A nucleic acid aptamer that specifically binds to the surface of the heart, its preparation method, and its application.

Abstract

This invention provides a nucleic acid aptamer that specifically binds to the surface of the heart, its preparation method, and its application, belonging to the field of molecular biology. The nucleic acid aptamer sequence of this invention is the DNA sequence shown in SEQ ID NO: 1. This invention also includes single-stranded DNA molecules with the same specific recognition function obtained by modifying, deleting, adding, or substituting bases on the nucleic acid aptamer sequence of this invention. The nucleic acid aptamer obtained by this invention possesses high specificity and affinity.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a nucleic acid aptamer that specifically binds to the surface of the heart, its preparation method, and its application. Background Technology

[0002] Nucleic acid aptamers are nucleic acid molecules with specific structures and functions obtained through in vitro screening technology (SELEX), capable of binding to target substances with high specificity and selectivity. Compared with traditional recognition molecules, nucleic acid aptamers have many advantages, such as high affinity, strong specificity, good biocompatibility, and ease of synthesis and modification, and have attracted widespread attention in fields such as biomedicine and food safety. In particular, nucleic acid aptamers are widely used in scientific research as excellent drug targeting carriers; however, currently, there are no nucleic acid aptamers specifically targeting the surface of the heart. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a nucleic acid aptamer that specifically binds to the surface of the heart and can specifically bind to cardiomyocytes.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] The present invention provides a nucleic acid aptamer that specifically binds to the surface of the heart, the sequence of which is shown in SEQ ID NO: 1.

[0006] Preferably, a position on the nucleotide sequence of the nucleic acid aptamer is phosphorylated, oxymethylated, methylated, aminoized, thiolated, fluorinated, or isotopized.

[0007] Preferably, the nucleotide sequence of the nucleic acid aptamer is coupled with a fluorescent group, a quenching group, biotin, nanomaterials, a thiol group, an amino group, or an enzyme.

[0008] This invention provides a nucleic acid aptamer that specifically binds to the surface of the heart, and a single-stranded DNA molecule with the same specific recognition function obtained by deleting, adding or replacing nucleotides in the above nucleic acid aptamer.

[0009] The present invention also provides a method for screening the above-mentioned nucleic acid aptamers, using single-stranded oligonucleotide fragments as screening materials, H9C2 cells as positive screening targets, and HUVEC cells as negative screening targets.

[0010] Preferably, the single-chain oligonucleotide fragment sequence is SEQ ID NO: 2.

[0011] This invention provides a nucleic acid aptamer derivative that specifically binds to the surface of the heart. The derivative is obtained by modifying the above-mentioned nucleic acid aptamer by amination, thiolation or isotopization, and has the same function as the nucleic acid aptamer.

[0012] The present invention also provides the application of the above-mentioned nucleic acid aptamers or the above-mentioned nucleic acid aptamer derivatives in cardiac targeted research.

[0013] Preferably, the cardiac-targeting drug carrier is prepared using the nucleic acid aptamer or the nucleic acid aptamer derivative.

[0014] The present invention also provides the application of the above-mentioned nucleic acid aptamers or the above-mentioned nucleic acid aptamer derivatives in the preparation of cardiac targeted drugs.

[0015] The beneficial effects of this invention are:

[0016] This invention is the first to obtain nucleic acid aptamers targeting the surface of the heart. The invention utilizes cell-SELEX technology and flow cytometry to obtain aptamer candidates, selects nucleic acid aptamers through flow cytometry and confocal fluorescence microscopy, and further verifies the high specificity and affinity of the obtained nucleic acid aptamers using flow cytometry and confocal fluorescence microscopy, ensuring the practicality of the obtained nucleic acid aptamers. Attached Figure Description

[0017] Figure 1 Electrophoresis diagram of PCR products;

[0018] Figure 2 Flow cytometry is used to monitor the screening process;

[0019] Figure 3 Schematic diagram of sequencing results;

[0020] Figure 4 The binding of the three sequences H1-3 (from left to right) to H9C2;

[0021] Figure 5 Schematic diagram of the secondary structure of nucleic acid aptamer H3;

[0022] Figure 6 Analysis of Kd values ​​of H3 at 4℃ (left figure) and 37℃ (right figure);

[0023] Figure 7 The experimental results of H3 were observed using a confocal microscope at 4℃ and 37℃, respectively.

[0024] Figure 8 The binding of different cells to aptamer H3;

[0025] Figure 9 Serum stability test results. Detailed Implementation

[0026] The present invention provides a nucleic acid aptamer that specifically binds to the surface of the heart. The sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1, consisting of 68 bases, specifically 5'-CACGGATCCTGACAAGACACGATAATCTGTGTTGGACACAGTCTGTTG TCATGGGCCAGCTCCGTCCG-3'.

[0027] This invention also provides a method for screening the aforementioned nucleic acid aptamers: using a 10OD single-stranded oligonucleotide fragment library as screening material, two rounds of screening and amplification are first performed with the positive screening target, rat cardiomyocytes (H9C2). This ensures the abundance of the target fragment while minimizing the loss of ssDNA (target fragment) that can bind to the target, making the screening results as reliable as possible. A negative screening process is then added to give the screened ssDNA better specificity. Considering the potential subsequent applications of the screening results, the negative screening target is human umbilical vein endothelial cells (HUVECs).

[0028] The single-stranded oligonucleotide fragment sequence described in this invention is shown in SEQ ID NO: 2, specifically 5′-CACGGATCCTGACAAG-(N40)-CAGCTCCGTCCG-3′, which consists of 68 bases. The two ends are fixed primer-binding regions of 28 bases each, corresponding to complementary primer sequences, and the middle is a random region of 40 bases, which is the region that binds to the target.

[0029] The nucleic acid aptamer of the present invention further includes a nucleic acid aptamer sequence obtained by phosphorylation, oxygen methylation, methylation, aminoation, thiolation, fluorination, or isotopization at a certain position on the nucleotide sequence shown in SEQ ID NO: 1. Preferably, 2′-O-methyl modification is used.

[0030] The nucleotide sequence of the nucleic acid aptamer described in this invention may also be coupled with a fluorescent group, a quenching group, biotin, nanomaterials, a thiol group, an amino group, or an enzyme.

[0031] The present invention also provides a nucleic acid aptamer that specifically binds to the surface of the heart, and a single-stranded DNA molecule with the same specific recognition function obtained by deleting, adding or replacing nucleotides in the nucleic acid aptamer.

[0032] The present invention also provides nucleic acid aptamer derivatives that specifically bind to the surface of the heart, which are nucleic acid aptamer derivatives with the same function as the nucleic acid aptamer described in the present invention, obtained by amylation, thiolation or isotopization modification of the nucleic acid aptamer described in the present invention.

[0033] The present invention also provides the application of the nucleic acid aptamer or nucleic acid aptamer derivative in cardiac targeted research, preferably using the nucleic acid aptamer or the nucleic acid aptamer derivative to prepare cardiac targeted drug carriers.

[0034] The present invention also provides the application of the nucleic acid aptamer or nucleic acid aptamer derivative in the preparation of cardiac targeted drugs.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Unless otherwise specified, the following examples are all conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0037] Example 1

[0038] This embodiment provides a method for screening nucleic acid aptamers:

[0039] (1) Ordered sequences DL, FS, LT, FP, RP1, and RP2 from Sangon Biotech (Shanghai) Co., Ltd. (except for DL, which is 10 OD, the rest are 2 OD).

[0040] DL (SEQ ID NO: 2) is a DNA library; FS (SEQ ID NO: 3) is a fluorescent substrate with the sequence 5'-ACTCTTCCTAGCTATGGTTCGATCAAGA-3', where the 13th T base is labeled with the FAM fluorescent group and the 15th T base is labeled with the quencher group DABCYL; LT (SEQ ID NO: 4) is a ligation template with the sequence 5'-CTAGGAAGAGTCGGACGGAGCTG-3', used to help phosphorylated FS ligate to DL; FP (SEQ ID NO: 5) is a front primer with the sequence 5'-CACGGATCCTGACAAG-3', consistent with the binding region of the first half of the DL primer; RP1 (SEQ ID NO: 6) is the back primer 1 with the sequence 5'-CGGACGGAGCTG-3', consistent with the binding region of the second half of the DL primer; RP2 (SEQ ID NO: 6) is the second primer 1 with the sequence 5'-CGGACGGAGCTG-3', consistent with the binding region of the second half of the DL primer; NO:7) is the second back primer, with the sequence 5'-AAAAAAAAAAAAAAAAAAAAAAACGGACGGAGCTG-3', where positions 20 and 21 are linked by S9 (a triethylene Glycol), which guides the intermediate modification iSP9 during primer synthesis. The half of the iSP9 is complementary to the latter half of the DL, allowing 20 adenine deoxyribonucleotides to be added to the complementary fragment of the target fragment, resulting in a difference in molecular weight between the two fragments, which can then be separated by denaturing polyacrylamide gel electrophoresis.

[0041] (2) First round of screening: 10 OD of DL was dissolved in Binding Buffer and placed on ice until ready for use. The cells were then cultured in 100 mm × 20 mm culture dishes until 90% H9C2 cells (positive screening target) were reached. The culture medium was discarded, and the cells were washed with Washing Buffer. The prepared Binding Buffer solution was added, and the cells were incubated at 4°C for 1 hour (50 rpm). The incubation solution was then discarded, and the cells were washed with Washing Buffer to remove and recover the DL bound to the surface of H9C2 cells. The Washing Buffer was discarded, and 500 ml of DEPC was added to the culture dish. H2O, scrape adherent cells off with a clean, ready-to-use cell scraper (operate on ice), collect the suspension into a 1.5 ml de-enzyme EP tube, place it in a preheated 95°C metal bath, remove the EP tube after 15 min, cool it on ice, then transfer it to a high-speed centrifuge pre-cooled to 4°C, balance the mixture, centrifuge at 13000 rpm for 10 min at 4°C, remove the EP tube, and transfer the supernatant to a 1.5 ml de-enzyme EP tube. The supernatant contains DL bound to the surface of H9C2 cells.

[0042] PCR amplification was performed twice: First round PCR: 1 μL template (DNA recovered before PCR amplification, DL), 0.5 μL front primer (FP), 0.5 μL back primer 1 (RP1), 12.5 μL 2×Taq MasterMix, 10.5 μL DEPC H2O; Second round PCR: 1 μL template (product of first round PCR), 0.5 μL front primer (FP), 0.5 μL back primer 2 (RP2), 12.5 μL 2×Taq MasterMix, 10.5 μL DEPC H2O. The amplification products were verified by agarose gel electrophoresis. Residual amplification products were separated by denatured PAGE, and the single-stranded DNA in the upper band (DL1, i.e., target fragment 1) was recovered for later use.

[0043] (3) Second round of screening: Replace DL with DL1 and repeat the first round of screening in step (2) to obtain DL2, and determine the concentration as 101.3 ng / μl.

[0044] (4) Third round of screening: Dissolve FS and LT in an appropriate amount of DEPC Water, and then perform 5' end phosphorylation of FS according to the concentration of DL2:

[0045] Table 1. FS 5' end phosphorylation system (total volume 50 μL)

[0046]

[0047] The criteria for determining A are as follows: The amount of DL added to the ligation reaction system in each round is maintained at 50 pmol. Based on this, the amount of FS in each round is determined according to the ratio of DL to FS phosphorylation product of 1:1.25, ensuring that the amount of FS phosphorylation product is greater than that of DL during the ligation reaction. Mix at low speed, react in a 37°C metal bath for 30 min, inactivate the enzyme in a 95°C metal bath for 5 min, and then allow to stand for 5 min until it returns to room temperature.

[0048] DL2, the phosphorylated product of FS, and LT were linked together in a molar ratio of DL:FS:LT = 1:1.25:1.5.

[0049] Table 2. Connection reaction system (total volume 100 μL)

[0050]

[0051] The criteria for determining B are as follows: the amount of DL added to the linkage reaction system in each round is maintained at 50 pmol; LT is maintained at 1 μL and is not varied proportionally; the mixture is stirred at low speed and reacted at 22°C for 2 h.

[0052] The ligation product was purified and recovered by denature PAGE, dissolved in Binding Buffer at a concentration of 50 pmol, and incubated on ice. HUVECs (negative screening target) cultured to 90% confluence in 60 mm × 15 mm culture dishes were then removed from the culture medium, washed with Washing Buffer, and the prepared Binding Buffer solution was added. The cells were incubated at 4°C with shaking for 1 hour (50 rpm), the incubation solution was recovered, and the cells were incubated on ice. H9C2 cells (positive screening target) cultured to 90% confluence in 100 mm × 20 mm culture dishes were then removed from the culture medium, washed with Washing Buffer, and the prepared incubation solution was added. The cells were incubated at 4°C with shaking for 1 hour (50 rpm), the incubation solution was discarded, and the cells were washed with Washing Buffer to remove and recover DL2 bound to the surface of H9C2 cells (method as above). PCR amplification was performed twice (method as above), and the amplification products were verified by agarose gel electrophoresis. Figure 1 (Lane 1 is DEPC H2O; Lane 2 is low molecular weight marker; Lane 3 is the first round PCR product, fragment size 68bp; Lane 4 is the second round PCR product, fragment size 88bp). After determining that the size and purity of the recovered PCR products are appropriate, the remaining amplification products are separated by denature PAGE, and the single-stranded DNA in the upper band is recovered for later use (DL3).

[0053] (5) Fourth to tenth rounds of screening: Subsequent screening steps are the same as in the third round. The ligation product refers to the combination of DL and FS in each round. Starting from the fourth round, reduce the number of cells for positive screening (use 100mm×20mm culture dishes for rounds 1-3, and 60mm×15mm culture dishes for round 4), and increase the number of cells for negative screening (use 60mm×15mm culture dishes for rounds 1-3, and 100mm×20mm culture dishes for round 4). At the same time, extend the washing time for peeling and recovering DL and increase the amount of Washing Buffer in each round, reduce the incubation time for positive screening, and increase the serum content in the Binding Buffer during positive screening. The screening conditions are shown in Table 1. The negative screening conditions remain unchanged in each round (incubation time 1h, serum content in Binding Buffer 10%).

[0054] Table 1 Changes in screening criteria for each round

[0055]

[0056]

[0057] The sequences of each DL stage (DL1 to DL10) are uncertain. As the number of screening rounds increases, the sequences with strong affinity and specificity to the target H9C2 in each DL stage are enriched, while the remaining sequences are gradually removed.

[0058] (6) The screening process was detected by flow cytometry, and the screening process was monitored by flow cytometry. Figure 2 To ensure the selection direction is correct, Figure 2 The left side shows that as the number of screening rounds increases, the binding ability of the FAM-labeled enriched DNA library to H9C2 becomes stronger. The black, red, green, and blue parts are the flow cytometry results of the binding of the random library, the 8th round, the 9th round, and the 10th round enriched DNA library to H9C2, respectively. Figure 2 The right side shows the binding of the corresponding DNA library to HUVEC.

[0059] (7) The DL10 obtained in the tenth round was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results are as follows: Figure 3 . Figure 3 In the sequence, H1 is shown as SEQ ID NO: 8, H2 is shown as SEQ ID NO: 9, H3 is shown as SEQ ID NO: 1; H4 is shown as SEQ ID NO: 10, H5 is shown as SEQ ID NO: 11, H6 is shown as SEQ ID NO: 12, H7 is shown as SEQ ID NO: 13, H8 is shown as SEQ ID NO: 14, and H9 is shown as SEQ ID NO: 15.

[0060] (8) Select the three most frequent ones (H1~H3) for subsequent affinity experiments (through fluorescence intensity analysis):

[0061] One million H9C2 cells were collected as samples, and another one million HUVECs were collected as negative controls.

[0062] 1. Select cells in good growth condition (using a small dish as an example). When the growth abundance reaches more than 90%, add 1 mL of PBS and wash 3 times. Digest with 0.5 mL of enzyme-free digestion solution at room temperature for a certain period of time (H9C 23 min; HUVEC 10 min, covering the bottom of the dish). Add 1-1.5 mL of PBS to stop the digestion. Gently pipette the cells to make a single-cell suspension. Transfer the suspension to a 15 mL centrifuge tube and centrifuge (1000 rpm, 5 min). Discard the supernatant, add 1 mL of washing buffer to resuspend the cells, and transfer to a 1.5 mL enzyme-free EP tube (4℃, 1150 rpm, 4 min) for washing.

[0063] 2. Wash the cells twice again with washing buffer (4℃, 1150 rpm, 4 min), resuspend in 800 μL of washing buffer, count the cells, and adjust the cell suspension to 100 × 10⁻⁶. 4 / mL, aliquoted into flow cytometry tubes (200μL per tube, 3 replicates). Add 500nM nucleic acid aptamer to each tube (dissolved in 200μL binding buffer). A negative control group was also included, with 500nM random DL added to this tube (dissolved in 200μL binding buffer). After mixing, incubate at 4°C in the dark for 30 min (final concentrations of both ordered nucleic acid aptamer and random DL in the incubation solution were 250nM).

[0064] 3. After incubation, wash twice more with washing buffer (4℃, 1150 rpm, 4 min). Resuspend each tube of cells in 200 μL of binding buffer and perform instrumental analysis, counting 20,000 cells.

[0065] Affinity experiment results as follows Figure 4 The colored portion represents the binding of the three most frequent sequences H1, H2, and H3 (from left to right) to H9C2 in the sequencing results; the black portion is a random library. Flow cytometry determined that H3 in the sequencing results has the highest affinity for the target H9C2 and is the optimal aptamer, which is the nucleic acid aptamer described in this invention. The sequence is shown in SEQ ID NO: 1.

[0066] The secondary structure diagram of H3 is obtained through NUPACK, such as... Figure 5 As shown.

[0067] (9) Analysis using Kd value:

[0068] The difference from step (8) is that in step 2, each tube is added with an appropriate concentration (a concentration gradient of 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 0 nM) of H3 (dissolved in 200 μL of binding buffer). A negative control group is also provided; this tube is added with 500 nM of randomized DL (dissolved in 200 μL of binding buffer, with the same concentration gradient as above), mixed well, and incubated at 4°C and 37°C in the dark for 30 min.

[0069] The relative fluorescence intensity was calculated by subtracting the average fluorescence intensity of the corresponding negative control from the average fluorescence intensity of the measured values, and then using the formula Y = BmaxX / (Kd + X) (where X represents the aptamer concentration (mol / L), Y represents the fluorescence intensity corresponding to each concentration, and Bmax represents the maximum fluorescence intensity). Results are shown below. Figure 6 (4℃ (left), 37℃ (right)) indicates that H3 has good affinity for the target at both 4℃ and 37℃.

[0070] (10) Confocal fluorescence microscopy analysis of the effect of temperature change on the affinity between aptamers and targets:

[0071] Cell slides:

[0072] 1. Culture cells (H9C2 and HUVEC) to 80% saturation, then wash three times with 2 mL PBS;

[0073] 2. Digest with 1 mL of pancreatic enzyme;

[0074] 3. Rinse the cells with 2 mL of whole culture, then repeatedly blow the mixture to mix.

[0075] 4. Add climbing slides to the 24-well plate. Before adding, moisten the bottom of the plate with a small amount of whole culture. Then, carefully remove a climbing slide with tweezers, quickly heat both sides, and carefully lift the 24-well plate near an alcohol lamp. Place the climbing slide into the well and gently press it down a couple of times with tweezers to ensure it adheres firmly to the bottom. After placing the climbing slide, add 1 mL of whole culture and about 10 μL of digested cells to the whole culture. Gently pipette to mix. Incubate at 37°C in a 5% CO2 incubator for 1–2 days.

[0076] 5. Once the cells have adhered to the plate and grown to 60-80% of the bottom area of ​​each well in the 24-well plate, discard the whole culture in the 24-well plate and wash three times with 1 mL PBS.

[0077] 6. According to the experimental purpose, add 200 μL of the optimal conformation nucleic acid aptamer H3 with a final concentration of 250 nM, and incubate at 4℃ and 37℃ for the corresponding time (30 min);

[0078] 7. Aspirate the incubation solution and wash three times with 1 mL of PBST;

[0079] 8. Fixed:

[0080] a. Add 200-400 μL of 4% paraformaldehyde to each well and fix for 10-15 min, then discard the paraformaldehyde;

[0081] b. Wash 3 times with 1 mL PBST (shake slowly on a shaker, 3 times, 5 min each time), then discard the PBST;

[0082] c. Add 10 μL of DAPI to the new well, place the slide face down, and immerse it in DAPI for 5 min;

[0083] d. Add 1 mL PBST and wash 3 times (shake slowly on a shaker, 3 times, 5 min each time). In the last wash of PBST, remove the slide with the front side facing up (do not discard PBST, directly remove the slide) and air dry for 30 min.

[0084] 9. Anti-quenching agent: Add 4-6 μL of anti-fluorescence quenching agent to the glass slide, and place the slide face down with the anti-quenching agent in contact;

[0085] 10. Sealing the film: Seal the edges of the film with nail polish;

[0086] The experimental results were observed using a confocal microscope. Figure 7 This indicates that H3 has good affinity for the target at both 4℃ and 37℃.

[0087] (11) Aptamer sequence specificity analysis

[0088] One million cells were collected from each sample. The binding of aptamer H3 to human primary hepatic sinusoidal endothelial cells (HHSEC), normal human liver epithelial cells (HL7702), human primary pulmonary artery endothelial cells (HPAEC), normal human lung epithelial cells (BEAS2B), human primary splenic microvascular endothelial cells (HSEC), and rat cardiomyocytes (H9C2) was detected by flow cytometry, following the same procedure as in step (8). Results are shown below. Figure 8 (From left to right: top layer consists of HHSEC cells, HL7702 cells, and HPAEC cells; bottom layer consists of BEAS2B cells, HSEC cells, and H9C2 cells). From Figure 8 It can be seen that H3 has high specificity for cardiomyocytes.

[0089] (12) Stability test of aptamer sequence in serum

[0090] H3 cells modified with 2′-O-methyl and unmodified H3 cells were ordered from Sangon Biotech (Shanghai) Co., Ltd. These were added to complete culture medium (containing 10% fetal bovine serum) and mixed thoroughly. The aptamer sequence concentration was 3 μM. The mixture was incubated at 37℃ for 0, 2, 4, 6, 8, 12, 24, 36, and 48 h. The aptamers were then recovered, and the degradation of the aptamer sequence was observed by 3% agarose gel electrophoresis. The results are shown below. Figure 9 (The upper layer is unmodified H3, and the lower layer is 2′-O-methyl modified H3, 68bp). The results show that the 2′-O-methyl modified H3 can maintain structural integrity more persistently and is more stable in the in vivo environment.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer that specifically binds to the surface of the heart, characterized in that, The nucleic acid aptamer sequence is SEQ ID NO:

1.

2. The nucleic acid aptamer according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid aptamer is phosphorylated, oxymethylated, methylated, aminated, thiolated, fluorinated, or isotopized at a certain position.

3. The nucleic acid aptamer according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid aptamer is coupled with a fluorescent group, a quencher group, biotin, nanomaterials, a thiol group, an amino group, or an enzyme.

4. A modified nucleic acid aptamer that specifically binds to the surface of the heart, characterized in that, A nucleic acid aptamer having the same function as the nucleic acid aptamer obtained by modifying the nucleic acid aptamer according to any one of claims 1 to 3 by amination, thiolation or isotopization.

5. The use of the nucleic acid aptamer according to any one of claims 1 to 3 or the modified nucleic acid aptamer according to claim 4 in the preparation of cardiac-targeted drug carriers.

6. The use of the nucleic acid aptamer according to any one of claims 1 to 3 or the modified nucleic acid aptamer according to claim 4 in the preparation of cardiac targeted drugs.

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