Cas13a protein ssDNA aptamer Cas13a-02 and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a Cas13a protein ssDNA nucleic acid aptamer Cas13a-02 and application of the Cas13a protein ssDNA nucleic acid aptamer Cas13a-02. The Cas13a protein ssDNA nucleic acid aptamer Cas13a-02 is obtained through six rounds of screening, and library affinity determination shows that along with progressive increase of screening rounds, the library binding capacity is higher, and the sixth round of library has very high affinity; according to the Cas13a protein ssDNA nucleic acid aptamer Cas13a-02, a high-throughput sequencing result is analyzed, 96 to-be-detected monoclone is selected, single-concentration primary screening is performed on the 96 to-be-detected monoclone through SPR, the binding capacity (binding value) of a plurality of monoclone strips is successfully obtained to be larger than 30 RU, it is preliminarily judged that the binding capacity of the monoclone strips is good, concentration gradient KD detection is further performed on the Cas13a-02, and KD of the Cas13a-02 is 2.497 nM.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a Cas13a protein ssDNA aptamer Cas13a-02 and its applications. Background Technology

[0002] Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules obtained through exponential enrichment ligand system evolution techniques. They can bind to target molecules with high specificity and affinity. Compared with traditional antibodies, nucleic acid aptamers have many advantages, such as simple chemical synthesis, high stability, ease of modification, and non-immunogenicity. Therefore, they show great application potential in fields such as biosensing, disease diagnosis, drug delivery, and targeted therapy.

[0003] As an important nuclease, Cas13a protein has attracted much attention in gene editing and nucleic acid detection. Detection technologies based on Cas13a protein have extremely high sensitivity and specificity, enabling rapid detection of trace amounts of nucleic acid. They have been widely used in pathogen detection, early cancer diagnosis, and other fields.

[0004] Therefore, developing ssDNA aptamers targeting the Cas13a protein has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a Cas13a protein ssDNA aptamer Cas13a-02 and its applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention: A Cas13a protein ssDNA aptamer, Cas13a-02.

[0007] The second technical solution of this invention: The above-mentioned method for screening the Cas13a protein ssDNA aptamer Cas13a-02 includes the following steps: 1) Immobilization of BSA protein with carboxyl magnetic beads; 2) Carboxyl magnetic beads immobilize Cas13a protein; 3) Screening; 4) Preparation of single chains.

[0008] Furthermore, the carboxyl magnetic beads immobilize the BSA protein, specifically including the following steps: ① Take carboxyl magnetic beads, wash them 4 times with ultrapure water, use a magnet to fish them out, and remove the supernatant; ② Take NHS and EDC, thaw at room temperature, add NHS to EDC, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ①, incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, and wash twice with DPBS. ③ Take BSA protein, add NaAC solution (pH=4.2), mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ②, incubate on a shaker at room temperature, use a magnet to fish, and remove the supernatant. ④ Take ethanolamine and add it to the carboxyl magnetic beads obtained in step ③. Incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, wash with DPBS 4 times, and label it as MB-BSA. Store it in a refrigerator at 4°C for later use.

[0009] Furthermore, the carboxyl magnetic beads immobilize the Cas13a protein, specifically including the following steps: ① Take carboxyl magnetic beads, wash them 4 times with ultrapure water, use a magnet to fish them out, and remove the supernatant; ② Take NHS and EDC, thaw at room temperature, add NHS to EDC, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ①, incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, and wash twice with DPBS. ③ Take Cas13a protein, add NaAC solution, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ②, incubate on a shaker at room temperature, use a magnet to fish, and remove the supernatant. ④ Add ethanolamine to the carboxyl magnetic beads obtained in step ③, incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, wash 4 times with DPBS, label it MB-Cas13a, and store it in a refrigerator at 4°C for later use.

[0010] Furthermore, the screening specifically includes the following steps: ① Take lib2-76nt library powder, centrifuge at 14000g for 10min, then add DPBS, vortex to dissolve, centrifuge at 14000g for 10min, and aliquot into PCR tubes; ② Place the PCR tube containing the lib2-76nt library from step ① into a PCR instrument for annealing. After annealing, immediately place it in an ice-water bath to equilibrate to room temperature. The reversion procedure is: 95℃ for 10 min; ③ Take the small peptide and add it to the lib2-76nt library that has undergone renaturation in step ②, and mix well; ④ Add the mixed solution from step ③ to the MB-BSA obtained in step 1), mix slowly by pipetting, incubate on a shaker at room temperature, and use a magnet to collect the solution. The supernatant is labeled as pool-. Then wash 4 times with 200 μL DPBS, and use a magnet to collect the solution each time. The supernatants from the 4 washes are labeled as wash1-, wash2-, wash3-, and wash4-, respectively. ⑤ Add the carboxyl magnetic beads obtained in step ④ to ultrapure water, boil in a water bath, use a magnet to fish, and label the supernatant as Elution-; ⑥ Add the pool- obtained in step ④ to the MB-Cas13a obtained in step 2), mix slowly by blowing, incubate on a shaker at room temperature, and then use a magnet to clean the mixture. After that, wash the mixture 4 times with DPBS and use a magnet to clean the mixture. The supernatants from the 4 washes are recorded as wash1+, wash2+, wash3+ and wash4+, respectively. ⑦ Add the carboxyl magnetic beads obtained in step ⑥ to ultrapure water, boil in a water bath, use a magnet to fish, and label the supernatant as Elution+; ⑧ Take an 8-tube Roche PCR tube, add 30 μL Q-PCR mix, 1 μL Elution- and 1 μL Elution+ to each well, and perform quantitative real-time PCR; The quantitative PCR program was as follows: 95℃ for 2 min, 95℃ for 0.5 min, 60℃ for 0.5 min, 72℃ for 0.5 min, 25 cycles.

[0011] Furthermore, the preparation of the single chain specifically includes the following steps: ① Add the PCR mix to the remaining Elution+ in step 3), transfer it to a centrifuge tube, mix well, then add EM90 oil, vortex to obtain an emulsion; ②Aliquot the emulsion obtained in step ① into PCR tubes, perform PCR amplification, and recover the ePCR product; The PCR program was: 95℃ for 2 min, 95℃ for 1 min, 60℃ for 1 min, 72℃ for 1 min, 25 cycles; ③ Transfer the ePCR product recovered in step ② to a centrifuge tube, fill it with n-butanol, mix well, centrifuge at 10000g for 10min, take the lower layer of ePCR product, transfer it to a centrifuge tube, add urea loading buffer, mix well, and heat in a PCR instrument at 95℃ for 10min. ④ Denaturing PAGE electrophoresis to separate single strands, n-butanol to concentrate ssDNA, and 3.5KD dialysis bag with DPBS overnight to dialyze ssDNA.

[0012] Furthermore, to enhance the accessibility of the document library, it needs to undergo six rounds of screening.

[0013] The second technical solution of this invention: The above-mentioned Cas13a protein ssDNA aptamer Cas13a-02 is used in the recognition, immobilization, enrichment of Cas13a protein and the construction of related nucleic acid detection or biosensing systems.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses a Cas13a protein ssDNA aptamer, Cas13a-02, obtained through six rounds of screening. Library affinity assays show that the library binding ability increases with each screening round, and the sixth-round library exhibits very strong affinity. This invention discloses a Cas13a protein ssDNA aptamer, Cas13a-02. By analyzing high-throughput sequencing results, 96 monoclonal antibodies were selected for testing. Single-concentration screening was performed using SPR, and several monoclonal antibodies with binding values ​​greater than 30 RU were successfully obtained. It was initially determined that these monoclonal antibodies had good binding ability. Further concentration gradient KD detection was performed on Cas13a-02, and its KD was 2.497 nM. Attached Figure Description

[0015] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a schematic diagram illustrating the screening principle of the present invention; Figure 2 This is a graph showing the retention rate of the six-round screening library in this invention. Figure 3 The graph shows the results of the library affinity assay. Figure 4 This is a graph showing the results of high-throughput sequencing. Figure 5 The binding curve of a single clone to the Cas13a protein; Figure 6 The binding curves of a single clone with other proteins carrying his-tag; Figure 7 The image shows the results of concentration gradient detection for monoclonal Cas13a-02. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0017] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Example 1 Screening of Cas13a protein ssDNA aptamer Cas13a-02 1. First round of screening 1) Immobilization of BSA protein with carboxyl magnetic beads ① Take 300μL of carboxyl magnetic beads, wash them 4 times with 200μL of ultrapure water, use a magnet to fish them out, and remove the supernatant; ② Take 100 μL NHS and 100 μL EDC, thaw at room temperature, add NHS to EDC, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ①, incubate on a shaker at room temperature for 20 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, shake the carboxyl magnetic beads well), magnetize, remove the supernatant, and wash twice with 200 μL DPBS; ③ Take 10 μL of BSA protein (concentration of 10 mg / mL), add 80 μL of NaAC solution (pH=4.2), mix well, and then add the mixed solution to the carboxyl magnetic beads obtained in step ②. Incubate on a shaker at room temperature for 60 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, shake the carboxyl magnetic beads well), and remove the supernatant by magnet fishing. ④ Take 100 μL of ethanolamine (concentration of 1 mol·L⁻¹) -1 Add the (pH=8.5) to the carboxyl magnetic beads obtained in step ③, incubate on a shaker at room temperature for 10 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, shake the carboxyl magnetic beads evenly), magnetize, remove the supernatant, wash 4 times with 200 μL DPBS, record as MB-BSA, and store in a refrigerator at 4℃ for later use. 2) Immobilization of Cas13a protein with carboxyl magnetic beads ① Take 50μL of carboxyl magnetic beads, wash them 4 times with 200μL of ultrapure water, use a magnet to fish them out, and remove the supernatant; ② Take 50 μL NHS and 50 μL EDC, thaw at room temperature, add NHS to EDC, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ①, incubate on a shaker at room temperature for 20 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, shake the carboxyl magnetic beads well), magnetize, remove the supernatant, and wash twice with 200 μL DPBS; ③ Take 5 μL of Cas13a protein (concentration of 2 mg / mL), add 10 μL of NaAC solution (pH=4.2), mix well, and then add the mixed solution to the carboxyl magnetic beads obtained in step ②. Incubate on a shaker at room temperature for 60 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, shake the carboxyl magnetic beads well), and remove the supernatant by magnet fishing. ④ Take 100 μL of ethanolamine (concentration of 1 mol·L⁻¹) -1 Add (pH=8.5) to the carboxyl magnetic beads obtained in step ③, incubate on a shaker at room temperature for 10 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, shake the carboxyl magnetic beads evenly), magnetize, remove the supernatant, wash 4 times with 200 μL DPBS, record as MB-Cas13a, and store in a refrigerator at 4℃ for later use. 3) Screening ① Take 10 μL of lib2-76nt library dry powder (concentration of 10 μmol·L⁻¹) -1 Centrifuge at 14000g for 10 min, then add 137 μL of DPBS, vortex to dissolve, centrifuge at 14000g for 10 min, and aliquot into PCR tubes; The library information for lib2-76nt is shown in Table 1. Table 1 Document Information

[0022] ② Place the PCR tube containing the lib2-76nt library from step ① into a PCR instrument for annealing. After annealing, immediately incubate in an ice-water bath for 5 minutes to equilibrate to room temperature. The reversion procedure is: 95℃ for 10 min; ③ Take 10 μL of 6His peptide (concentration of 0.1 mg / mL) and add it to the lib2-76nt library that has undergone renaturation in step ②, and mix well; ④ Add the mixed solution from step ③ to 50 μL of MB-BSA obtained in step 1), mix slowly by pipetting, and incubate on a shaker at room temperature for 40 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, shake the carboxyl magnetic beads to mix), and use a magnet to collect the supernatant as pool-. Then wash 4 times with 200 μL of DPBS, and use a magnet to collect the supernatant from each wash. The supernatants from the 4 washes are recorded as wash1-, wash2-, wash3-, and wash4-, respectively. ⑤ Add the carboxyl magnetic beads obtained in step ④ to 200 μL of ultrapure water, boil in a water bath for 10 min, and use a magnet to collect the supernatant. Record the supernatant as Elution-. ⑥ Add the pool- obtained in step ④ to 50 μL of MB-Cas13a obtained in step 2), mix slowly by pipetting, and incubate on a shaker at room temperature for 40 min (if the carboxyl magnetic beads aggregate during the incubation on a shaker at room temperature, the carboxyl magnetic beads need to be shaken evenly), and then use a magnet to pick up the supernatant. After that, wash 4 times with 200 μL of DPBS, and pick up the supernatant with a magnet each time. The supernatant from the 4 washes is recorded as wash1+, wash2+, wash3+ and wash4+, respectively. ⑦ Add the carboxyl magnetic beads obtained in step ⑥ to 100 μL of ultrapure water, boil in a water bath for 10 min, and use a magnet to collect the supernatant. Record the supernatant as Elution+. ⑧ Take an 8-tube Roche PCR tube, add 30 μL Q-PCR mix, 1 μL Elution- and 1 μL Elution+ to each well, and perform quantitative real-time PCR; The quantitative PCR program was as follows: 95℃ for 2 min, 95℃ for 0.5 min, 60℃ for 0.5 min, 72℃ for 0.5 min, 25 cycles; The preparation of the Q-PCR mix is ​​shown in Table 2. Table 2 Preparation of Q-PCR mix

[0023] 4) Preparation of single chains ① Add 2 mL of PCR mix to the remaining Elution+ in step 3), transfer to a 50 mL centrifuge tube, mix well, then add 8 mL of EM90 oil, vortex to obtain an emulsion; The preparation of the ePCR mix is ​​shown in Table 3. Table 3 Preparation of PCR mix

[0024] ② Aliquot the emulsion obtained in step ① into PCR tubes, 90 μL per tube, perform PCR amplification, and recover the ePCR product; The PCR program was: 95℃ for 2 min, 95℃ for 1 min, 60℃ for 1 min, 72℃ for 1 min, 25 cycles; ③ Transfer the ePCR product recovered in step ② to a 10 mL centrifuge tube, fill it with n-butanol, mix well, centrifuge at 10000g for 10 min, take 90 μL of the lower layer ePCR product, transfer it to a centrifuge tube, add 100 μL of urea loading buffer, mix well, and heat the PCR instrument at 95℃ for 10 min. ④ Denaturing PAGE electrophoresis to separate single strands, n-butanol to concentrate ssDNA, and 3.5KD dialysis bag with DPBS overnight to dialyze ssDNA; 2. Multiple rounds of screening The specific screening steps are the same as in step 1, the only difference being the screening criteria shown in Table 4; Table 4 Filtering Criteria

[0025] Example 1: Screening principle as follows Figure 1 As shown.

[0026] The retention rates of the six-round screening library in Example 1 are shown in Table 5. Table 5 Library Retention Rate

[0027] Example 1: Analysis of Retention Rate of Library After Six Rounds of Screening (as follows) Figure 2 As shown; Depend on Figure 2 It can be seen that the library gradually enriched itself in multiple rounds of screening, with the proportion of high-affinity sequences increasing in each round, reaching the optimal enrichment state in the sixth round, proving that the screening system is effective and the affinity is significantly improved.

[0028] Effect verification I. SPR test for library affinity 1) Chip coupling ① Replace the SPR chip with a new CM5 chip and replace the running buffer with PBS; ② Use a NaOH solution containing SDS (concentration of 50 mmol·L⁻¹) -1 The chip was cleaned twice, then rinsed with NaOH solution (concentration 50 mmol·L⁻¹). -1 Clean once; ③ Take NHS and EDC, thaw at room temperature, mix them in equal volumes, and inject via SPR (injection flow rate 5 μL / min, injection time 10 min). ④ The Cas13a protein was treated with sodium acetate (concentration 10 mmol·L⁻¹). -1 Dilute to 50 μg / mL (pH=4.0), inject via SPR (injection flow rate 5 μL / min, injection time 2 min), and couple to Fc4 channel; ⑤ The CD38 protein (with a his-tag) was treated with sodium acetate (concentration of 10 mmol·L⁻¹). -1 Dilute to 50 μg / mL (pH=4.0), inject via SPR (injection flow rate 5 μL / min, injection time 2 min), and couple to Fc2 channel; ⑥ Take 100 μL of ethanolamine (concentration 1 mol·L⁻¹) -1 (pH=8.5), the chip was sealed, and SPR was used for injection (injection flow rate 5 μL / min, injection time 5 min). 2) Library affinity determination ① Dilute the PBS, pool6, pool4, and pool0 (synthetic libraries) obtained from six screenings in Example 1 to 500 nmol·L⁻¹ with DPBS. -1 95℃ for 10 minutes, then immediately ice water bath for 5 minutes; ②SPR injection (Fc2-1): Flow rate 10 μL / min, injection (PBS) for 3 min, wait 2 min, regeneration (1 mol·L⁻¹) - 1 NaCl) 1 min; ③ SPR injection (Fc2-1): Flow rate 10 μL / min, injection (pool6) 3 min, wait 2 min, regeneration (1 mol·L⁻¹) - 1 NaCl) 1 min; ④SPR injection (Fc2-1): Flow rate 10 μL / min, injection (pool4) 3 min, wait 2 min, regeneration (1 mol·L⁻¹) - 1 NaCl) 1 min; ⑤ SPR injection (Fc2-1): Flow rate 10 μL / min, injection (pool0) 3 min, wait 2 min, regeneration (1 mol·L⁻¹) - 1 NaCl) 1 min.

[0029] 3) Analyze the SPR measurement results ① The Cas13a protein conjugation amount was 7075 RU, and the CD38 protein conjugation amount was 2184 RU. ② The selected libraries pool4 and pool6 showed strong binding to Cas13a protein but no binding to CD38 protein. ③pool0 has a certain degree of weak binding to both CD38 and Cas13a proteins. ④ The library has a very strong user-friendly appeal. Library affinity assay results are as follows Figure 3 As shown; Depend on Figure 3 It can be seen that as the number of screening rounds increases, the document integration capability becomes stronger, and the sixth round of documents has a very strong affinity.

[0030] II. High-throughput sequencing 1) Add 10 μL of single-stranded template (concentration of 0.5 μmol·L⁻¹) -1 Add the contents to 400 μL of PCR mix and aliquot 100 μL into eight-tube PCR amplification in each well using a Bio-rad instrument. The PCR amplification program was as follows: 95℃ pre-denaturation for 1 min, 95℃ denaturation for 60 s, 60℃ for 60 s, 72℃ for 60 s, 25 cycles; The PCR amplification tag primer information is shown in Table 6; Table 6. Tag Primer Information

[0031] 2) After PCR amplification, 20 μL of each sample was taken for preservation and electrophoresis detection, and the rest was mixed and aliquoted into two 15 mL centrifuge tubes and concentrated with n-butanol to a volume of about 100 μL. 3) Recover the double strands using the UNIQ-10 oligonucleotide purification kit: ① High salt adsorption Sample: Binding buffer = 1:10 (0.1 mL sample, 1 mL binding buffer), mix well, add 550 μL of sample to each of the two adsorption columns, centrifuge at 8000 rpm for 1 min, and repeatedly load the filtrate onto the column and centrifuge three times. Wash the adsorption column with washing buffer (check that the correct amount of anhydrous ethanol has been added to the wash solution before first use) 500 μL each time, centrifuge at 10000 rpm for 1 min, and repeatedly load the filtrate onto the sample and centrifuge three times; discard the waste liquid in the collection tube, put the adsorption column into the same collection tube, and centrifuge at 14000 rpm for 2 min. Transfer the mixture to the adsorption column, let it stand at room temperature for 2 minutes, centrifuge at 8000 rpm for 2 minutes, discard the liquid in the collection tube, and put the adsorption column into the same collection tube. ② Low-salt elution Place the adsorption column into a clean 1.5 mL centrifuge tube, add 100 μL of Elution buffer (Tris-EDTA, Elution buffer is 2.5 mM Tris-HCl, pH=8.5; TE or water (pH>7.0) can be used instead of Elution buffer; preheating to 60℃ can further improve the yield), let stand at room temperature for 5 min, centrifuge at 12000 rpm for 1 min, repeat the elution twice, and measure the concentration; after centrifugation, add the liquid from adsorption columns 2, 3, and 4 to adsorption column 1 and elute again to obtain DNA, i.e., the monoclonal detection sample; The obtained dsDNA solution was designated Cas13a, with a concentration of 738 μg / ml. The high-throughput sequencing results are as follows: Figure 4 As shown in the figure, from left to right: aerosol, p2, p3, p4, marker, and Cas13a.

[0032] III. Selection of Synthesized Monoclonal Cells 1) The obtained raw sequencing results were analyzed using software to extract sequences according to the tag primers, resulting in a total of 1,277,723 sequences, of which 537,077 were sequence types; Note: The extracted result is a random sequence, i.e. the primer regions at both ends have been removed. When synthesizing, primer sequences need to be added before and after the extracted sequence. 2) Analyze the obtained sequences, select the sequences to be tested, synthesize single clones, the library is a closed loop library, and when synthesizing single clones, remove the open loop regions at both ends of the library; 3) Information on the synthesized monoclonal antibodies is shown in Table 7; Table 7 Monoclonal Information

[0033] IV. Monoclonal Single-Concentration Binding Ability Detection and Specificity Detection 1) Chip coupling ① Replace the SPR chip with a new CM5 chip and replace the running buffer with PBS; ② Use a NaOH solution containing SDS (concentration of 50 mmol·L⁻¹) -1 The chip was cleaned twice, then rinsed with NaOH solution (concentration 50 mmol·L⁻¹). -1 Clean once; ③ Take NHS and EDC, thaw at room temperature, mix them in equal volumes, and inject via SPR (injection flow rate 5 μL / min, injection time 10 min) to activate Fc3 and Fc4 channels; ④ The Cas13a protein was treated with sodium acetate (concentration 10 mmol·L⁻¹). -1 Dilute to 20 μg / mL (pH=4.0), inject via SPR (injection flow rate 5 μL / min, injection time 10 min), and couple to the Fc4 channel. The Cas13a protein coupling amount is 5882 RU. ⑤ Take 100 μL of ethanolamine (concentration 1 mol·L⁻¹) -1 (pH=8.5), the chip was sealed, and the Fc3 and Fc4 channels were sealed by SPR injection (injection flow rate 5 μL / min, injection time 5 min). 2) Monoclonal binding capacity assay ① Dilute the monoclonal antibody to 1 μmol·L⁻¹ using PBS. -1 200 μL, set the injection program; ② First inject PBS at a flow rate of 30 μL / min; inject for 3 min, wait for 1 min, then regenerate (2 mol·L⁻¹). -1 NaCl) 1 min; ③ Inject samples sequentially according to the single-clone sequence number: flow rate 30 μL / min; injection time 3 min, wait 1 min, regeneration (2 mol·L⁻¹) - 1 NaCl) 1 min; The binding curves of monoclonal antibodies to Cas13a protein are shown below. Figure 5 As shown; The binding curves of monoclonal antibodies with other proteins carrying his-tag are as follows: Figure 6 As shown; Depend on Figure 5 and Figure 6 It can be seen that the monoclonal antibody binds strongly only to Cas13a, but not to other His-tag proteins, indicating that it has high specificity and stable high affinity.

[0034] The binding and stability values ​​of a single clone are shown in Table 8. Table 8 Binding and Stability Values

[0035] V. Monoclonal Concentration Gradient KD Detection 1) Chip coupling ① Replace the SPR chip with a new CM5 chip and replace the running buffer with PBS; ② Use a NaOH solution containing SDS (concentration of 50 mmol·L⁻¹) -1 The chip was cleaned twice, then rinsed with NaOH solution (concentration 50 mmol·L⁻¹). -1 Clean once; ③ Take NHS and EDC, thaw at room temperature, mix them in equal volumes, and inject via SPR (injection flow rate 5 μL / min, injection time 5 min) to activate Fc1 and Fc3 channels; ④ The Cas13a protein was treated with sodium acetate (concentration 10 mmol·L⁻¹). -1 Dilute to 50 μg / mL (pH=4.0), inject via SPR (injection flow rate 10 μL / min, injection time 5 min), and couple to the Fc2 channel. The Cas13a protein coupling amount is 6811.5 RU. ⑤ Take 100 μL of ethanolamine (concentration 1 mol·L⁻¹) -1 (pH=8.5), the chip was sealed, and SPR was used for injection (injection flow rate 5 μL / min, injection time 5 min). ⑥Fc4 is linked to other proteins with his-tags; 2) Monoclonal Concentration Gradient Detection ① Dilute monoclonal Cas13a-02 to 1000 nmol·L⁻¹ using PBS -1 500 nmol·L -1 250 nmol·L -1 125 nmol·L -1 62.5 nmol·L -1 and 31.25 nmol·L -1 There are 6 concentrations in total, 200 μL for each concentration, and the injection program is set. ② First-pass PBS: Channels Fc3-Fc1, flow rate 30 μL / min; injection 3 min, wait 5 min, regeneration (2 mol·L⁻¹) - 1 NaCl) 1 min; ③ Inject samples sequentially according to the single-clone sample number: channels Fc3-Fc1 and Fc4-Fc1, flow rate 30 μL / min; inject for 3 min, wait for 2 min, regenerate (2 mol·L⁻¹) -1 NaCl) 1 min.

[0036] The results of monoclonal Cas13a-02 concentration gradient detection are as follows: Figure 7 As shown; Depend on Figure 7It can be seen that the concentration gradient KD of monoclonal Cas13a-02 is 2.497 nM.

[0037] Based on the screening and identification data from the project, and with reference to the accompanying figures, it is evident that the binding reaction between the Cas13a-02 monoclonal molecule and the Cas13a protein exhibits high specificity, strong stability, and nanomolar-level high affinity. The accompanying figures clearly show a concentration-dependent binding curve, with a fitted dissociation constant KD value of 2.497 nM, meeting the screening criteria for high-affinity molecules. The binding signal (binding value) detected by SPR far exceeds the 30 RU screening threshold, and the binding-dissociation curve shows no significant drift, indicating extremely low non-specific binding and excellent complex stability. Furthermore, the enrichment trend chart of the library in the accompanying figures shows that Cas13a-02 was continuously enriched in six rounds of screening. Combined with the project's results of "increasing retention rate in each round of positive screening, small library size, and accurate determination of affinity," this further validates its superior position in enriched libraries. The sequence shows high compatibility with the Cas13a protein binding site, with no risk of cross-reaction, making it a highly reliable Cas13a binding molecule for subsequent application development.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A Cas13a protein ssDNA aptamer, Cas13a-02.

2. A method for screening the Cas13a protein ssDNA aptamer Cas13a-02 as described in claim 1, characterized in that, The screening method includes the following steps: 1) Immobilization of BSA protein with carboxyl magnetic beads; 2) Carboxyl magnetic beads immobilize Cas13a protein; 3) Screening; 4) Preparation of single chains.

3. The screening method according to claim 2, characterized in that, The immobilization of BSA protein by the carboxyl magnetic beads specifically includes the following steps: ① Take carboxyl magnetic beads, wash them 4 times with ultrapure water, use a magnet to fish them out, and remove the supernatant; ② Take NHS and EDC, thaw at room temperature, add NHS to EDC, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ①, incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, and wash twice with DPBS. ③ Take BSA protein, add NaAC solution (pH=4.2), mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ②, incubate on a shaker at room temperature, use a magnet to fish, and remove the supernatant. ④ Take ethanolamine and add it to the carboxyl magnetic beads obtained in step ③. Incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, wash with DPBS 4 times, and label it as MB-BSA. Store it in a refrigerator at 4°C for later use.

4. The screening method according to claim 2, characterized in that, The carboxyl magnetic beads immobilize the Cas13a protein, specifically including the following steps: ① Take carboxyl magnetic beads, wash them 4 times with ultrapure water, use a magnet to fish them out, and remove the supernatant; ② Take NHS and EDC, thaw at room temperature, add NHS to EDC, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ①, incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, and wash twice with DPBS. ③ Take Cas13a protein, add NaAC solution, mix well, then add the mixed solution to the carboxyl magnetic beads obtained in step ②, incubate on a shaker at room temperature, use a magnet to fish, and remove the supernatant. ④ Add ethanolamine to the carboxyl magnetic beads obtained in step ③, incubate on a shaker at room temperature, use a magnet to pick up the beads, remove the supernatant, wash 4 times with DPBS, label it MB-Cas13a, and store it in a refrigerator at 4°C for later use.

5. The screening method according to claim 2, characterized in that, The screening process specifically includes the following steps: ① Take lib2-76nt library powder, centrifuge at 14000g for 10min, then add DPBS, vortex to dissolve, centrifuge at 14000g for 10min, and aliquot into PCR tubes; ② Place the PCR tube containing the lib2-76nt library from step ① into a PCR instrument for annealing. After annealing, immediately place it in an ice-water bath to equilibrate to room temperature. The reversion procedure is: 95℃ for 10 min; ③ Take the small peptide and add it to the lib2-76nt library that has undergone renaturation in step ②, and mix well; ④ Add the mixed solution from step ③ to the MB-BSA obtained in step 1), mix slowly by pipetting, incubate on a shaker at room temperature, and use a magnet to collect the solution. The supernatant is labeled as pool-. Then wash 4 times with 200 μL DPBS, and use a magnet to collect the solution each time. The supernatants from the 4 washes are labeled as wash1-, wash2-, wash3-, and wash4-, respectively. ⑤ Add the carboxyl magnetic beads obtained in step ④ to ultrapure water, boil in a water bath, use a magnet to fish, and label the supernatant as Elution-; ⑥ Add the pool- obtained in step ④ to the MB-Cas13a obtained in step 2), mix slowly by blowing, incubate on a shaker at room temperature, and then use a magnet to clean the mixture. After that, wash the mixture 4 times with DPBS and use a magnet to clean the mixture. The supernatants from the 4 washes are recorded as wash1+, wash2+, wash3+ and wash4+, respectively. ⑦ Add the carboxyl magnetic beads obtained in step ⑥ to ultrapure water, boil in a water bath, use a magnet to fish, and label the supernatant as Elution+; ⑧ Take an 8-tube Roche PCR tube, add 30 μL Q-PCR mix, 1 μL Elution- and 1 μL Elution+ to each well, and perform quantitative real-time PCR; The quantitative PCR program was as follows: 95℃ for 2 min, 95℃ for 0.5 min, 60℃ for 0.5 min, 72℃ for 0.5 min, 25 cycles.

6. The screening method according to claim 2, characterized in that, The preparation of the single chain specifically includes the following steps: ① Add the PCR mix to the remaining Elution+ in step 3), transfer it to a centrifuge tube, mix well, then add EM90 oil, vortex to obtain an emulsion; ②Aliquot the emulsion obtained in step ① into PCR tubes, perform PCR amplification, and recover the ePCR product; The PCR program was: 95℃ for 2 min, 95℃ for 1 min, 60℃ for 1 min, 72℃ for 1 min, 25 cycles; ③ Transfer the ePCR product recovered in step ② to a centrifuge tube, fill it with n-butanol, mix well, centrifuge at 10000g for 10min, take the lower layer of ePCR product, transfer it to a centrifuge tube, add urea loading buffer, mix well, and heat in a PCR instrument at 95℃ for 10min. ④ Denaturing PAGE electrophoresis to separate single strands, n-butanol to concentrate ssDNA, and 3.5KD dialysis bag with DPBS overnight to dialyze ssDNA.

7. The screening method according to claim 2, characterized in that, To enhance the accessibility of the document library, it needs to undergo six rounds of screening.

8. The application of the Cas13a protein ssDNA aptamer Cas13a-02 as described in claim 1 in the recognition, immobilization, enrichment of Cas13a protein and the construction of related nucleic acid detection or biosensing systems.

Images