Nucleic acid aptamer targeting camkii alpha and screening method and application thereof

Abstract

This invention discloses a nucleic acid aptamer targeting CaMKIIα, its screening method, and its applications. The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The screening method employs SELEX technology based on magnetic beads, using a seven-round gradient pressure screening process to obtain aptamers with high affinity and high specificity. The pressure strategy includes a gradient decrease in the concentration of the added library, a gradient shortening of the incubation time, upgrading the screening medium from ethanolamine to a control peptide, and a gradient incorporation of inactivated serum to simulate a physiological environment. Flow cytometry verification shows that the aptamer can specifically bind to CaMKIIα protein at 4°C, room temperature, and 37°C. The aptamer of this invention exhibits high specificity, high affinity, and good temperature stability, and can be used for CaMKIIα detection, related disease diagnosis, and targeted therapy.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a nucleic acid aptamer targeting calmodulin-dependent kinase II α (CaMKIIα), its screening method, and its applications. The nucleic acid aptamer is suitable for the accurate detection of CaMKIIα, as well as for the preparation of diagnostic kits or targeted therapeutic drugs for diseases related to abnormal CaMKIIα expression. Background Technology

[0002] Nucleic acid aptamers are single-stranded DNA or RNA molecules obtained through systematic evolution of ligands using exponential enrichment (SELEX) technology. Due to their high specificity and affinity binding to target molecules, ease of chemical synthesis and modification, strong in vitro stability, non-immunogenicity, low production cost, and good tissue penetration, they have broad application prospects in fields such as biological detection, disease diagnosis, and targeted drug delivery. They are widely considered an ideal alternative to traditional antibody molecules and have been successfully applied to the specific recognition and detection of various protein, small molecule, and cellular targets. Currently, drug development targeting CaMKⅡα mainly focuses on cardiovascular diseases, with activity inhibition as the primary direction. Representative inhibitors include small molecule inhibitors (such as KN-93, KN-62, AS105, and NP202), peptide inhibitors (such as CaMKIItide, CN21, and CN19), and antisense oligonucleotides (ASOs). Small molecule inhibitors generally suffer from insufficient tissue selectivity, significant off-target effects, and short in vivo half-life; peptides and ASOs, although highly specific, have not yet achieved clinical translation; antibodies, however, are limited in their application in organisms due to their long preparation cycle, high production cost, susceptibility to temperature and pH, and difficulty in penetrating cell membranes and the blood-brain barrier.

[0003] To date, no research on nucleic acid aptamers targeting CaMKⅡα has been reported. Therefore, screening for and obtaining a high-affinity nucleic acid aptamer targeting CaMKⅡα is of great significance for advancing the precision diagnosis and treatment of related diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a nucleic acid aptamer targeting CaMKⅡα, its screening method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A nucleic acid aptamer targeting CaMKⅡα, wherein the nucleotide sequence of the nucleic acid aptamer is selected from any of the following:

[0007] (1) 5'-GCACGGACACAAGAACAAAGAGGGGGACTGCTCGGGATTGCGGATACTTGGTGTTCCTTGTGCTGCCTTTGTTCTG-3';

[0008] (2) 5'-AAGAACAAAGAGGGGGACTGCTCGGGATTGCGGATACTTGGTGTTCCTTGTGCTGC-3';

[0009] (3) A derivative sequence that has at least 80% homology with the nucleotide sequence shown in (1) or (2) and has a specific binding function of CaMKⅡα.

[0010] A method for screening nucleic acid aptamers targeting CaMKⅡα includes the following steps:

[0011] (1) Provide a random nucleic acid library, wherein the nucleotide sequence of the random nucleic acid library is 5'-GCACGGACACAAGAACAAAG-N (36) -CTTGTGCTGCCTTTGTTCTG-3', where N represents any one of the bases A, T, C, and G;

[0012] (2) Prepare functionalized magnetic beads, including positive screening magnetic beads coupled with CaMKⅡα protein, reverse screening magnetic beads for removing non-specific adsorption background, and reverse screening control magnetic beads for excluding cross-reactive sequences of homologous domains.

[0013] (3) After the random nucleic acid library is subjected to renaturation treatment, it is subjected to multiple rounds of gradient screening. In the first round of screening, the reverse screening step is not performed, and the library after renaturation treatment is directly incubated with positive screening magnetic beads. From the second round onwards, the reverse screening step is performed. The library after renaturation treatment is first incubated with reverse screening magnetic beads or reverse screening control magnetic beads, and the unbound components are collected and then incubated with positive screening magnetic beads. In each round of screening, the nucleic acid sequences bound to the positive screening magnetic beads are collected by hot elution, and the single-stranded DNA secondary library is prepared by emulsion PCR amplification and denaturing PAGE electrophoresis using this as a template for the next round of screening.

[0014] (4) Real-time quantitative PCR was used to monitor the retention rate of each round of screening and to evaluate the library enrichment efficiency;

[0015] (5) High-throughput sequencing was performed on the enriched library. By cluster analysis and abundance statistics, the top 36 candidate aptamer sequences in terms of abundance were selected by comprehensively considering the sequence copy number, enrichment trajectory and secondary structure prediction results.

[0016] (6) The surface plasmon resonance technique was used to determine the affinity of candidate aptamers and optimize their truncation to obtain nucleic acid aptamers targeting CaMKⅡα;

[0017] Furthermore, the control peptides coupled to the reverse screening control magnetic beads include ISHRSTVASCMHRQETVDCLKKF and NARRKLKGAILTTMLATRNFSG.

[0018] Furthermore, the multi-round screening adopts a phased gradient pressure strategy, including one or more of the following pressure methods:

[0019] (a) The concentration of the input library was gradually reduced in stages;

[0020] (b) The incubation time for positive screening and / or negative screening is gradually shortened in stages;

[0021] (c) The screening medium is gradually upgraded as the screening rounds progress, transitioning from ethanolamine screening to control peptide screening;

[0022] (d) Inactivated human serum was added to the library to simulate the physiological environment;

[0023] Furthermore, the multi-round screening is carried out in a total of 7 rounds. In the first round, no reverse screening step is performed. In the second and third rounds, ethanolamine reverse screening magnetic beads are used to maintain the basic screening pressure. From the fourth round onwards, the library input concentration is gradually reduced, the incubation time is shortened, and inactivated human serum is added to increase the screening pressure. From the fifth round onwards, the reverse screening medium is upgraded to control peptide reverse screening magnetic beads.

[0024] Furthermore, the primers used in the real-time quantitative PCR described in step (4) are the forward primer 5'-GCACGGACACAAGAACAAAG-3' and the reverse primer 5'-CAGAACAAAGGCAGCACAAG-3';

[0025] Furthermore, the barcode forward primers used to distinguish samples from each round in the high-throughput sequencing described in step (5) are selected from the following sequences:

[0026] 5'-gcaacaGCACGGACACAAGAACAAAG-3',

[0027] 5'-gccaacGCACGGACACAAGAACAAAG-3',

[0028] 5'-gcgaagGCACGGACACAAGAAAAAG-3',

[0029] 5'-gctaatGCACGGACACAAGAAAAAG-3',

[0030] 5'-ggaacgGCACGGACACAAGAAAAAG-3',

[0031] 5'-ggtactGCACGGACACAAGACAAAG-3'.

[0032] The above-mentioned nucleic acid aptamers are used in the preparation of CaMKⅡα detection reagents or diagnostic kits.

[0033] The above-mentioned nucleic acid aptamers are used in the preparation of therapeutic drugs targeting CaMKⅡα-related diseases.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention adopts SELEX screening technology based on magnetic beads. Through a multi-round gradient pressure strategy, including the gradient reduction of the concentration of the input library, the gradient shortening of the incubation time, the upgrade of the screening medium from ethanolamine to the control peptide, and the introduction of inactivated serum to simulate the physiological environment, the targeted enrichment of CaMKⅡα nucleic acid aptamers with high affinity and high specificity is achieved, the screening efficiency is significantly improved, and the overall enrichment effect of 7 rounds of screening is significant.

[0036] (2) The full-length aptamer 36#L and the truncated aptamer 36#S obtained by screening in this invention can specifically bind to CaMKⅡα protein. The equilibrium dissociation constant KD value of the truncated aptamer 36#S reaches 80.82nM, the affinity is about 3 times higher than that of the full-length sequence, and the molecular weight is smaller, making it easier to chemically synthesize and deliver in vivo.

[0037] (3) The aptamer of the present invention has high specificity and high affinity. The truncated sequence 36#S maintains stable target binding activity in the entire temperature range of 4~37℃. The binding rate reaches 51.6% after 10 minutes of incubation at 37℃ and there is no obvious dissociation after 24 hours of incubation. At the same time, there is no non-specific adsorption in serum and peptide screening environment, showing excellent environmental adaptability and binding stability.

[0038] (4) Compared with existing antibodies and small molecule inhibitors, the nucleic acid aptamers of the present invention have advantages such as low preparation cost, small batch-to-batch variation, strong thermal stability, no immunogenicity, easy chemical modification and delivery across the blood-brain barrier, and have broad application prospects in the diagnosis and targeted treatment of CaMKⅡα-related central nervous system diseases. Attached Figure Description

[0039] Figure 1 Fixed target screening flowchart.

[0040] Figure 2 : Amplification curves of real-time PCR for each round of screening.

[0041] Figure 3 Screening retention rate analysis results chart.

[0042] Figure 4SPR sensor map of CaMKⅡα protein coupling (channels 3 and 4, used for initial screening of candidate aptamers and 36#L affinity determination).

[0043] Figure 5 SPR sensing map of CaMKⅡα protein coupling (channels 1 and 2, used for 36#S affinity assay).

[0044] Figure 6 SPR sensing map of 36#L monoclonal (full length) binding to CaMKⅡα protein (channel 4 difference minus channel 3).

[0045] Figure 7 : Fitting curve of KD value for 36#L monoclonal (full length)

[0046] Figure 8 SPR sensing map of 36#S monoclonal (truncated) binding to CaMKⅡα protein (channel 2 difference minus channel 1).

[0047] Figure 9 :36#S monoclonal (truncated) KD value fitting curve.

[0048] Figure 10 Flow cytometry analysis of the binding activity of CaMKⅡα aptamer with different protein magnetic beads.

[0049] Figure 11 Flow cytometry analysis of the binding activity of CaMKⅡα aptamer to CaMKⅡα protein magnetic beads (37℃).

[0050] Figure 12 Flow cytometry analysis of the binding activity of CaMKⅡα aptamer to CaMKⅡα protein magnetic beads (25℃).

[0051] Figure 13 Flow cytometry analysis of the binding activity of CaMKⅡα aptamer to CaMKⅡα protein magnetic beads (4℃).

[0052] Figure 14 : 36#S secondary structure prediction diagram. Detailed Implementation

[0053] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0054] The core reagents, materials, and instruments involved in the implementation method are as follows:

[0055] Document Information:

[0056] The initial screening library was a Lib24-76nt random nucleic acid library with the nucleotide sequence 5'-GCACGGACACAAGAACAAAG-N. (36) -CTTGTGCTGCCTTTGTTCTG-3', where each end contains a fixed primer binding region of 20 bases, and the middle contains a random sequence region of 36 bases (N represents any one of A, T, C, G) to ensure the sequence diversity of the library.

[0057] Primer information:

[0058] qPCR monitoring primers (universal for all rounds, used to monitor library enrichment efficiency):

[0059] Forward primer Lib24 S1: 5'-GCACGGACACAAGAACAAAG-3',

[0060] Reverse primer Lib24 A2: 5'-CAGAACAAAGGCAGCACAAG-3';

[0061] Secondary library amplification primers (used for ePCR amplification of elution+, and preparation of the next round of library input):

[0062] The first round of screening used:

[0063] Fluorescently labeled forward primer Lib24 S1-FAM: 5'-FAM-GCACGGACACAAGAACAAAG-3',

[0064] polyA-modified reverse primer Lib24 A2-polyA: 5'-AAAAAAAAAAAAAAAAAAAAAAAAAAA-Spacer18-CAGAACAAAGGCAGCACAAG-3';

[0065] The second to seventh rounds of screening used forward primers with barcode labels (the reverse primer remained Lib24 A2-polyA), and the corresponding primers for each round are shown below. The barcode sequence (lowercase letters) is located at the 5' end of the forward primer and is used to distinguish samples from each round during subsequent high-throughput sequencing:

[0066] Second round: Lib24-S1001: 5'-gcaacaGCACGGACACAAGAACAAAG-3'.

[0067] Third round: Lib24-S1002: 5'-gccaacGCACGGACACAAGAACAAAG-3',

[0068] Round 4: Lib24-S1003: 5'-gcgaagGCACGGACACAAGAACAAAG-3',

[0069] Fifth round: Lib24-S1004: 5'-gctaatGCACGGACACAAGAACAAAG-3',

[0070] Round 6: Lib24-S1005: 5'-ggaacgGCACGGACACAAGAACAAAG-3',

[0071] Round 7: Lib24-S1006: 5'-ggtactGCACGGACACAAGAACAAAG-3'.

[0072] Buffer information: DPBS buffer, with the following composition: 137mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 8mM Na2HPO4, 1mM CaCl2, 0.5mM MgCl2; pH 7.2~7.4.

[0073] Experimental materials: Dynabead TM MyOne TM Carboxylic acid magnetic beads (Thermo, catalog number 65011); Q-PCR mix (Aptamy, catalog number ESE-201801); ePCR mix (Aptamy); SELEX single-chain preparation kit (long and short chain method) (Aptamy); ePCR microdroplet generating oil (Aptamy, catalog number EPO-100); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (0.4M, Sigma-Aldrich, catalog number 800907); N-hydroxysuccinimide (NHS) solution (0.1M, Sigma-Aldrich, catalog number 130672); ethanolamine solution (1M, pH 8.5); Real-time PCR instrument (Roche LC96); PCR instrument (Bio-Rad / T100); Nucleic acid analyzer (Eppendorf BioPhotometer, Plus, 6132, Bio Photometer); CaMKⅡα protein (MCE, catalog number HY-P7S0425).

[0074] The reverse screening magnetic beads (MB-ethanolamine) involved in the embodiments are used to remove non-specific adsorption background, and their preparation method includes the following steps:

[0075] a. Take 50 μl of Dynabead TM MyOne TMCarboxylic acid magnetic beads were thoroughly resuspended and washed with 200 μl ddH2O, followed by magnetic separation and discarding of the supernatant; this washing step was repeated 4 times.

[0076] b. Mix 50 μl of 0.1 M NHS solution and 50 μl of 0.4 M EDC solution, then add the mixture to the magnetic beads treated in step a. Incubate on a shaker at room temperature for 20 min to activate the carboxyl groups. After incubation, magnetically separate the mixture and discard the supernatant. Wash once with 200 μl of DPBS buffer.

[0077] c. Add 100 μl of 1M ethanolamine solution (pH 8.5) to the magnetic beads obtained in step b, and incubate on a shaker at room temperature for 10 min to block unreacted activation sites. After incubation, magnetically separate and discard the supernatant. Wash four times with 200 μl of DPBS buffer, and then resuspend in 50 μl of DPBS buffer to obtain the reverse screening magnetic bead (MB-ethanolamine) solution for later use.

[0078] The positive sieve magnetic beads (MB-CaMKⅡα) involved in the implementation are used for specific target capture, and their preparation method includes the following steps:

[0079] a. Take 50 μl of Dynabead TM MyOne TM Carboxylic acid magnetic beads were thoroughly resuspended and washed with 200 μl ddH2O, followed by magnetic separation and discarding of the supernatant; this washing step was repeated 4 times.

[0080] b. Mix 50 μl of 0.1 M NHS solution and 50 μl of 0.4 M EDC solution, then add the mixture to the magnetic beads treated in step a. Incubate on a shaker at room temperature for 20 min to activate the carboxyl groups. After incubation, magnetically separate the mixture and discard the supernatant. Wash once with 200 μl of DPBS buffer.

[0081] c. Mix 20 μl of 1 mg / mL CaMKⅡα protein solution with 200 μl of 10 mM NaAC solution (pH 4.0), then add the mixture to the magnetic beads treated in step b. Incubate on a shaker at room temperature for 60 min to immobilize the target protein on the surface of the magnetic beads via covalent coupling. After incubation, magnetically separate the mixture and discard the supernatant.

[0082] d. Add 100 μl of 1M ethanolamine solution (pH 8.5) to the magnetic beads obtained in step c, and incubate on a shaker at room temperature for 10 min to block unreacted activation sites. After incubation, magnetically separate and discard the supernatant. Wash four times with 200 μl of DPBS buffer, and then resuspend in 50 μl of DPBS buffer to obtain the positive sieve magnetic bead (MB-CaMKⅡα) solution for later use.

[0083] The reverse screening control magnetic beads (MB-peptide) involved in the implementation method are used to exclude cross-reactive sequences of homologous domains, and their preparation method includes the following steps:

[0084] a. Take 50 μl of Dynabead TM MyOne TM Carboxylic acid magnetic beads were thoroughly resuspended and washed with 200 μl ddH2O, followed by magnetic separation and discarding of the supernatant; this washing step was repeated 4 times.

[0085] b. Mix 50 μl of 0.1 M NHS solution and 50 μl of 0.4 M EDC solution, then add the mixture to the magnetic beads treated in step a. Incubate on a shaker at room temperature for 20 min to activate the carboxyl groups. After incubation, magnetically separate the mixture and discard the supernatant. Wash once with 200 μl of DPBS buffer.

[0086] c. Take 10 μl of a mixture of peptide 1 and peptide 2 (the total concentration of peptide 1 and peptide 2 in the mixture is 1 mg / mL, and the concentration ratio of the two peptides is 1:1; the amino acid sequence of peptide 1 is ISHRSTVASCMHRQETVDCLKKF, and the amino acid sequence of peptide 2 is NARRKLKGAILTTMLATRNFSG), add 200 μl of 10 mM NaAc buffer (pH 4.0), mix well, and then transfer to the magnetic beads treated in step b. Incubate on a shaker at room temperature for 60 min to immobilize the peptides on the surface of the magnetic beads using a covalent coupling reaction. After incubation, magnetically separate and discard the supernatant.

[0087] d. Add 100 μl of 1M ethanolamine solution (pH 8.5) to the magnetic beads obtained in step c, and incubate on a shaker at room temperature for 10 min to block unreacted activation sites. After incubation, magnetically separate and discard the supernatant, wash four times with 200 μl of DPBS buffer, and then resuspend in 50 μl of DPBS buffer to obtain the reverse screening control magnetic bead (MB-peptide) solution for later use.

[0088] Example 1:

[0089] A method for screening nucleic acid aptamers targeting CaMKⅡα includes the following steps:

[0090] S1: First Round of Screening

[0091] S1-1: Nucleic Acid Library Preprocessing

[0092] Take one vial of Lib24-76bp nucleic acid library powder with a total volume of 1 OD, centrifuge at 14000 rpm for 10 min, then add 140 μl of DPBS buffer to dissolve thoroughly, obtaining the library solution. Place the library solution in a PCR instrument for renaturation to promote a stable spatial conformation; the renaturation program is set as follows: 95℃ for 10 min, 4℃ for 5 min, 25℃ for 10 min. Dilute the renaturated library solution to 5 μM with DPBS buffer for later use.

[0093] S1-2: Positive screening (no reverse screening is performed in this round; the document library directly enters the positive screening process).

[0094] The renatured library solution (280 μM) was transferred to 50 μL of MB-CaMKⅡα magnetic beads solution. The mixture was gently pipetted and incubated on a shaker at room temperature for 60 min. Magnetic separation was then performed, and the supernatant (containing unbound target sequences) was collected. Subsequently, the magnetic beads were washed to remove non-specifically bound sequences: 200 μL of DPBS buffer was added each time, the beads were resuspended by pipetting, and the supernatant was collected after magnetic separation. This step was repeated four times, and the collected supernatants were designated Wash1+, Wash2+, Wash3+, and Wash4+, respectively. After washing, 200 μL of DPBS buffer was added to the magnetic beads, and the mixture was boiled for 10 min to allow the aptamers specifically bound to the CaMKⅡα target protein to dissociate through thermal denaturation. After the boiling water bath, the supernatant was collected after magnetic separation and designated as elution+. This fraction represents the target-specific nucleic acid library enriched in this round of screening.

[0095] S1-3: Preparation and purification of single-stranded DNA (preparation of the second-round library)

[0096] S1-3-1: Emulsion PCR (ePCR) amplification

[0097] Add all elution+ components to 2 mL of ePCR Mix (formula shown in the table below) and vortex for 30 seconds to mix thoroughly. Add 8 mL of ePCR microdroplet-generating oil to the mixture and vortex at 2000–3000 rpm for 1 min to form a homogeneous emulsion. Then aliquot the emulsion into PCR tubes (100 μl per tube) and place them in a PCR instrument. Amplify according to the following program: 95°C for 3 min; 95°C for 1 min, 60°C for 1 min, 72°C for 1 min, for a total of 30 cycles; 72°C for 10 min; store at 4°C. After amplification, recover and combine the PCR amplification products from the emulsions in each tube.

[0098]

[0099] S1-3-2: Demulsification and Concentration of n-Butanol

[0100] The recovered emulsion PCR amplification product was aliquoted into two centrifuge tubes. 50–100 μl of ultrapure water was added to each tube, and the mixture was gently pipetted to mix. 8 mL of n-butanol was added to each tube, the tubes were capped, and the tubes were inverted approximately 10 times to ensure complete demulsification. The tubes were centrifuged at 7500 g for 10 min. After centrifugation, the liquid in the tubes separated into three layers: a clear n-butanol phase on top, a residual oily emulsion in the middle, and an aqueous phase containing the amplification product at the bottom. The upper n-butanol phase and the residual oily emulsion in the middle were carefully removed using a pipette, retaining the bottom aqueous phase. Five times the volume of n-butanol was added to the aqueous phase, and the mixture was inverted to mix until the liquid became slightly turbid. The mixture was then centrifuged at 12000 rpm for 2 min, and the upper n-butanol phase was removed. This concentration process was repeated until the volume of the aqueous phase was reduced to approximately 100 μl, yielding the concentrated emulsion PCR amplification product.

[0101] S1-3-3: Denaturing PAGE electrophoresis separation of single strands

[0102] Add an equal volume of 2×TBE urea loading buffer to the concentrated emulsion PCR amplification product, vortex for 10 seconds, heat in a boiling water bath for 10 minutes to fully denature and dissociate the double-stranded DNA, remove and briefly centrifuge for 5 seconds, then immediately place on ice to cool for 5 minutes to obtain a cooled denatured sample. Load the cooled denatured sample into the wells of a pre-prepared 8% denaturing PAGE gel, and simultaneously add a fluorescently labeled single-stranded DNA molecular weight standard (Fluorescent ssDNA ladder) to the side channel as a reference. Electrophoresis is performed at a constant voltage of 300-400V until the target band migrates to the bottom 1 / 3 of the gel. Observe under a handheld UV lamp or transmitted UV lamp, and according to the migration position of the fluorescent molecular weight standard, cut out the corresponding 76nt single-stranded DNA target band, and place the cut strip into a 1.5mL centrifuge tube. Add 300μl of sterile ultrapure water to the tube, heat in a 65℃ metal bath, and invert and mix every 2-3 minutes until the gel completely melts into a clear liquid. Add an equal volume of water-saturated n-butanol to the completely melted gel solution, vortex for 30 seconds, mix thoroughly, and centrifuge at 12,000 rpm for 1 minute at room temperature. Remove the upper n-butanol phase. Repeat this concentration operation until the aqueous phase volume is concentrated to 100-200 μl. Then, open the cap and let stand at room temperature for 5 minutes to evaporate any residual trace amounts of n-butanol, obtaining the single-stranded DNA library solution.

[0103] S1-3-4: Dialysis purification and concentration determination

[0104] The single-stranded DNA library solution obtained in the previous step was dialyzed overnight at 4°C using a micro-nucleic acid dialysis membrane with a molecular weight cutoff of 3.5 kDa in 40 mL of DPBS buffer. The dialyzed single-stranded DNA library solution was then centrifuged at 12,000 rpm for 5 min, and the supernatant was collected. The concentration and purity of the supernatant were determined using a nucleic acid analyzer, and the samples were labeled as "Round 1 Library" and stored at -20°C for use as the input library for the second round of screening.

[0105] S1-4: qPCR monitoring

[0106] S1-4-1: Sample Processing

[0107] The Wash1+, Wash2+, Wash3+, Wash4+, and elution+ fractions collected during the positive screening process were centrifuged at 12000 rpm for 2 min to remove any possible residual magnetic beads. The supernatant from each tube was collected after centrifugation and used as template for qPCR detection.

[0108] S1-4-2: qPCR amplification

[0109] Take an 8-tube Roche PCR tube, add 30 μl of Q-PCR mix (formula shown in the table below) to each well, and then add 2 μl of the supernatant of the corresponding component as a template. Place the PCR tube in a real-time PCR instrument and perform amplification according to the following program: 95℃ for 2 min; 95℃ for 0.5 min, 60℃ for 0.5 min, 72℃ for 0.5 min, for a total of 30 cycles.

[0110]

[0111] S1-4-3: Standard Curve and Quantitative Calculation

[0112] A serially diluted Lib24-76nt standard library of known concentrations was used as a standard, and qPCR amplification was performed simultaneously. A standard curve was plotted with the logarithm of the standard concentration (logC, C in pM) on the x-axis and the corresponding Cq value on the y-axis. The resulting standard curve equation is: Y = -0.2688x + 4.7397 (R² + π / 4)². 2 =0.9927). Based on the Cq values ​​of each analyte, substitute them into the standard curve equation to calculate the DNA concentration in each analyte. Then, based on the volume of each analyte, convert it to the number of DNA molecules (number of molecules = concentration (pM) × 10). -12 ×Volume (L)×6.02×10 23 ).

[0113] S1-4-4: Retention Rate Calculation

[0114] Based on the quantitative results, the positive screening retention rate was calculated using the following formula: Positive screening retention rate = elution + number of DNA molecules in the sample / total number of DNA molecules in the input library. The calculated positive screening retention rate for the first round of screening was 5.62E-05.

[0115] S2: Second Round of Screening

[0116] S2-1: Complexity of Input Documents

[0117] The libraries selected in the second round of screening were placed in a PCR instrument for renaturation treatment, following the same procedure as the first round of screening. The renaturated libraries were then diluted to 800 nM with DPBS buffer for later use.

[0118] S2-2: Reverse screening (introduced for the first time)

[0119] 100 μL of the renatured 800 nM library was added to 50 μL of MB-ethanolamine magnetic bead solution, gently mixed by pipetting, and incubated on a shaker at room temperature for 60 min. After incubation, magnetic separation was performed, and the supernatant was collected and denoted as Pool-. Pool- represents the library after the removal of the magnetic bead matrix and non-specific adsorbed sequences, and is used entirely for subsequent positive screening. To evaluate the reverse screening efficiency, the magnetic beads adsorbed with non-specific sequences were washed: four times with 200 μL of DPBS buffer, and the wash solutions were denoted as Wash1-, Wash2-, Wash3-, and Wash4-, respectively. Then, 200 μL of DPBS buffer was added to the washed magnetic beads, and the mixture was eluted in a boiling water bath for 10 min. After magnetic separation, the supernatant was collected and denoted as elution-.

[0120] S2-3: Positive sieve

[0121] All pool-components collected by reverse sieving were transferred to 50 μl of positive sieve magnetic beads (MB-CaMKⅡα) solution, gently mixed by pipetting, and incubated on a shaker at room temperature for 60 min. After incubation, magnetic separation was performed, and the supernatant (containing unbound target sequences) was collected. The magnetic beads were washed four times sequentially with 200 μl of DPBS buffer, and the supernatants were labeled Wash1+, Wash2+, Wash3+, and Wash4+, respectively. 200 μl of DPBS buffer was added to the washed magnetic beads, and the mixture was incubated in a boiling water bath for 10 min. After magnetic separation, the supernatant was collected and labeled elution+.

[0122] S2-4: Preparation and purification of single-stranded DNA (preparation of the third round of library input)

[0123] Using the elution+ component obtained from this round of forward screening as a template, the procedure was performed according to the steps in S1-3 of the first round, "Preparation and Purification of Single-Stranded DNA," with the following difference: During emulsion PCR amplification, the forward primer was replaced from Lib24 S1-FAM with the corresponding barcode-tagged forward primer Lib24-S1001, while the reverse primer remained Lib24 A2-polyA. The remaining reaction system, amplification program, microdroplet preparation, and product purification procedures were the same as in the first round. After determining the concentration and purity using a nucleic acid analyzer, the sample was labeled "Second Round Library" and stored at -20℃ for use as a library in the third round of screening.

[0124] S2-5: qPCR monitoring

[0125] Following the steps of the first round of "qPCR monitoring," qPCR quantitative analysis was performed on each component (Wash1-, Wash2-, Wash3-, Wash4-, elution-, Wash1+, Wash2+, Wash3+, Wash4+, and elution+) collected during this round of reverse and forward screening. Reverse screening retention rate = number of DNA molecules in elution- / total number of DNA molecules in the input library; calculated, the forward screening retention rate for the second round was 1.54E-04, and the reverse screening retention rate was 8.53E-04.

[0126] S3: Third Round of Screening

[0127] S3-1: Complexity of Input Documents

[0128] The libraries selected in the third round of screening were placed in a PCR instrument for renaturation treatment, following the same procedure as the first round of screening. The renaturated libraries were then diluted to 800 nM with DPBS buffer for later use.

[0129] S3-2: Reverse screening

[0130] The operation is the same as the second round of "reverse screening" steps.

[0131] S3-3: Positive sieve

[0132] The operation is the same as the second round of "positive screening" steps.

[0133] S3-4: Preparation and purification of single-stranded DNA

[0134] Using the elution+ component obtained from this round of forward screening as a template, the procedure was performed according to the steps in S1-3 of the first round, "Preparation and Purification of Single-Stranded DNA," with the following difference: During emulsion PCR amplification, the forward primer was replaced from Lib24 S1-FAM with the corresponding barcode-tagged forward primer Lib24-S1002, while the reverse primer remained Lib24 A2-polyA. The remaining reaction system, amplification program, microdroplet preparation, and product purification procedures were the same as in the first round. After determining the concentration and purity using a nucleic acid analyzer, the sample was labeled "Third Round Library" and stored at -20℃ for use as a library in the fourth round of screening.

[0135] S3-5: qPCR monitoring

[0136] Following the steps of the first round of "qPCR monitoring," qPCR quantitative analysis was performed on all components (Wash1-, Wash2-, Wash3-, Wash4-, elution-, Wash1+, Wash2+, Wash3+, Wash4+, and elution+) collected during this round of reverse and forward screening. The calculated retention rate for the third round of screening was 3.39E-03 for the forward screening and 1.48E-03 for the reverse screening.

[0137] S4: Fourth Round of Selection

[0138] S4-1: Complexity of Input Documents

[0139] The input libraries selected in the fourth round of screening were placed in a PCR instrument for renaturation, following the same procedure as the first round of screening. The renaturated input libraries were then diluted to 400 nM with DPBS buffer and 5 vol% inactivated human serum was added for later use.

[0140] S4-2: Reverse screening

[0141] The operation was basically the same as the second round of "reverse screening" steps, except that: the concentration of the input library after renaturation treatment was 400 nM and contained 5 vol% inactivated human serum; the incubation time was 50 min.

[0142] S4-3: Positive sieve

[0143] The operation is basically the same as the second round of "positive screening" steps, except that the incubation time is 50 minutes.

[0144] S4-4: Preparation and Purification of Single-Stranded DNA

[0145] Using the elution+ component obtained from this round of forward screening as a template, the procedure was performed according to the steps in S1-3 of the first round, "Preparation and Purification of Single-Stranded DNA," with the following difference: During emulsion PCR amplification, the forward primer was replaced from Lib24 S1-FAM with the corresponding barcode-tagged forward primer Lib24-S1003, while the reverse primer remained Lib24 A2-polyA. The remaining reaction system, amplification program, microdroplet preparation, and product purification procedures were the same as in the first round. After determining the concentration and purity using a nucleic acid analyzer, the sample was labeled "Fourth Round Library" and stored at -20℃ for use as a library in the fifth round of screening.

[0146] S4-5: qPCR monitoring

[0147] Follow the same steps as in the first round of "qPCR monitoring". The calculated retention rate for the positive screening in the fourth round was 1.89E-02, and the retention rate for the negative screening was 2.49E-03.

[0148] S5: Fifth Round of Screening

[0149] S5-1: Complexity of Input Documents

[0150] The input libraries selected in the fifth round of screening were placed in a PCR instrument for renaturation treatment, following the same procedure as the first round of screening. The renaturated input libraries were then diluted to 400 nM with DPBS buffer and 5 vol% inactivated human serum was added for later use.

[0151] S5-2: Reverse screening

[0152] The operation was basically the same as the second round of "reverse screening" steps, except that: the concentration of the input library after renaturation treatment was 400 nM and contained 5 vol% inactivated human serum; the reverse screening magnetic bead (MB-ethanolamine) solution was replaced with the reverse screening control magnetic bead (MB-peptide) solution; and the incubation time was 40 min.

[0153] S5-3: Positive sieve

[0154] The operation is basically the same as the second round of "positive screening" steps, except that the incubation time is 40 minutes.

[0155] S5-4: Preparation and Purification of Single-Stranded DNA

[0156] Using the elution+ component obtained in this round of forward screening as a template, the procedure was performed according to the steps in S1-3 of the first round, "Preparation and Purification of Single-Stranded DNA," with the difference that during emulsion PCR amplification, the forward primer was replaced from Lib24 S1-FAM with the corresponding barcode-tagged forward primer Lib24-S1004, while the reverse primer remained Lib24 A2-polyA. The remaining reaction system, amplification program, microdroplet preparation, and product purification procedures were the same as in the first round. After determining the concentration and purity using a nucleic acid analyzer, the sample was labeled "Fifth Round Library" and stored at -20℃ for use as a library in the sixth round of screening.

[0157] S5-5: qPCR monitoring

[0158] Follow the same steps as in the first round of "qPCR monitoring". The calculated retention rate for the positive screening in the fifth round was 1.42E-02, and the retention rate for the negative screening was 5.29E-03.

[0159] S6: Sixth Round of Selection

[0160] S6-1: Complexity of Input Documents

[0161] The input libraries selected in the sixth round of screening were placed in a PCR instrument for renaturation treatment, following the same procedure as the first round of screening. The renaturated input libraries were then diluted to 400 nM with DPBS buffer and 10 vol% inactivated human serum was added for later use.

[0162] S6-2: Reverse screening

[0163] The operation was basically the same as the second round of "reverse screening" steps, except that: the concentration of the input library after renaturation treatment was 400 nM and contained 10 vol% inactivated human serum; the reverse screening magnetic bead (MB-ethanolamine) solution was replaced with the reverse screening control magnetic bead (MB-peptide) solution; and the incubation time was 35 min.

[0164] S6-3: Positive sieve

[0165] The operation is basically the same as the second round of "positive screening" steps, except that the incubation time is 35 minutes.

[0166] S6-4: Preparation and Purification of Single-Stranded DNA

[0167] Using the elution+ component obtained in this round of forward screening as a template, the procedure was performed according to the steps in S1-3 of the first round, "Preparation and Purification of Single-Stranded DNA," with the following difference: During emulsion PCR amplification, the forward primer was replaced from Lib24 S1-FAM with the corresponding barcode-tagged forward primer Lib24-S1005, while the reverse primer remained Lib24 A2-polyA. The remaining reaction system, amplification program, microdroplet preparation, and product purification procedures were the same as in the first round. After determining the concentration and purity using a nucleic acid analyzer, the sample was labeled "Sixth Round Library" and stored at -20℃ for use as the input library in the seventh round of screening.

[0168] S6-5: qPCR monitoring

[0169] Follow the same steps as in the first round of "qPCR monitoring". The calculated retention rates for the positive screening (1.68E-02) and the negative screening (8.98E-03) in the sixth round were as follows.

[0170] S7: Seventh Round of Selection

[0171] S7-1: Complexity of Input Documents

[0172] The input libraries selected in the seventh round of screening were placed in a PCR instrument for renaturation treatment, following the same procedure as the first round of screening. The renaturated input libraries were then diluted to 250 nM with DPBS buffer.

[0173] S7-2: Reverse screening

[0174] The operation was basically the same as the second round of "reverse screening" steps, except that: the concentration of the input library after renaturation treatment was 250 nM; the reverse screening magnetic bead (MB-ethanolamine) solution was replaced with the reverse screening control magnetic bead (MB-peptide) solution; and the incubation time was 30 min.

[0175] S7-3: Positive sieve

[0176] The operation is basically the same as the second round of "positive screening" steps, except that the incubation time is 30 minutes.

[0177] S7-4: Preparation and Purification of Single-Stranded DNA

[0178] Using the elution+ component obtained from this round of forward screening as a template, the procedure was performed according to the steps in S1-3 of the first round, "Preparation and Purification of Single-Stranded DNA," with the following difference: During emulsion PCR amplification, the forward primer was replaced from Lib24 S1-FAM with the corresponding barcode-tagged forward primer Lib24-S1006, while the reverse primer remained Lib24 A2-polyA. The remaining reaction system, amplification program, microdroplet preparation, and product purification procedures were the same as in the first round. After determining the concentration and purity using a nucleic acid analyzer, the sample was labeled "Seventh Round Library" and stored at -20℃ for later use.

[0179] S7-5: qPCR monitoring

[0180] The procedure was followed as described in the first round of "qPCR monitoring". The calculated retention rate for the seventh round of positive screening was 5.16E-02, and the retention rate for negative screening was 3.74E-02. This completes the seven rounds of screening. The positive screening retention rate increased significantly from 5.62E-05 in the first round to 5.16E-02 in the seventh round, indicating a substantial increase in overall enrichment, demonstrating effective enrichment of sequences specifically binding to CaMKⅡα.

[0181] S8: High-throughput sequencing and candidate sequence analysis

[0182] S8-1: Sequencing Sample Preparation

[0183] Use 20 μl (500 nM) of the high-purity single-stranded DNA library obtained from each round of screening as templates, and add it to 400 μl of PCR mix (the PCR mix formula is the same as the Q-PCR mix in S1-4-2, but the forward primers are replaced with the corresponding barcode-tagged forward primers for each round, i.e., Lib24-S1001 for the second round, Lib24-S1002 for the third round, Lib24-S1003 for the fourth round, Lib24-S1004 for the fifth round, Lib24-S1005 for the sixth round, and Lib24-S1006 for the seventh round). The reverse primer is Lib24 A2 for all rounds. Perform PCR amplification to introduce sequencing tags according to the following program: 95℃ for 1 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for a total of 20 cycles; 72℃ for 5 min; store at 4℃. After amplification, the PCR products from each round of samples were mixed and aliquoted into two centrifuge tubes. Purification was performed using the UNIQ-10 oligonucleotide purification kit (Shanghai Sangon Biotech, catalog number: B511143). The purified sequencing library was then subjected to high-throughput sequencing using the Illumina-NovaSeq 6000 sequencing system platform.

[0184] S8-2: Data Analysis and Candidate Sequence Screening

[0185] Bioinformatics clustering analysis and abundance statistics were performed on over 130,000 valid sequences obtained from sequencing. Considering sequence copy number, enrichment trajectory, and secondary structure prediction results, the top 36 abundant sequences were selected as candidate aptamers, which were then synthesized by a commercial company for subsequent affinity identification. Specific information on the 36 candidate sequences is shown in the table below:

[0186]

[0187] S8-3: Affinity Identification and Optimization of Candidate Aptamers

[0188] The affinity of 36 synthesized candidate aptamers was determined and truncation optimized using surface plasmon resonance (SPR) technology (Biacore T200, GE Healthcare).

[0189] S8-3-1: Chip Activation and Protein Coupling

[0190] The S-series CM5 chip (GE Healthcare, catalog number: 29149603) was installed in the SPR instrument. DPBS buffer was used as the run buffer at a flow rate of 10 μl / min, and the chip was equilibrated until baseline stability was achieved. The experimental channels (channels 3 and 4) were activated using an activation mixture (equal volumes of EDC (400 mM) and NHS (100 mM)) at a flow rate of 10 μl / min. CaMKⅡα protein was diluted to 10 μg / mL with 10 mM sodium acetate aqueous solution at pH 4.0 and injected into the activated channel 4 for coupling, with a coupling amount of 7529 RU, serving as the detection channel. Channel 3 was directly blocked with 1M ethanolamine (pH 8.5) after activation, serving as the reference channel. After injection, 1M ethanolamine (pH 8.5) was also injected into channel 4 for blocking at a flow rate of 10 μl / min for 10 min.

[0191] S8-3-2: Initial screening of candidate aptamers

[0192] Thirty-six candidate aptamers were diluted to 1 μM with DPBS buffer and sequentially flowed through channels 3 and 4. The injection flow rate was 25 μl / min, with a binding time of 1.5 min; the dissociation flow rate was 25 μl / min, with a dissociation time of 1.5 min. After each sample test, the chip was regenerated with 1 M NaCl solution at a flow rate of 25 μl / min for 1 min. The data is the result of subtracting the signal from the signal in channel 3 from the signal in channel 4. The results showed that aptamer C036 (i.e., 36#) had the strongest binding signal with CaMKⅡα and was selected for further investigation.

[0193]

[0194] S8-3-3: Affinity determination of full-length aptamer 36#L

[0195] The 36#L aptamer (SEQ ID NO.1, full length 76 nt, sequence 5'-GCACGGACACAAGAACAAAGAGGGGGACTGCTCGGGATTGCGGATACTTGGTGTTCCTTGTGCTGCCTTTGTTCTG-3') was diluted with DPBS buffer to 11 concentration gradients: 2 μM, 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM, and 1.953125 nM. The chip was then reactivated and the protein conjugated (steps as in S8-3-1). The CaMKⅡα protein was diluted to 10 μg / mL with a 10 mM sodium acetate aqueous solution at pH 4.0 and injected into the activated channel 4 for coupling, with a coupling amount of 3849 RU, serving as the detection channel. Channel 3 was directly blocked by 1M ethanolamine (pH 8.5) after activation, serving as the reference channel. The protein was injected into channels 3 and 4 sequentially in ascending order of concentration. The injection flow rate was 30 μl / min, with a binding time of 2.5 min and a dissociation time of 2.5 min. After each concentration test, the chip was regenerated using 4 mM NaOH solution at a flow rate of 20 μl / min for 1 min. Using Biacore T200 software, binding-dissociation curves were fitted in 1:1 binding Kinetics mode (data processed by subtracting channel 3 from channel 4 difference), and the equilibrium dissociation constant KD value for the binding of 36#L with CaMKⅡα was calculated to be 247.9 nM.

[0196] S8-3-4: Design and affinity determination of truncated aptamer 36#S

[0197] Based on the secondary structure prediction results of 36#L, a truncated form 36#S (SEQ ID NO.2, full length 56nt, sequence 5'-AAGAACAAAGAGGGGGACTGCTCGGGATTGCGGATACTTGGTGTTCCTTGTGCTGC-3') was designed. The chip was reactivated and protein-coupled (steps same as S8-3-1). CaMKⅡα protein was diluted to 10 μg / mL with 10 mM sodium acetate aqueous solution at pH 4.0 and injected into the activated Channel 2 for coupling, with a coupling amount of 10548 RU, serving as the detection channel; Channel 1, after activation, was directly blocked with 1M ethanolamine (pH 8.5) and served as the reference channel. Dilute 36#S with DPBS buffer to 12 concentration gradients: 5 μM, 2.5 μM, 1.25 μM, 625 nM, 312.5 nM, 156.25 nM, 78.125 nM, 39.0625 nM, 19.53 nM, 9.765 nM, 4.8825 nM, and 2.441 nM. Flow through channels 1 and 2 sequentially in ascending order of concentration. Injection flow rate: 25 μL / min; binding time: 3 min; dissociation time: 4 min. After each concentration test, regenerate the chip with 2.5 mM NaOH solution at a flow rate of 20 μL / min for 45 s. Using Biacore T200 software, the binding-dissociation curve was fitted in 1:1 binding Kinetics mode (data was processed by subtracting channel one from channel two difference). The equilibrium dissociation constant KD value of 36#S binding with CaMKⅡα was calculated to be 80.82 nM, and the affinity was increased to about 3 times compared with the full-length 36#L.

[0198] In SPR assays, the binding-dissociation curves of the aptamer and CaMKⅡα protein exhibited typical specific binding characteristics. After regeneration, the chip response value recovered to the baseline level, indicating that the binding of the aptamer to the target is reversible and stable. The affinity of the truncated 36#S sequence was approximately 3 times higher than that of the full-length 36#L sequence, indicating that the truncated sequence retained the core binding domain and, due to its reduced molecular weight and more rigid structure, exhibited superior target binding kinetics.

[0199] S8-4: Flow cytometry verification

[0200] The binding ability of the candidate aptamer to CaMKⅡα-coupled magnetic beads (MB-CaMKⅡα) was rapidly detected by flow cytometry, further verifying the binding stability and specificity of the truncated aptamer 36#S to the CaMKⅡα protein.

[0201] S8-4-1: Experimental Materials

[0202] Carboxylated magnetic beads (Dynabead) TM MyOneTM Thermo (catalog number 65011), CaMKIIα protein (1 mg / mL), control protein SDF-1 (0.1 mg / mL), 0.4 M EDC aqueous solution, 0.1 M NHS aqueous solution, DPBS buffer, coupling buffer (pH 4.0, 10 mM NaAc buffer), blocking reagent (1 M ethanolamine.HCl, pH 8.5), FAM-modified CaMKIIα aptamer (36#S-FITC), flow cytometer.

[0203] S8-4-2: Preparation of Protein Magnetic Beads

[0204] Take 50 μl of carboxyl magnetic beads and wash them four times with 200 μl of ddH2O. After each wash, magnetically separate and discard the supernatant. Add 100 μl of EDC aqueous solution and 100 μl of NHS aqueous solution (mix the NHS aqueous solution into the EDC aqueous solution) to the washed magnetic beads and incubate on a shaker at room temperature for 25 min to activate the carboxyl groups. After incubation, magnetically separate and discard the supernatant, add 50 μl of ddH2O to resuspend the magnetic beads, and divide them into two equal portions (25 μl each). Take 10 μl of CaMKⅡα protein (1 mg / mL) and add it to 400 μl of NaAc buffer (pH 4.0), mix well, and add it to one portion of the activated magnetic beads (experimental group); take 100 μl of 0.1 mg / mL SDF-1 protein and add it to 310 μl of NaAc buffer (pH 4.0), mix well, and add it to the other portion of the activated magnetic beads (control group). Mix the magnetic beads and protein with a pipette and incubate on a vertical rotary mixer at room temperature for 60 min. After coupling, the supernatant was discarded after magnetic separation. 100 μl of 1M ethanolamine·HCl solution (pH 8.5) was added to the magnetic beads, and the mixture was incubated at room temperature for 20 min on a vertical rotary mixer to block unreacted activation sites on the surface of the magnetic beads. After blocking, the magnetic beads were washed four times with 100 μl of DPBS buffer.

[0205] S8-4-3: Aptamer Incubation and Flow Cytometry Detection

[0206] 100 μl of 500 nM FAM fluorescently labeled 36#S aptamer solution was added to both the experimental group magnetic beads coupled with CaMKⅡα protein and the control group magnetic beads coupled with SDF-1 protein. Incubation was performed at different temperatures for specified times: 12 h at 4℃; 1 h, 2 h, 4 h, 12 h, and 24 h at room temperature; and 10 min, 30 min, 1 h, 2 h, 4 h, 12 h, and 24 h at 37℃. After incubation, each group of magnetic beads was washed twice with 100 μl of DPBS, diluted to 1 mL with DPBS, and analyzed by flow cytometry.

[0207] S8-4-4: Flow Cytometry Results

[0208] At 4℃ for 12 hours, the fluorescence positivity rate of the control group (SDF-1 coupled magnetic beads) was only 0.2%, while the fluorescence positivity rate of the experimental group (CaMKⅡα coupled magnetic beads) reached 93.3%. This indicates that the aptamer can achieve efficient and specific binding to CaMKⅡα protein in a low-temperature environment, and the binding complex has excellent stability, making it suitable for low-temperature detection and storage.

[0209] At room temperature: The fluorescence positivity rate of the control group (incubated for 12 hours) was only 0.12%. The fluorescence positivity rate of the experimental group showed a pattern of first rising, briefly falling, continuously rising and finally stabilizing: 21.8% after 1 hour of incubation, reaching a peak of 44.8% after 2 hours, briefly falling to 30.7% after 4 hours, and then remaining stable with prolonged incubation time, reaching 75.3% after 12 hours and still maintaining 75.8% after 24 hours, with no significant activity decay.

[0210] Under simulated physiological temperature conditions of 37℃, the fluorescence positivity rate of the control group (incubated for 24 hours) was only 0.2%. The experimental group exhibited excellent rapid binding characteristics, with a fluorescence positivity rate of 51.6% after 10 minutes of incubation. Subsequently, it entered the conformational rearrangement stage of aptamer-target binding, during which the fluorescence positivity rate slightly decreased and remained stable over a long period: 39.4% at 30 minutes, 42.5% at 1 hour, and still maintained a high level of 33.5% at 24 hours, with no significant dissociation, demonstrating good potential stability for in vivo application.

[0211] In summary, the aptamers specifically bound to CaMKⅡα protein at 4℃, room temperature, and 37℃. Under different test temperatures and corresponding incubation times, the fluorescence positivity rate of the control group was no higher than 0.2%, effectively eliminating interference from non-specific binding of the magnetic bead matrix, buffer components, and control protein. The experimental group showed stable target binding activity at different test temperatures. Combined with SPR technology verification results, both the full-length sequence 36#L (KD=247.9nM) and the truncated sequence 36#S (KD=80.82nM) exhibited nanomolar-level high target affinity. 36#S, in particular, demonstrated excellent binding specificity and temperature stability through multi-temperature gradient verification, further confirming that this series of aptamers possesses high specificity, high affinity, and good structural and functional stability.

Claims

1. A nucleic acid aptamer targeting CaMKⅡα, characterized in that: The nucleotide sequence of the nucleic acid aptamer is selected from any of the following: (1) 5'-GCACGGACACAAGAACAAAGAGGGGGACTGCTCGGGATTGCGGATACTTGGTGTTCCTTGTGCTGCCTTTGTTCTG-3'; (2) 5'-AAGAACAAAGAGGGGGACTGCTCGGGATTGCGGATACTTGGTGTTCCTTGTGCTGC-3'; (3) A derivative sequence that has at least 80% homology with the nucleotide sequence shown in (1) or (2) and has a specific binding function of CaMKⅡα.

2. A method for screening nucleic acid aptamers targeting CaMKⅡα, characterized in that: Includes the following steps: (1) Provide a random nucleic acid library, wherein the nucleotide sequence of the random nucleic acid library is 5'-GCACGGACACAAGAACAAAG-N (36) -CTTGTGCTGCCTTTGTTCTG-3', where N represents any one of the bases A, T, C, and G; (2) Prepare functionalized magnetic beads, including positive screening magnetic beads coupled with CaMKⅡα protein, reverse screening magnetic beads for removing non-specific adsorption background, and reverse screening control magnetic beads for excluding cross-reactive sequences of homologous domains. (3) After the random nucleic acid library is subjected to renaturation treatment, it is subjected to multiple rounds of gradient screening. In the first round of screening, the reverse screening step is not performed, and the library after renaturation treatment is directly incubated with positive screening magnetic beads. From the second round onwards, the reverse screening step is performed. The library after renaturation treatment is first incubated with reverse screening magnetic beads or reverse screening control magnetic beads, and the unbound components are collected and then incubated with positive screening magnetic beads. In each round of screening, the nucleic acid sequences bound to the positive screening magnetic beads are collected by hot elution, and the single-stranded DNA secondary library is prepared by emulsion PCR amplification and denaturing PAGE electrophoresis using this as a template for the next round of screening. (4) Real-time quantitative PCR was used to monitor the retention rate of each round of screening and to evaluate the library enrichment efficiency; (5) High-throughput sequencing was performed on the enriched library. By cluster analysis and abundance statistics, the top 36 candidate aptamer sequences in terms of abundance were selected by comprehensively considering the sequence copy number, enrichment trajectory and secondary structure prediction results. (6) The surface plasmon resonance technique was used to determine the affinity of candidate aptamers and optimize their truncation to obtain nucleic acid aptamers targeting CaMKⅡα.

3. The screening method according to claim 2, characterized in that: The control peptides coupled to the reverse screening control magnetic beads include ISHRSTVASCMHRQETVDCLKKF and NARRKLKGAILTTMLATRNFSG.

4. The screening method according to claim 2, characterized in that: The multi-round screening adopts a phased gradient pressure strategy, including one or more of the following pressure methods: (a) The concentration of the input library was gradually reduced in stages; (b) The incubation time for positive screening and / or negative screening is gradually shortened in stages; (c) The screening medium is gradually upgraded as the screening rounds progress, transitioning from ethanolamine screening to control peptide screening; (d) Inactivated human serum was incorporated into the library to simulate the physiological environment.

5. The screening method according to claim 4, characterized in that: The multi-round screening is conducted in 7 rounds. In the first round, no reverse screening step is performed. In the second and third rounds, ethanolamine reverse screening magnetic beads are used to maintain the basic screening pressure. From the fourth round onwards, the concentration of the library input is gradually reduced, the incubation time is shortened, and inactivated human serum is added to increase the screening pressure. From the fifth round onwards, the reverse screening medium is upgraded to control peptide reverse screening magnetic beads.

6. The screening method according to claim 2, characterized in that: The primers used in step (4) for real-time quantitative PCR are the forward primer 5'-GCACGGACACAAGAACAAAG-3' and the reverse primer 5'-CAGAACAAAGGCAGCACAAG-3'.

7. The screening method according to claim 2, characterized in that: In step (5), the barcode forward primers used to distinguish samples from each round in the high-throughput sequencing are selected from the following sequences: 5'-gcaacaGCACGGACACAAGAACAAAG-3', 5'-gccaacGCACGGACACAAGAACAAAG-3', 5'-gcgaagGCACGGACACAAGAAAAAG-3', 5'-gctaatGCACGGACACAAGAAAAAG-3', 5'-ggaacgGCACGGACACAAGAAAAAG-3', 5'-ggtactGCACGGACACAAGACAAAG-3'.

8. The use of the nucleic acid aptamer according to claim 1 in the preparation of CaMKⅡα detection reagents or diagnostic kits.

9. The use of the nucleic acid aptamer according to claim 1 in the preparation of therapeutic drugs targeting CaMKⅡα-related diseases.

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