A pac1 aptamer and use thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a PAC1 nucleic acid aptamer and application thereof. The application uses protein SELEX technology to screen four nucleic acid aptamers with small molecular weight, stability and easy modification, and capable of recognizing PAC1 with high specificity, and the specific nucleotide sequences are as shown in SEQ ID NO. 1-4. The four nucleic acid aptamers are verified by examples to have high affinity with PAC1, do not specifically bind to other proteins, have no immunogenicity, are easy to synthesize and store, and an oligonucleotide sequence with homology of more than 60% to the nucleic acid aptamer or a nucleic acid aptamer treated by truncation or modification can still efficiently bind to PAC1. The PAC1 nucleic acid aptamer provided by the application can be used as a small molecule targeted probe of PAC1, and provides a new idea and new tool for tumor targeted diagnosis and treatment and antiviral treatment, fills the gap of the prior art, and has important clinical value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a PAC1 nucleic acid aptamer and its applications. Background Technology

[0002] Activated cellular phosphatase 1 (PAC1), also known as dual-specific phosphatase 2 (DUSP2), is a mitogen-induced threonine / tyrosine phosphatase. PAC1 is the only member of the DUSP family specifically expressed in T-cell-rich tissues and can serve as a novel immune checkpoint to negatively regulate T-cell anti-tumor responses. PAC1 depletion can enhance effector T-cell activity and prevent tumor growth in mice. In the tumor microenvironment, highly expressed PAC1 effector cells transition to a depleted state and are positively correlated with poor patient prognosis. As an epigenetic checkpoint for depleted T cells, PAC1 is a potential candidate for drug development in cancer immunotherapy. Since there are no small-molecule inhibitors or targeted drugs for PAC1, the development of small-molecule probes targeting PAC1 is of great significance for enhancing T-cell function to promote anti-tumor immunity and antiviral therapy.

[0003] Aptamers, also known as chemical antibodies, are single-stranded oligonucleotides that fold into unique three-dimensional conformations to bind to their corresponding targets. Aptamers possess advantages such as non-immunogenicity, small molecular weight, strong structural flexibility, high chemical stability, and ease of synthesis and modification. Currently, aptamers are widely used in sensing, purification, diagnostics, drug delivery, and targeted therapy. These oligonucleotides can serve as ligand blockers or agonists, or as carriers for delivering imaging agents and precision medicine drugs. However, nucleic acid aptamer sequences targeting PAC1 have not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a PAC1 nucleic acid aptamer that recognizes PAC1 with high affinity and high specificity.

[0005] This invention provides a PAC1 nucleic acid aptamer, wherein the PAC1 nucleic acid aptamer is selected from any one or more of the following:

[0006] (a) Nucleic acid aptamer PA5, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0007] (b) Nucleic acid aptamer PA2, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0008] (c) Nucleic acid aptamer PA4, the nucleotide sequence of which is shown in SEQ ID NO.3;

[0009] (d) Nucleic acid aptamer PA12, the nucleotide sequence of which is shown in SEQ ID NO.4;

[0010] (e) Oligonucleotide sequences that share more than 60% homology with any of the nucleic acid aptamers in (a) to (d);

[0011] The truncated sequence of the nucleic acid aptamer PA5 described in (f)(a);

[0012] (g) The sequence obtained by modifying any one of the nucleic acid aptamers in (a) to (d).

[0013] Preferably, the modification includes fluorescent modification or biotin modification.

[0014] The present invention also provides the application of the PAC1 nucleic acid aptamer described in the above technical solution in the preparation of products for detecting PAC1 protein.

[0015] The present invention also provides the application of the PAC1 nucleic acid aptamer described in the above technical solution in products for diagnosing and / or treating tumors.

[0016] This invention also provides the application of the PAC1 nucleic acid aptamer described in the above technical solution in the preparation of antiviral therapeutic products.

[0017] Beneficial effects:

[0018] This invention utilizes protein SELEX technology to screen and obtain four small, stable, easily modified nucleic acid aptamers capable of highly specific recognition of PAC1. The specific nucleotide sequences are shown in SEQ ID NO. 1–4. Examples have verified that these four nucleic acid aptamers have high affinity for PAC1, do not specifically bind to other proteins, are non-immunogenic, easy to synthesize and store, and possess oligonucleotide sequences with more than 60% homology to the aptamers, or truncated or modified aptamers that still efficiently bind to PAC1. The PAC1 nucleic acid aptamers provided by this invention can serve as small-molecule targeting probes for PAC1, offering new ideas and tools for tumor targeted diagnosis and treatment, as well as antiviral therapy, filling a gap in existing technologies and possessing significant clinical value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1To monitor the enrichment process of the enriched library binding to PAC1 protein or blank nickel beads using flow cytometry; where A represents the change in fluorescence intensity on the surface of PAC1-beads with increasing screening rounds; and B represents the change in fluorescence intensity on the surface of Ni-beads with increasing screening rounds.

[0021] Figure 2 To create a sequence homology comparison plot in the library after 8 rounds of screening;

[0022] Figure 3 Flow cytometry results of the binding of candidate aptamers PA1-12 to PAC1-beads;

[0023] Figure 4 Fluorescence imaging results of candidate aptamers PA2, PA4, PA5 and PA12 binding to PAC1-beads;

[0024] Figure 5 The graph shows the results of the equilibrium dissociation constant (Kd value) determination for PA2, PA4, PA5 and PA12;

[0025] Figure 6 Flow cytometry results showing the binding of the truncated PA5 sequence to the target protein;

[0026] Figure 7 Confocal fluorescence imaging results of the binding of the truncated PA5 sequence to PAC1-positive cells;

[0027] Figure 8 The results of PA5 detecting PAC1 in a complex system. Detailed Implementation

[0028] This invention provides a PAC1 nucleic acid aptamer, wherein the PAC1 nucleic acid aptamer is selected from any one or more of the following:

[0029] (a) Nucleic acid aptamer PA5, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0030] (b) Nucleic acid aptamer PA2, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0031] (c) Nucleic acid aptamer PA4, the nucleotide sequence of which is shown in SEQ ID NO.3;

[0032] (d) Nucleic acid aptamer PA12, the nucleotide sequence of which is shown in SEQ ID NO.4;

[0033] (e) Oligonucleotide sequences that share more than 60% homology with any of the nucleic acid aptamers in (a) to (d);

[0034] The truncated sequence of the nucleic acid aptamer PA5 described in (f)(a);

[0035] (g) The sequence obtained by modifying any one of the nucleic acid aptamers in (a) to (d).

[0036] In this invention, the PAC1 nucleic acid aptamer is preferably selected from any one of nucleic acid aptamers PA2, PA4, PA5, and PA12, or an oligonucleotide sequence with more than 60% homology to any one of the nucleic acid aptamers PA2, PA4, PA5, or PA12, or a truncated sequence of the nucleic acid aptamer PA5, or a sequence obtained by modifying any one of the nucleic acid aptamers PA2, PA4, PA5, or PA12.

[0037] The nucleotide sequences of the nucleic acid aptamers PA5, PA2, PA4, and PA12 described in this invention are as follows:

[0038] Nucleic acid aptamer PA5 (SEQ ID NO.1): 5'-ATCCAGAGTGACGCAGCACCTA CATTCACCGTCTCACTTCTCCCCTCTCGTTCCCCTCTGGACACGGTGGCTTA GT-3';

[0039] Nucleic acid aptamer PA2 (SEQ ID NO.2): 5'-ATCCAGAGTGACGCAGCACCCC ACCCGCACGTCATTTCCACCCTTCTCTACTTCTCTCTGGACACGGTGGCTTA GT-3';

[0040] Nucleic acid aptamer PA4 (SEQ ID NO.3): 5'-ATCCAGAGTGACGCAGCACCTT ACATCCCGCATACAGCCCGCTCACACCCTCCCTTAGTGGACACGGTGGCTT AGT-3';

[0041] Nucleic acid aptamer PA12 (SEQ ID NO.4): 5'-ATCCAGAGTGACGCAGCAGTG CTCGTGCCCCCGTACCCTTGCTCTAGACCTCTCCCTCTGGACACGGTGGCT TAGT-3'.

[0042] In this invention, the oligonucleotide sequences that have a homology of more than 60% with any one of the nucleic acid aptamers PA2, PA4, PA5 and PA12 are preferably as shown in SEQ ID NO.13 to SEQ ID NO.19, and the specific sequence information is shown in Table 2.

[0043] In this invention, the truncated sequence of the nucleic acid aptamer PA5 preferably includes any one of PA5a, PA5b, PA5c, or PA5d; the nucleotide sequences of PA5a, PA5b, PA5c, and PA5d are specifically as follows:

[0044] PA5a (SEQ ID NO.5): 5'-ATCCAGAGTGACGCAGCACCTACATTCACCGTCTCACTTCTCCCCTCTCGTTCCCCTC-3'

[0045] PA5b (SEQ ID NO.6): 5'-CCTACATTCACCGTCTCACTTCTCCCCTCTCGTTCCCCTCTGGACACGGTGGCTTAGT-3'

[0046] PA5c (SEQ ID NO.7): 5'-CCTACATTCACCGTCTCACTTCTCCCCTCTC GTTCCCCTC-3'

[0047] PA5d (SEQ ID NO. 8): 5'-TTCACCGTCTCACTTCTCCCCTCTCGTT-3'.

[0048] In this invention, the modification methods include fluorescent modification or biotin modification, and the fluorescent modification preferably includes FAM modification.

[0049] This invention utilizes protein SELEX technology and, through eight rounds of screening, obtained four small, stable, and easily modifiable nucleotide sequences. Any one of these nucleotide sequences, or an oligonucleotide sequence with more than 60% homology to any other nucleotide sequence, or a truncated sequence of any nucleotide sequence, or a sequence obtained by modifying any nucleic acid aptamer, can highly specifically recognize PAC1. Furthermore, these sequences exhibit high affinity, do not specifically bind to other proteins, are non-immunogenic, and are easy to synthesize and store, making them suitable as small-molecule targeting probes for PAC1.

[0050] The functions of the PAC1 nucleic acid aptamer, molecular probe, and kit provided by this invention, and any application of the aforementioned PAC1 nucleic acid aptamer, molecular probe, or kit in the preparation of products for detecting PAC1 protein, products for diagnosing and / or treating tumors, and products for the preparation of antiviral therapies, are all within the scope of protection of this invention. The PAC1 nucleic acid aptamer provided by this invention offers new ideas and tools for targeted tumor diagnosis and treatment, as well as antiviral therapy, and has significant clinical value.

[0051] To further illustrate the present invention, a PAC1 nucleic acid aptamer and its applications provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] PAC1 protein aptamer screening and cloning

[0054] 1. Document Preprocessing

[0055] The initial screening library was a single-stranded oligonucleotide sequence library with a total length of 76 nt, with constant primer sequences of 18 nt at each end to facilitate sequence amplification, and a 40 nt random nucleic acid library sequence in the middle (5'-ATCCAGAGTGACGCAGCA-N40-TGGACACGGTGGCTTAGT-3', SEQ ID NO.9). The random nucleic acid library powder was dissolved and heated to 95-100℃ for denaturation for 5-10 min, and then immediately placed on ice to cool for 5-10 min to form a relatively stable secondary structure (denoted as the pretreated random nucleic acid library). The library needs to be pretreated before each round of screening and then put into the screening process.

[0056] 2. Preparation of screening targets

[0057] The nickel bead suspension (GE Healthcare) was added to 500 μL of protein buffer (PBS containing 30 mmol / L imidazole) and equilibrated three times to obtain a protein buffer-nickel bead suspension mixture. PAC1 protein (UniProt database) was added to 100 μL of the protein buffer-nickel bead suspension mixture at a mass ratio of nickel beads to PAC1 protein (UniProt database) of 1:12. After thorough mixing, the mixture was incubated at 4°C on a shaker for 1 hour. After incubation, the protein was washed three times with protein buffer to obtain the target protein immobilized on the nickel beads, denoted as PAC1-beads. For screening, PAC1-beads were used as the positive selection target, and bare nickel beads (Ni-beads) were used as the negative selection target.

[0058] 3. Reverse screening and forward screening

[0059] PAC1 aptamers were screened using protein SELEX technology. The pre-processed random nucleic acid library was subjected to eight rounds of screening. The first three rounds involved only positive screening, while the latter five rounds involved a combination of reverse and positive screening (one round consisted of one reverse and one positive screening sequentially). The specific steps of the entire aptamer screening process are as follows:

[0060] 3.1 First 3 rounds of positive screening:

[0061] 3.1.1 Add PAC1-beads to the pretreated random nucleic acid library obtained in step 1, incubate with shaking at 4°C, wash 3 times with washing buffer, and discard the supernatant; collect PAC1-beads into EP tubes, denature at 95-100°C for 5-10 min, centrifuge at high speed, and retain the supernatant.

[0062] The supernatant was used as a template for the first PCR amplification, wherein,

[0063] The first PCR amplification system (total volume 1500 μL) consisted of: 150 μL 10×PCR Buffer, 120 μL dNTP mixture, 37.5 μL each of forward and reverse primers, 1000 μL template, 5 μL rTaq, and 150 μL ddH2O. Amplification was performed on a PCR instrument under the following conditions: pre-denaturation at 95–100℃ for 5–10 min; denaturation at 95–100℃ for 30–40 s, annealing at 45–55℃ for 30–40 s, extension at 72℃ for 30–40 s, for a total of 10 cycles; final extension at 72℃ for 5–15 min; and final incubation at 4℃. Forward primer: 5'-ATCCAGAGTGACGCAGCA-3' (SEQ ID NO. 10); Reverse primer: 5'-TGGACACGGTGGCTTAGT-3' (SEQ ID NO. 11).

[0064] Using the product of the first PCR amplification as a template, a second PCR amplification was performed, in which...

[0065] The second PCR amplification system (total volume 2000 μL) consisted of: 200 μL 10×PCR Buffer, 160 μL dNTP mixture, 50 μL each of forward and reverse primers, 200 μL template, 10 μL rTaq, and 1520 μL ddH2O (the forward primer used in this case must be fluorescently labeled, and the reverse primer must be biotinylated). Amplification was performed on the PCR instrument under the following conditions: 95–100℃ pre-denaturation for 5–10 min; 95–100℃ denaturation for 30–40 s, 45–55℃ annealing for 30–40 s, and 72℃ extension for 30–40 s, for a total of 10 cycles (this number of cycles was determined after optimization); 72℃ extension for 5–15 min; and finally, permanent incubation at 4℃. Upstream primer: 5'-ATCCAGAGTGACGCAGCA-3' (SEQ ID NO.10); Downstream primer: 5'-TGGACACGGTGGCTTAGT-3' (SEQ ID NO.11).

[0066] The 10×PCR Buffer and dNTPs used in the above PCR experiments were purchased from TOYOBO; Taq DNA polymerase was purchased from TAKARA. PCR primers were purchased from Beijing Qingke Xinyue Biotechnology Co., Ltd. and Beijing Ruiboxingke Biotechnology Co., Ltd.

[0067] Fluorescently labeled single-stranded DNA sequences were isolated from the second PCR product using streptavidin microspheres. The streptavidin microspheres could remove the biotin-labeled complementary strand, thereby obtaining the FITC-labeled target strand, i.e., a secondary random nucleotide library.

[0068] 3.1.2 Repeated screening: The secondary nucleotide library obtained in step 3.1.1 is repeated twice, that is, three rounds of positive screening are completed, and a third round of secondary nucleotide library is obtained.

[0069] 3.2 The last 5 rounds of reverse screening + forward screening:

[0070] 3.2.1 Starting from the fourth round of screening, a reverse screening operation is performed before each round of forward screening: After equilibrating the nickel beads with washing buffer, the pretreated third-round secondary nucleotide library is added, and after co-incubation at 4°C, the supernatant is collected and labeled as the reverse screening product of that round.

[0071] 3.2.2 Perform forward screening on the reverse screening products of this round according to step 3.1.1 to obtain the secondary random nucleotide library of this round.

[0072] 3.2.3 Repeated Screening: The secondary random nucleotide library obtained in step 3.2.2 is subjected to the same operations as in 3.2.1 and 3.2.2 four times each, i.e., four rounds of reverse screening + forward screening. In these four rounds of reverse screening, the nucleotide library added in the next round of reverse screening is the secondary nucleotide library prepared in the previous round of forward screening; in these four rounds of forward screening, the nucleotide library added in the next round of forward screening is the secondary nucleotide library prepared in the previous round of reverse screening. After repeated screening, the 8th round of enriched random nucleotide library is finally obtained.

[0073] In this embodiment, to improve the affinity and specificity of nucleic acid aptamers, the screening pressure was gradually increased during the screening process, and the enrichment was monitored using flow cytometry. The results are as follows: Figure 1 The specific control conditions for the screening process are shown in Table 1.

[0074] Table 1. Pressure regulation conditions for PAC1 aptamer screening

[0075]

[0076] according to Figure 1 It can be seen that the fluorescence intensity on the surface of PAC1-beads gradually increases with the increase of the number of screening rounds. Figure 1 (A); The fluorescence intensity on the Ni-beads surface does not change significantly with the increase of the number of screening rounds. Figure 1 (B)

[0077] 4. Library cloning and sequencing and acquisition of candidate aptamers

[0078] After eight rounds of screening, using the screened products as templates, PCR amplification was performed using the upstream primer: 5'-ATCCAGAGTGACGCAGCA-3' (SEQ ID NO.10) and the downstream primer: 5'-TGGACACGGTGGCTTAGT-3' (SEQ ID NO.11). The PCR products were then cloned using the TA clone technique, and 50 clones were randomly selected for sequencing, yielding 48 valid sequencing results. Analysis of these 48 valid sequencing results, based on homology comparison of their primary and secondary structures, classified them into six sequence families. The results are as follows: Figure 2 As shown in Table 2, two candidate sequences with homology of more than 60% were selected from each of the six families for further verification and named PA1 to PA12 respectively.

[0079] Table 2. Pressure regulation conditions for PAC1 aptamer screening

[0080]

[0081] Example 2

[0082] 1. Binding ability of nucleic acid aptamers to targets

[0083] (1) The 12 nucleic acid aptamers obtained in Example 1 were labeled with FAM fluorescence and then respectively with 2×10⁻⁶ 5 Ni-beads and PAC1-beads were incubated on ice in the dark for 30 min (final concentration 250 nmol / L). After washing three times thoroughly with 500 μL of washing buffer (composed of Tris-HCl 10 mmol / L, NaCl 150 mmol / L, KCl 5 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, and the remainder water), the nickel beads were resuspended in 500 μL of binding buffer (obtained by dissolving 50 μL of Tween-20 in 100 mL of washing buffer). Untreated nickel beads served as a negative control. The binding ability of the 12 nucleic acid aptamers obtained in Example 1 was investigated using flow cytometry, and the results are as follows: Figure 3 .

[0084] according to Figure 3 As can be seen, all 12 candidate aptamers could bind to the PAC1 protein compared to the negative control. Among them, aptamers PA3 and PA11 showed weaker binding affinity, while PA1, PA2, PA4, PA5, PA6, PA7, PA8, PA9, PA10, and PA12 showed stronger binding affinity. Comparing the binding of each candidate aptamer to bare nickel beads, it was observed that candidate aptamers PA1, PA6, PA7, PA8, PA9, PA10, and PA11 also exhibited some non-specific binding with bare nickel beads. Notably, the fluorescence curves of PA6 after incubation with Ni-beads and P-beads almost overlapped, indicating poor specificity for this candidate aptamer. Based on the above analysis, we selected PA2, PA4, PA5, and PA12 as representative candidate aptamers for further screening and optimization.

[0085] (2) The binding of nucleic acid aptamers PA2, PA4, PA5, PA12 to PAC1 was verified using fluorescence microscopy. The sample processing was the same as in step (1) above. The initial screening library (5'-ATCCAGAGTGACGCAGCA-N40-TGGACACGGTGGCTTAGT-3', SEQ ID NO.9) was used as a control. The fluorescence imaging results are shown in [Figure 1]. Figure 4 .

[0086] according to Figure 4It can be seen that no fluorescence signal was observed after the four nucleic acid aptamers were incubated with Ni-beads, but fluorescence was observed on the surface of the nickel beads after incubation with PAC1-beads. No fluorescence signal was also observed after the initial library labeled with FAM was incubated with Ni-beads and PAC1-beads respectively, indicating that each candidate aptamer showed strong binding ability.

[0087] (3) Following the procedure in step (1) of Example 2, the affinity of nucleic acid aptamers PA2, PA4, PA5, and PA12 for PAC1 was detected. Flow cytometry was used to detect the four candidate aptamer samples prepared in parallel at five concentration gradients (10 nM, 20 nM, 500 nM, 100 nM, 200 nM), and the median fluorescence intensity of the FAM-labeled aptamer binding to the target protein under each concentration condition was recorded. After subtracting the intrinsic fluorescence background of the P-beads samples that were not incubated with any aptamer, the recorded data were imported into Sigmaplot software for analysis, and then analyzed according to the single-point adsorption model formula Y = B. max The equilibrium dissociation constant, i.e., the Kd value, of each candidate aptamer was calculated by fitting the X / (Kd+X) curve. The results are as follows: Figure 5 As shown.

[0088] according to Figure 5 It can be seen that all four nucleic acid aptamers can bind to PAC1. The Kd values ​​of PA2, PA4, PA5 and PA12 are 86.10±24.74nM, 118.27±14.44nM, 61.33±17.08nM and 92.92±40.59nM respectively. PA5 has the smallest Kd value. Combined with the detection results of step (1), PA5 has the strongest affinity for PAC1 protein. Compared with the other three nucleic acid aptamers, PA5 has a stronger ability to specifically bind. Therefore, in the following experiments, aptamer PA5 will be selected for in-depth study.

[0089] 2. Adaptor truncation optimization

[0090] (1) In various functional studies, the longer the DNA, the worse its stability. Furthermore, some base sequences in aptamers do not have the function of binding to target proteins; these redundant base sequences may create steric hindrance, which is detrimental to the binding of aptamers to target proteins. Therefore, this study further optimized the PA5 sequence, mainly by shortening the primer ends, that is, removing all or part of the primer-binding regions at both ends of the original sequence. The optimized and shortened aptamer sequences are shown in Table 3.

[0091] Table 3 PA5 Optimized Truncated Sequence Information

[0092]

[0093] (2) Examination of the binding ability of PA5 optimized sequence

[0094] a. Using PA5 as a positive control and bare nickel beads as a blank control, the binding ability of the positive control, blank control, and optimized PA5 sequences was examined by flow cytometry. The results are shown in [Figure number missing]. Figure 6 .

[0095] according to Figure 6 It can be seen that, compared with the original PA5 sequence, the binding ability of PA5a, PA5b, and PA5d to the PAC1 target protein is weakened. However, the binding ability of PA5c to the PAC1 target protein is basically the same as that of the original PA5 sequence. This indicates that the optimized truncated PA5 sequences provided by this invention can all bind to the PAC1 target protein, with the optimized PA5c sequence showing the best binding effect. This demonstrates that, to a certain extent, redundant sequences that do not play a specific binding role have been removed, while retaining the key region for target binding.

[0096] b. The optimized aptamer PA5c with FAM fluorescent labeling at the 5' end was incubated with ordinary Jurkat cells and Jurkat cells stably expressing mcherry-PAC1 (construction method referred to Nat Immunol. 2020 Mar; 21(3):287-297.). The fluorescence was observed using a fluorescence confocal imager. The results are as follows: Figure 7 As shown.

[0097] according to Figure 7 It can be seen that the fluorescently labeled nucleic acid aptamer PA5c has obvious co-localization with the mcherry-PAC1 fusion protein and can specifically recognize Jurkat cells that stably express mcherry-PAC1, indicating that the nucleic acid aptamer PA5c provided by the present invention can specifically bind to PAC1 in cells.

[0098] Example 3

[0099] Detecting PAC1 in complex systems

[0100] 1. Experimental grouping and processing:

[0101] Experimental group: The cell lysate of Jurkat cells stably expressing PAC1-Flag (J-PAC1-Flag) was incubated with 5' biotin-labeled PA5c at 4°C for 4 hours to obtain a mixture; the mixture was then incubated with streptavidin beads (SA-beads) at 4°C for 3 hours.

[0102] Control group 1: Cell lysate of Jurkat cells (J-PAC1-Flag) stably expressing PAC1-Flag was incubated with a 5' biotin-labeled initial screening library (5'-ATCCAGAGTGACGCAGCA-N40-TGGACACGGTGGCTTAGT-3', SEQ ID NO.9) at 4°C for 4 hours to obtain a mixture; the mixture was then incubated with streptavidin beads (SA-beads) at 4°C for 3 hours.

[0103] Control group 2: The cell lysate of Jurkat cells (J-Mock-Flag) stably expressing the control vector was incubated with 5' biotin-labeled PA5c at 4°C for 4 hours to obtain a mixture; the mixture was then incubated with streptavidin beads (SA-beads) at 4°C for 3 hours.

[0104] Control group: The cell lysate of Jurkat cells (J-PAC1-Flag) that stably expressed PAC1-Flag was incubated at 4°C for 4 hours and then incubated with streptavidin beads (SA-beads) at 4°C for 3 hours.

[0105] 2. After incubation in each treatment group in step 1, wash three times, collect the beads, and perform heat denaturation on the incubated samples. Finally, perform Western blot analysis, using anti-Flag tag antibody to detect proteins. The results are as follows: Figure 8 As shown, where Figure 8 The input detection results indicate the presence of PAC1 protein in the reaction system.

[0106] according to Figure 8 As can be seen, the target band of PAC1 was detected in samples with J-PAC1-Flag cell lysates containing aptamer PA5c and SA-beads, while the target band was not detected in samples containing pure streptavidin beads (SA-beads) or samples containing a selection library sequence and streptavidin beads (SA-beads). Similarly, samples with J-Mock-Flag cell lysates containing aptamer PA5c and SA-beads also did not show the target band. This indicates that aptamer PA5c can specifically bind to the PAC1 protein in complex systems and can be used for the detection of the PAC1 protein.

[0107] As can be seen from the above, the PAC1 nucleic acid aptamer provided by this invention has a high affinity for PAC1, is non-immunogenic, easy to synthesize and store, and can be used as a small molecule targeting probe for PAC1 to detect PAC1 protein.

[0108] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A PAC1 nucleic acid aptamer, wherein the PAC1 nucleic acid aptamer is selected from any one or more of the following: (a) Nucleic acid aptamer PA5, nucleotide sequence as shown in SEQ ID NO.1; (b) The truncated sequence of the nucleic acid aptamer PA5 described in (a) has the nucleotide sequence shown in SEQ ID NO.7; (c) A sequence obtained by modifying (a) or (b) a nucleic acid aptamer; wherein the modification is fluorescent modification or biotin modification.

2. The use of the PAC1 nucleic acid aptamer according to claim 1 in the preparation of products for detecting PAC1 protein.

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