in part why the N501Y mutant is more infectious. Therefore, studies of antibody neutralization, ACE2 binding and virus entry patterns of the N501Y mutant are important aspects of preventing the spread of SARS-CoV-2.
The aptamer is an oligonucleotide single strand screened by a systematic evolution technique (SELEX) for exponential enrichment of ligands, which is capable of forming a specific three-dimensional structure by intramolecular folding. The aptamer has various structures, so that the aptamer has the characteristics of high affinity, strong specificity, various target molecules and the like. Therefore, the aptamer shows wide application prospects in the aspects of in-vivo molecular imaging, target exploration, drug development and the like.
Antibodies neutralize the ability of viruses to infect human cells by targeting to the same epitope as the viral surface protein. The aptamer is also called as a chemical antibody, has the similar function as an antibody, and can neutralize viruses and play an antiviral role. Meanwhile, compared with the traditional antibody, the aptamer has the advantages of small molecular weight, easiness in modification, convenience in preparation, no immunogenicity, short preparation period, capability of being artificially synthesized and the like, and a series of processes such as animal immunization, feeding, protein extraction and purification and the like are omitted, so that the aptamer has greater application potential compared with the research and application of the antibody.
Therefore, the aptamer with high binding affinity is obtained by screening aiming at the novel coronavirus SARS-CoV-2 Spike RBD (N501Y) mutant strain, and a rapid diagnosis method is developed, which has important significance for the detection and neutralization treatment of the novel coronavirus rapid mutant strain.
Disclosure of Invention
The invention aims to provide a nucleic acid aptamer which has high specificity, small molecular weight, stable chemical property, easy preservation and can be specifically combined with SARS-CoV-2 Spike RBD (N501Y) protein and application thereof.
The 501 th residue of the spike protein Receptor Binding Domain (RBD) of SARS-CoV-2 was mutated from Asn to Tyr and named as the N501Y mutant. The mutant N501Y was reported to spread rapidly first in england, south africa and brazil, and the mutant N501Y was more infectious than the non-mutant. Aiming at the emerging N501Y mutant strain, the invention mainly obtains two aptamers for detecting SARS-CoV-2 Spike RBD (N501Y) Protein by the Protein-SELEX technology.
The sequences are respectively as follows:
NY 36: 5'-AACACGACACTAGTTCGGCTCGCGAACCCATCGCACGTCGTG-3' and NY27: 5'-AAGCCCACATGCCTGTGACCATCGAACAGCAGCAGCGTCGTG-3' and; the two aptamers for detecting SARS-CoV-2 Spike RBD (N501Y) protein are short chain nucleic acid sequences of 42 bases, and the binding condition is mainly detected by Surface Plasmon Resonance (SPR).
The SARS-CoV-2 Spike RBD (N501Y) protein is bonded on the surface of the biosensor, and then the selected single-stranded nucleic acid solution is injected into and flows through the surface of the biosensor. Binding between the protein and the aptamer causes an increase in the surface quality of the biosensor, resulting in an increase in the refractive index in the same proportion, and a change in the reaction between the protein and the aptamer is observed. And judging by combining the strength condition through a sensor and signal amplification.
In addition, a fluorescent group, a therapeutic substance, biotin or an enzyme labeling group can be connected to the nucleic acid sequence, so that the application of the aptamer in diagnosis and treatment of new coronavirus cells, living bodies and the like is promoted. The aptamer is prepared into a preparation for detecting the N501Y mutant strain.
The invention is based on Protein-SELEX technology, and SARS-CoV-2 Spike RBD (N501Y) Protein is used as screening sample to carry out two rounds of screening. Selecting nucleic acid sequences with abundance of more than 150 from the screening result sequences, satisfying the conditions in different evolutionary tree families, and selecting 50 candidate sequences from the nucleic acid sequences. The binding capacity of the candidate sequences is further detected by SPR experiments subsequently, and the invention is finally obtained.
The invention provides a detection or diagnosis kit, which comprises the aptamer.
The invention provides the application of the aptamer in preparing a detection kit of SARS-CoV-2 Spike RBD (N501Y) protein or a diagnostic reagent of SARS-CoV-2 Spike RBD (N501Y) protein.
The aptamer can be specifically combined with SARS-CoV-2 Spike RBD (N501Y) protein to play a role in virus neutralization, thereby playing an antiviral effect.
The invention has the beneficial effects that: the invention screens out the aptamer which can be combined with SARS-CoV-2 Spike RBD (N501Y) protein specificity through the external screening technique, named NY27 and NY36, and the combination affinity experiment can find that the affinity of the two aptamers to SARS-CoV-2 Spike RBD (N501Y) protein is more than one order of magnitude higher than that of the ordinary RBD protein, and the aptamer has high affinity and strong practicability, can be used for the rapid pathogen diagnosis of SARS-CoV-2 mutant strain, also can be used as small molecule drug for the treatment of SARS-CoV-2.
The aptamer obtained by screening the mutant N501Y protein for the first time plays a role similar to an antibody by utilizing the neutralizing activity of the obtained aptamer to the mutant N501 y. The novel crown virus specific protein target is combined to neutralize the toxicity of the novel crown virus, so that a neutralizing antibody of a novel crown mutant strain is developed, and the method has important significance for the detection and neutralization treatment of the rapid mutant strain of the novel crown virus for developing a rapid diagnosis method. The aptamer has the characteristics similar to or even more superior than a protein antibody, is mainly reflected in that the aptamer has specificity and high affinity, low toxicity and low immunogenicity, can be synthesized and modified in vitro, is convenient to store and transport and the like. Meanwhile, the aptamer of the invention is a short-chain nucleic acid sequence, and has the advantages of low in-vitro synthesis cost, short period and good reproducibility.
Drawings
FIG. 1 is a flow chart of X-aptamer library screening for SARS-CoV-2 Spike RBD (N501Y) protein;
FIG. 2 is a PCR amplification cycle number condition optimized gel-running chart;
FIG. 3 is a tree diagram of a family relationship of partially selected sequences;
FIG. 4 shows the affinity distribution (single concentration fit) of 50 nucleic acid aptamers to SARS-CoV-2 Spike RBD (N501Y) protein;
FIG. 5 shows the results of NY36 on multi-gradient KD of SARS-CoV-2 Spike RBD (N501Y) protein;
FIG. 6 shows the result of NY27 on multi-gradient KD of SARS-CoV-2 Spike RBD (N501Y) protein;
FIG. 7 shows the result of NY36 on multi-gradient KD of SARS-CoV-2 Spike RBD protein;
FIG. 8 shows the results of NY27 for SARS-CoV-2 Spike RBD protein multi-gradient KD.
Detailed Description
The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. All other embodiments, which can be made by those skilled in the art without any inventive work, based on the embodiments of the present invention, belong to the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were obtained from conventional biochemical reagent stores without specific indication.
Example 1: new coronavirus SARS-CoV-2 Spike RBD (N501Y) protein nucleic acid aptamer screening
Reagent: His-Tag magnetic beads (Invitrogen, Dynabeads. His-Tag Isolation & PullDown, 10103D), SARS-CoV-2 Spike RBD (N501Y) protein (purchased from Chinesia, 40592-V08B).
As shown in the schematic diagram of FIG. 1, based on Protein-SELEX technology, an X-Aptamer microsphere library with specific modification is selected for screening. Firstly, carrying out negative sieve incubation on the initial library and His-tag magnetic beads, and taking a microsphere library which is magnetically separated and reserved after incubation as a negative sieve sample. Collecting the aptamer microsphere library which is not combined with the magnetic beads, incubating with His-SARS-CoV-2 Spike RBD (N501Y) protein and His-tag magnetic beads, magnetically separating and collecting the aptamer microsphere library combined with the magnetic beads. Releasing the target aptamer enriched in the first round of screening from the microspheres through alkaline lysis and collecting the target aptamer through a balance filter column, wherein the obtained aptamer library is a first round of positive screening library. The first round of positive-screen bank was again incubated with His-SARS-CoV-2 Spike RBD (N501Y) protein and His-tag magnetic beads, and aptamers capable of binding to the protein were magnetically separated and collected as the second round of positive-screen bank. Aptamers that bind to magnetic beads and are partially "false positive" can be excluded by negative and two rounds of positive screening.
The sequences of the second round of positive and negative screen pools were subjected to pcr amplification under optimized conditions, and the number of partial rounds of cycling was optimized as shown in fig. 2. The optimal pcr conditions were chosen and extensive amplification and second-generation sequencing were performed. As shown in FIG. 3, the obtained sequencing results are obtained by sequencing the abundance values of 1618554 aptamer sequences, carrying out homology comparison on the sequences through MEGA7 software and constructing a evolutionary tree, and dividing the sequence comparison into different family trees. Finally, 50 candidate nucleic acid sequences are selected, and the sequences meet the selection conditions that the protein has high binding abundance value with the SARS-CoV-2 Spike RBD (N501Y) protein and is distributed in different family trees.
Example 2: screening aptamer with strong binding force with mutant protein of new coronavirus Spike RBD (N501Y) by SPR technology
1. Sample, experiment, model, reagent and consumable processing:
(1) model used in this experiment: biacore T200.
(2) S series CM5 chip: 10305570, purchased as GE Healthcare.
(3) The amino coupling kit (cat # BR-1000-50) was purchased from GE Healthcare.
(4) Sample dilution: the ligand protein was diluted with PBS (pH7.4). The analyte protein was diluted with PBSTM (pH 7.4).
2. The design and parameters of the SPR experimental method are shown in Table 1
TABLE 1 design and parameters of SPR experimental method for new coronavirus Spike RBD (N501Y)
Figure 618145DEST_PATH_IMAGE002
3. Experimental procedure
(1) The instrument was turned on according to Biacore T200 standard procedures.
(2) The Biacore T200 control software is opened to install the CM5 chip according to the standard flow.
(3) In preparation for the experiment, the buffer solution washes the flow path system inside the whole system at a higher flow rate.
(4) Appropriate procedures were set up according to the samples.
(5) To begin capture of ligand protein, the coupling buffer was PBSTM (pH7.4), sufficient volume of ligand protein Spike RBD (N501Y), 10 mM Sodium Acetate (pH4.5), EDC, NHS, blocking buffer was prepared and the coupling procedure was started, with a final ligand coupling of about 4900 RU.
(6) After the coupling is finished, the detection is started, the analyte binding time is set to be 180 s, and the flow rate is set to be 20 mu L/min; the dissociation time is 240 s, and the flow rate is 20 muL/min; the regeneration time was 30 s and the flow rate was 20. mu.L/min.
(7) 50 candidate sequences of 500nM and 200. mu.L were prepared and the assay was started.
(8) And analyzing results, and performing fitting analysis on data to obtain a final screening result.
(9) According to the screening result of the first 50 candidate sequences, two aptamers NY36 and NY27 are selected for multi-gradient KD detection.
(10) NY36 and NY27 (2000 nM, 1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.25 nM) were tested separately, setting the analyte binding time to 180 s and the flow rate to 20 μ L/min; the dissociation time is 240 s, and the flow rate is 20 muL/min; the regeneration time is 30 s, the flow rate is 20 mu L/min,
4. results of the experiment
The single concentration affinity detection results and specific numerical values of the 50 candidate nucleic acid sequences and SARS-CoV-2 Spike RBD (N501Y) protein at 500nM concentration are shown in FIG. 4 and Table 2.
TABLE 250 screening results of 500nM aptamer and SARS-CoV-2 Spike RBD (N501Y) protein
Serial number Sample name ka (1/Ms) kd (1/s) KD (M) Rmax(RU)
1 N501YSEQ36 2.34E+04 6.51E-04 2.78E-08 34.8
2 N501YSEQ27 3.41E+04 2.02E-03 5.93E-08 24.3
3 501 Seq5 6.29E+04 6.27E-02 9.97E-07 18.7
4 N501YSEQ73 5.21E+04 2.25E-03 4.32E-08 16.4
5 N501YSEQ43 4.91E+04 3.33E-03 6.78E-08 13.2
6 N501YSEQ30 2.17E+05 8.98E-03 4.13E-08 11.8
7 N501YSEQ25 3.40E+05 1.92E-02 5.64E-08 11.2
8 501 Seq52 4.91E+05 5.66E-02 1.15E-07 10.7
9 501 Seq7 1.27E+05 8.43E-03 6.62E-08 10.6
10 N501YSEQ29 8.14E+04 4.73E-03 5.82E-08 10.5
11 N501YSEQ70 6.23E+04 2.94E-03 4.72E-08 10.4
12 N501YSEQ34 5.21E+04 4.37E-03 8.40E-08 10.2
13 N501YSEQ28 1.66E+05 8.82E-03 5.30E-08 10.1
14 N501YSEQ21 3.23E+04 5.78E-03 1.79E-07 10.1
15 N501YSEQ48 7.90E+04 3.74E-03 4.74E-08 9.9
16 501 Seq26 5.87E+04 5.65E-03 9.62E-08 9.8
17 501 Seq17 9.53E+04 8.05E-03 8.45E-08 9.6
18 501 Seq16 6.96E+04 7.41E-03 1.06E-07 9.6
19 501 Seq41 2.90E+05 2.88E-02 9.94E-08 9.6
20 501 Seq14 7.75E+04 5.05E-03 6.52E-08 9.4
21 501 Seq72 2.77E+05 4.18E-02 1.51E-07 9.3
22 N501YSEQ19 1.71E+05 8.85E-03 5.18E-08 9.2
23 501 Seq9 3.19E+05 3.46E-02 1.09E-07 8.9
24 N501YSEQ59 5.18E+04 4.10E-03 7.91E-08 8.9
25 501 Seq4 8.83E+04 1.24E-02 1.40E-07 8.9
26 501 Seq8 8.89E+04 2.26E-02 2.54E-07 8.5
27 N501YSEQ38 4.55E+04 4.87E-03 1.07E-07 8.5
28 501 Seq20 6.71E+04 6.23E-03 9.28E-08 8.4
29 501 Seq6 2.46E+05 3.23E-02 1.31E-07 8.3
30 N501YSEQ61 5.21E+04 2.96E-03 5.69E-08 8.2
31 N501YSEQ23 5.41E+04 5.10E-03 9.43E-08 8.2
32 N501YSEQ56 1.00E+05 4.68E-03 4.67E-08 8
33 N501YSEQ62 5.59E+04 3.27E-03 5.85E-08 7.8
34 N501YSEQ31 2.35E+05 1.17E-02 4.97E-08 7.8
35 N501YSEQ18 8.10E+04 6.21E-03 7.67E-08 7.4
36 N501YSEQ32 7.97E+04 7.07E-03 8.87E-08 7.3
37 N501YSEQ33 5.85E+04 4.84E-03 8.26E-08 7.3
38 N501YSEQ65 4.21E+04 2.61E-03 6.22E-08 7.2
39 501 Seq37 5.01E+04 7.03E-03 1.40E-07 7.2
40 501 Seq12 5.58E+04 7.26E-03 1.30E-07 6.7
41 501 Seq15 5.99E+04 9.20E-03 1.53E-07 6.3
42 501 Seq13 6.12E+04 1.26E-02 2.06E-07 6.2
43 501 Seq22 6.34E+04 1.44E-02 2.27E-07 5.8
44 N501YSEQ53 5.74E+04 3.73E-03 6.49E-08 5.8
45 501 Seq24 6.58E+04 9.61E-03 1.46E-07 5.6
46 501 Seq11 6.61E+04 1.43E-02 2.17E-07 4.7
47 501 Seq10 1.32E+05 1.88E-03 1.42E-08 0
48 501 Seq1 1.47E+05 1.99E-03 1.35E-08 0
49 501 Seq2 1.56E+05 2.33E-03 1.49E-08 0
50 501 Seq3 1.43E+05 4.19E-03 2.92E-08 0
Two aptamers NY36 (corresponding to N501YSEQ36 in the table) and NY27 (corresponding to N501YSEQ27 in the table) with the highest comprehensive ranking are selected from the table for KD value determination. NY36 multi-gradient KD results are shown in figure 5, and NY27 multi-gradient KD results are shown in figure 6. The affinity of SARS-CoV-2 Spike RBD (N501Y) to NY36 was 2.00E-07M, and the affinity of SARS-CoV-2 Spike RBD (N501Y) to NY27 was 1.22E-07M. Comparative example 3 it can be found that the affinity of these two aptamers for SARS-CoV-2 Spike RBD (N501Y) protein is more than an order of magnitude higher than that of the ordinary RBD protein.
Example 3: the combination condition of NY27 and NY36 on the new coronavirus Spike RBD protein is detected by SPR technology
1. Sample, experiment, model, reagent and consumable treatments were as in example 2
2. SPR experimental method design and parameters are shown in Table 3
TABLE 3 design and parameters of SPR experimental method for new coronavirus Spike RBD protein
Figure 66444DEST_PATH_IMAGE004
3. Experimental procedure
(1) The machine is started according to the Biacore T200 instrument standard operation.
(2) Opening Biacore T200 control software and installing CM5 chip according to standard flow
(3) In preparation for the experiment, the buffer solution washes the flow path system inside the whole system at a higher flow rate.
(4) Appropriate procedures were set up according to the samples.
(5) To begin capture of ligand protein, the coupling buffer was PBSTM (pH7.4), sufficient volume of ligand protein Spike RBD (N501Y), 10 mM Sodium Acetate (pH4.5), EDC, NHS, blocking buffer was prepared and the coupling procedure was started, with a final ligand coupling of about 4900 RU.
(6) After the coupling is finished, the detection is started, NY27 and NY36 (2000 nM 1000nM 500nM 250nM 125nM 62.5nM 31.25 nM) are respectively detected, the binding time of the analyte is set to be 180 s, and the flow rate is set to be 20 muL/min; the dissociation time is 240 s, and the flow rate is 20 muL/min; the regeneration time is 30 s, the flow rate is 20 mu L/min,
4. results of the experiment
The multi-gradient KD result of NY36 for RBD protein is shown in FIG. 7, and the multi-gradient KD result of NY27 for RBD protein is shown in FIG. 8. The affinity of SARS-CoV-2 Spike RBD to NY36 was 1.13E-06M, and the affinity of SARS-CoV-2 Spike RBD protein to NY27 was 1.18E-06M. It can be seen that the nucleic acid aptamers NY36 and NY27 selected aiming at the N501y mutant strain have recognition capability for common RBD protein, but have recognition capability for common RBD protein which is more than one order of magnitude weaker than that of the aptamers for the mutant strain with strong recognition at the E-07 degree.
<110> university of Hunan
<120> nucleic acid aptamer for detecting novel coronavirus SARS-CoV-2 and application thereof
<160> 2
<210> 1
<211> 42
<212> DNA
<400> 1
AACACGACACTAGTTCGGCTCGCGAACCCATCGCACGTCGTG 42
<210> 2
<211> 42
<212> DNA
<400> 2
AAGCCCACATGCCTGTGACCATCGAACAGCAGCAGCGTCGTG 42

Claims (8)

1. An aptamer specifically binding to spike protein of SARS-CoV-2 virus, wherein the nucleotide sequence of the aptamer is:
5'-AACACGACACTAGTTCGGCTCGCGAACCCATCGCACGTCGTG-3' or
5′-AAGCCCACATGCCTGTGACCATCGAACAGCAGCAGCGTCGTG-3′。
2. The aptamer according to claim 1, wherein the label modification is attachment of a fluorescent, radioactive, therapeutic, biotin, or enzyme label to the aptamer.
3. The nucleic acid aptamer of claim 1 or 2, wherein the SARS-CoV-2 virus is the N501Y mutant strain.
4. A kit for detecting or diagnosing SARS-CoV-2 virus, comprising the nucleic acid aptamer according to any one of claims 1 to 3.
5. Use of the nucleic acid aptamer of any one of claims 1 to 3 for the preparation of a SARS-CoV-2 virus detection kit or a SARS-CoV-2 diagnostic reagent.
6. The use of claim 5, wherein the SARS-CoV-2 virus is the N501Y mutant strain.
7. Use of the aptamer of any one of claims 1 to 3 for the preparation of a product for the treatment and/or prevention of SARS-CoV-2 virus.
8. The use of claim 7, wherein the SARS-CoV-2 virus is the N501Y mutant strain.
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