Nucleic acid aptamers for maytansinoids and uses thereof

Abstract

The application discloses a nucleic acid aptamer of maytansine and application thereof, the nucleic acid aptamer is Seq2-X, and a DNA sequence of Seq2-X is shown as SEQ ID NO. 1. The nucleic acid aptamer can be combined with the target maytansine with high affinity and high specificity, the equilibrium dissociation constant is in the nanomolar range, the water solubility is good, and after being combined with the fat-soluble maytansine, the solubility of the drug can be improved. By utilizing the specific interaction between the nucleic acid aptamer and maytansine, the stability of maytansine drug can be improved, the cytotoxicity of maytansine is enhanced, and the treatment window is widened. By utilizing the nucleic acid aptamer as a drug delivery carrier of maytansine, the limitation of maytansine in clinical application can be improved, which has important significance for the design and development of maytansine drugs in clinical application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and relates to a nucleic acid aptamer and application thereof, in particular to a nucleic acid aptamer of an antitumor drug maytansine and application thereof. BACKGROUND

[0002] Maytansine is an antitumor drug containing a macrocyclic lactone structure, which can effectively inhibit the proliferation of malignant tumors, but its toxic side effects cannot be ignored. Maytansine has problems of poor water solubility, poor stability and narrow therapeutic window in clinical application. In order to solve these problems, people have constructed covalent conjugates of maytansine with antibodies or nucleic acid aptamers, or prepared maytansine into nanodrugs to solve the limitations of its clinical application. However, although these two methods have achieved obvious results, they are still limited by high production cost, complicated preparation process and difficulty in large-scale production. Therefore, the clinical application of maytansine still has great challenges.

[0003] Nucleic acid aptamer is a single-stranded oligonucleotide (single-stranded DNA or RNA) obtained by screening through the system evolution of ligand enrichment (SELEX), which can specifically bind to the target. Nucleic acid aptamer has affinity and specificity comparable to antibodies, and has more advantages than antibodies, such as small molecular weight, low immunogenicity, easy chemical synthesis, low cost, easy modification and flexible design, stable properties and easy storage. Different from the covalent coupling mode of nucleic acid aptamer and drug, the specific binding of nucleic acid aptamer to the target is realized by hydrogen bond, van der Waals force and other non-covalent interactions. Therefore, using nucleic acid aptamer as a carrier may bring new ideas and methods for drug delivery.

[0004] However, there is no related report on maytansine nucleic acid aptamer. Therefore, if a nucleic acid aptamer that specifically binds to maytansine can be obtained, the affinity between maytansine and its nucleic acid aptamer will be expected to solve the limitations of maytansine in clinical application, and thus provide a new research perspective for delivering the antitumor drug maytansine. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art, and to provide a nucleic acid aptamer of maytansine, which can specifically and highly bind to maytansine, has low immunogenicity, can be synthesized in batches, has good biocompatibility and stability, has small molecular weight and is easy to store; and the application of the nucleic acid aptamer in solving the limitations of maytansine in clinical application is also provided.

[0006] To solve the above technical problems, the present application provides a nucleic acid aptamer of maytansine, which is Seq2-X, and the DNA sequence of Seq2-X is shown as SEQ ID NO. 1.

[0007] The nucleic acid aptamer, further, the nucleic acid aptamer is a nucleic acid aptamer combined with biotin, digoxin, fluorescent substance, nanometer luminescent material or enzyme label on the DNA sequence of Seq2-X.

[0008] The nucleic acid aptamer, further, the fluorescent substance is FAM fluorescent group or Cy5 fluorescent group; the nanometer luminescent material is quantum dot or up-conversion nanoparticle; and the enzyme label is horseradish peroxidase or sucrose.

[0009] The nucleic acid aptamer, further, the nucleic acid aptamer is a nucleic acid aptamer Seq2-X, wherein a certain position on the nucleotide sequence of the nucleic acid aptamer Seq2-X is phosphorylated, methylated, aminated, sulfhydrylated or isotopically labeled under the premise that the overall structure of Seq2-X remains unchanged.

[0010] The nucleic acid aptamer, further, the amination is that an amino group -NH2 is labeled at the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq2-X; and the sulfhydrylation is that a sulfhydryl group -SH is labeled at the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq2-X.

[0011] The nucleic acid aptamer, further, the nucleic acid aptamer is a nucleic acid aptamer with a DNA sequence being any one of the following four sequences:

[0012] (1) having a homology of more than 60% with the nucleotide sequence of the nucleic acid aptamer Seq2-X;

[0013] (2) a sequence capable of hybridizing with the nucleotide sequence of the nucleic acid aptamer Seq2-X;

[0014] (3) an RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer Seq2-X;

[0015] (4) a derivative of Seq2-X, wherein the derivative is a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer Seq2-X, or a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer.

[0016] Based on the overall technical concept, the present application further provides an application of the nucleic acid aptamer in improving the solubility of maytansine.

[0017] Based on one general technical concept, the present application also provides an application of the nucleic acid aptamer in widening the therapeutic window of maytansine.

[0018] Based on one general technical concept, the present application also provides an application of the nucleic acid aptamer in improving the stability of maytansine.

[0019] Based on one general technical concept, the present application also provides an application of the nucleic acid aptamer in a drug carrier.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] (1) The present application provides a nucleic acid aptamer of the antitumor drug maytansine, which can bind to the target maytansine with high affinity and high specificity, and the equilibrium dissociation constant is in the nanomolar range.

[0022] (2) The present application provides a nucleic acid aptamer of the antitumor drug maytansine, which has less immunogenicity than protein antibodies, can be synthesized in large quantities in vitro, has a small molecular weight, can be modified and substituted at different sites, and has stable chemical properties, is easy to store, and is convenient for labeling. At the same time, the nucleic acid aptamer has good water solubility, and after being combined with the liposoluble drug maytansine, the solubility of the drug can be improved, and the toxic side effects caused by the addition of organic solvents (such as DMSO, etc.) or surfactants (such as Tween, etc.) can be avoided.

[0023] (3) The present application provides a nucleic acid aptamer of the antitumor drug maytansine, which can improve the stability of maytansine and enhance the cytotoxicity of maytansine to widen the therapeutic window by utilizing the specific interaction between the nucleic acid aptamer and maytansine. The nucleic acid aptamer of the present application can be used as a drug delivery carrier for maytansine to improve the limitations of clinical application of maytansine (improve solubility, improve stability, and widen the therapeutic window), which has important significance for the design and development of maytansine in clinical application. At the same time, the corresponding labeling, modification or substitution of the nucleic acid aptamer of the present application is conducive to the tracing of maytansine in vivo, which helps to reveal the mechanism of drug absorption, distribution, metabolism and excretion, and promotes the effectiveness and safety of drug therapy.

[0024] In addition to the objects, features and advantages described above, the present application has other objects, features and advantages. The present application will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0025] The accompanying drawings, which form a part of the present application, are included to provide a further understanding of the application, and are incorporated herein for purposes of illustrating the illustrative embodiments of the present application and the explanations provided herein, and are not intended as a limitation of the present application.

[0026] Figure 1 Structure diagram of the secondary structure simulation of the nucleic acid aptamer Seq2-X sequence by using the Mfold service in the experiment one of the present application.

[0027] Figure 2 The concentration optimization diagram of the incubation of the nucleic acid aptamer FAM-Seq2-X and the quencher probe Quencher in the experiment two of the present application, wherein the abscissa is the gradually increased quencher probe, and the ordinate is the fluorescence intensity.

[0028] Figure 3 The fluorescence recovery saturation curve after adding different concentrations of maytansine for incubation under the optimal FAM-Seq2-X and quencher quenching ratio in the experiment three of the present application, wherein the abscissa is the gradually increased maytansine, and the ordinate is the fluorescence intensity.

[0029] Figure 4 The investigation result of the specificity of the nucleic acid aptamer FAM-Seq2-X in the experiment four of the present application.

[0030] Figure 5 The result of the nucleic acid aptamer Seq2-X improving the solubility of maytansine under different incubation times in the experiment five of the present application.

[0031] Figure 6 The result of the nucleic acid aptamer Seq2-X improving the solubility of maytansine under different concentrations in the experiment six of the present application.

[0032] Figure 7 The investigation result of the nucleic acid aptamer Seq2-X widening the treatment window of maytansine in the experiment seven of the present application.

[0033] Figure 8 The investigation result of the nucleic acid aptamer Seq2-X improving the stability of maytansine in the experiment eight of the present application. DETAILED DESCRIPTION

[0034] The present application will be further described in the following with reference to the accompanying drawings and specific preferred embodiments, but the protection scope of the present application is not limited thereto. The present application will be described in detail with reference to the accompanying drawings and embodiments.

[0035] The materials and instruments used in the following embodiments are commercially available.

[0036] Embodiment 1

[0037] The present application provides a nucleic acid aptamer of maytansine, which is the nucleic acid aptamer Seq2-X, and the nucleotide sequence of the nucleic acid aptamer Seq2-X is the DNA fragment shown in SEQ ID NO. 1, which is specifically as follows:

[0038] 5'-CTCTCGGGACGACTTGTCATGTGTAGAAGGAGTGGGTTGAGATGTCGTCCC-3'.

[0039] The nucleic acid aptamer Seq2-X is mainly obtained by the following screening method:

[0040] (1) Synthesize the random single-stranded DNA library, primers and capture chain shown in the following sequence.

[0041] Random single-stranded DNA library Library:

[0042] 5'-GGAGGCTCTCGGGACGAC-N30-GTCGTCCCGATGCTGCAATCGTAA-3' (wherein N is any one of A, T, C, G four bases);

[0043] Forward primer: 5'-FAM-GGAGGCTCTCGGGACGAC-3';

[0044] Reverse primer: 5'-Biotin-TTACGATTGCAGCATCGGGACG-3';

[0045] Capture chain cDNA: 5'-GTCGTCCCGAGAGCCATA-BioTEG-3'.

[0046] (2) Screening the nucleic acid aptamer of maytansine by Capture-SELEX technology.

[0047] 2.1. Hybridization of library and capture chain: After the initial library or secondary library is dissolved and quantified, the hybridization ratio of the optimized library and capture chain (1:5) is obtained. In the first round of screening, 1 nmol of random single-stranded DNA library Library is dissolved in 5 nmol of capture chain cDNA in 250 μL of screening buffer (the composition of the screening buffer is: 1.8 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl and 2 mM MgCl2, pH 7.4), mixed well, and then the solution is divided into 5 tubes, each tube containing 50 μL. Slowly anneal and hybridize in a PCR instrument, and the annealing and hybridization program is: 95°C for 10 min; 60°C for 10 min; 25°C for 30 min; the secondary library and capture chain of each round of screening are annealed and hybridized in the PCR instrument according to the above hybridization ratio.

[0048] 2.2. Strep-tactin agarose bead packing: The spin minicolumns were first sterilized by UV light before use. Then, 600 μL of 250 nM random sequence (5'-GCGGAGCGTGGCAGG-3') was added to the treated columns and incubated for 10 min at room temperature on a test tube rotator shaker to block the non-specific adsorption sites in the spin minicolumns. After the blocking was completed, the spin minicolumns were washed with 600 μL of wash buffer (the composition of the wash buffer is 3.8 mM NaH2PO4, 16.2 mM Na2HPO4, and 150 mM NaCl, pH 7.5) for 3 times to remove the residual random sequence. Then, 250 μL of strep-tactin agarose beads were added to the spin minicolumns, and the protection solution was removed after centrifugation at 1000 x g for 30 s. The spin minicolumns were sequentially washed with 250 μL of wash buffer (the composition of the wash buffer is the same as above) for 3 times and 250 μL of selection buffer for 4 times.

[0049] 2.3. Library immobilization: After the spin minicolumns were packed with strep-tactin agarose beads, 250 μL of library-capture strand hybridization complex was added to the spin minicolumns. The end plug and the cap were covered, and the spin minicolumns were incubated on a test tube rotator shaker for 30 min (avoiding light, room temperature). The library immobilization efficiency was calculated by the following formula: immobilization efficiency = (C1-C2) / C1 x 100% (Note: the concentration of the library-capture strand hybridization complex solution before incubation with the strep-tactin agarose beads was C1, and the concentration of the solution in the outer tube after centrifugation after incubation of the library-capture strand hybridization complex solution with the strep-tactin agarose beads was C2).

[0050] 2.4. Background elution: After the library was immobilized, the spin minicolumns were washed with 250 μL of selection buffer each time to wash away the unbound and weakly bound library molecules. The number of washes was adjusted according to the selection pressure. The first round of selection was washed for 10 times, and the subsequent rounds were adjusted accordingly, with each wash being about 2 min. The solutions after each wash were collected and named as Wash#1, Wash#2, …, Wash#final (final indicates the last wash of each round of selection).

[0051] 2.5. Counter screen: The counter screen was performed from the 4th round of screening. During the counter screen, 250 μL of screening buffer containing 2.5% DMSO was added to the centrifugal micro-chromatography column, and the end plug and the cap were put on. The column was incubated on the tube rotator for a certain period of time, and the incubation time was adjusted according to the screening pressure. After the counter screen, the end plug was removed, and the column was fitted into a 1.5 mL outer tube. The solution in the outer tube was collected after centrifugation at 1000 x g for 1 min. The counter screen was repeated, and the solutions collected after the second counter screen were named Counter #1 and Counter #2, respectively.

[0052] 2.6. Washing after counter screen: After the counter screen, an additional washing step was performed before the positive screen, and the purpose was to wash out the DMSO and the ssDNA that dissociated during the counter screen. The number of washing steps was adjusted according to the screening pressure. During each washing step, 250 μL of screening buffer was added, and the solution was gently blown with a pipette tip. After blowing for about 2 min, the solution was collected after centrifugation at 1000 x g for 1 min. The solutions collected after each washing step were named W#1, W#2,..., W#final (final means the last washing step in each round of screening).

[0053] 2.7. Positive screen: During the positive screen, 250 μL of target maytansinoid (dissolved in 2.5% DMSO) was added to the centrifugal micro-chromatography column, and the end plug and the cap were put on. The column was incubated on the tube rotator for a certain period of time. The incubation time and the concentration of the target added were adjusted according to the screening pressure. After the incubation, the column was fitted into a 1.5 mL outer tube, and the solution in the outer tube was collected after centrifugation at 1000 x g for 1 min. The positive screen was repeated, and the solutions collected after the second positive screen were named Target #1 and Target #2, respectively.

[0054] 2.8. PCR amplification: The products of the two positive screens in 2.7 were combined, and 100 μL of the combined product was subjected to PCR amplification to obtain the PCR double-stranded product. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 62.7°C for 30 s, extension at 72°C for 30 s, and appropriate cycle number (optimized according to 3% agarose electrophoresis); and final extension at 72°C for 4 min. After the PCR, all the PCR double-stranded products were collected and combined into an EP tube.

[0055] 2.9. Preparation of single-stranded DNA: Take 150 μL of streptavidin agarose microbeads in a 1.5 mL low adsorption EP tube, centrifuge at 1000 x g for 30 s, and discard the supernatant. Then add 400 μL of washing buffer to wash the microbeads, and gently blow the microbeads with a gun head during the washing process. After sufficient blowing, centrifuge to remove the washing solution, and wash for a total of three times. Add the combined PCR product to the above pretreated microbeads, and incubate at room temperature in the dark for 30 min, and mix 6-10 times during the incubation to ensure that the biotinylated PCR product is fixed on the streptavidin agarose microbeads. After the incubation, centrifuge the sample at 1000 x g for 30 s, and discard the supernatant. Add 200 μL of washing buffer to wash, and wash for a total of five times to remove excess enzymes, primers, and double-stranded DNA that is not fixed. At the same time, the PCR buffer contains Mg 2+ , and multiple washing with the washing buffer without Mg 2+ can sufficiently achieve solvent exchange, and avoid the generation of Mg(OH)2precipitate due to the addition of NaOH solution in the subsequent operation. After the washing, add 300 μL of DNA denaturation solution (200 mM NaOH solution) to the microbeads, and alkali denaturation incubate in the dark for 15 min. Then centrifuge at 1000 x g for 30 s, and collect the supernatant. Further, take 80 μL, 70 μL, and 50 μL of DNA denaturation solution to treat the above microbeads in the same way. After the alkali denaturation, centrifuge and collect the supernatant for three times. Combine the four supernatants to obtain about 500 μL of single-stranded DNA library, and place it in a 1.5 mL EP tube.

[0056] 2.10. Desalination and concentration of single-stranded DNA: The large amount of NaOH introduced in the alkali denaturation process not only changes the pH of the screening buffer system, but also changes the ionic strength of the screening buffer. Therefore, the prepared single-stranded DNA cannot be directly used in the next round of screening, and needs to be desalted to remove the large amount of Na + and OH -The desalting operation is as follows: first, the desalting column is hung vertically on a support, the lids at the top and bottom of the desalting column are opened, and the liquid in the desalting column is allowed to flow out naturally by gravity; second, at least 10 mL of sterilized water is added to the desalting column (1 mL at a time, at least 10 times), the purpose being to drive away the gas remaining in the gel in the desalting column, so that the solvent can flow more uniformly and uniformly, and the separation effect is ensured; then, the 500 μL of the single-stranded DNA library obtained above is added to the desalting column, and the liquid that flows out naturally is discarded; finally, when the liquid has completely dripped by gravity, 1 mL of freshly prepared sterilized water is added to the desalting column, at which time the liquid that flows out is collected, and this is the single-stranded DNA after desalting. After desalting, the single-stranded DNA library obtained is vacuum-dried at 75°C using a vacuum concentration centrifugal dryer. After about 2.5 h of drying, single-stranded DNA powder (pale yellow) is obtained, which is sealed and stored in a -20°C refrigerator for standby use as the screening library for the next round.

[0057] 2.11. Cycle of screening: first, the dried single-stranded DNA powder is centrifuged to avoid loss of the library due to splashing of the powder when the lid is opened; second, an appropriate amount of screening buffer is added to the powder to dissolve it thoroughly; finally, the absorbance of the single-stranded DNA solution at 260 nm is measured using a UV spectrophotometer, and the amount of single-stranded DNA to be put into the next round of screening is calculated according to the absorbance value. Operations 2.1-2.10 are repeated until the screening library is fully enriched, and in this process, different screening pressures are controlled in different screening rounds, the purpose being to increase the specificity and affinity of the screened sequences. After enrichment is complete, the screening process is ended.

[0058] 2.12. Monitoring of the screening process: the screening process is monitored simultaneously using fluorescence and electrophoresis. The principle of fluorescence monitoring is as follows: the 5' end of the forward primer is modified with fluorescent dye FAM during the screening process, and after PCR amplification and alkaline hydrolysis to prepare a secondary library, the 5' end of the DNA library to be put into the screening process is modified with FAM from the second round of screening. Therefore, the amount of ssDNA eluted in each round of screening can be determined by detecting the fluorescence value of the fluorescent dye FAM.

[0059] Principle of electrophoresis monitoring: the volume of background elution, negative screening volume, volume of solution washing after negative screening, and positive screening volume in each round of screening process are all 250 μL. The solution after each operation is collected and marked, and then 5 μL of the collected solution is taken to prepare a PCR amplification system with a total volume of 50 μL. The specific reaction system is as follows: 25 μL of 2x PCR Mix, 2.5 μL of FAM-forward primer (10 μM), 2.5 μL of Biotin-reverse primer (10 μM), 5 μL of template DNA, and 15 μL of sterilized water. The reaction amplification conditions are as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 62.7 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 10 cycles of amplification; and 72 ℃ final extension for 4 min. After the completion of PCR, 3% agarose gel electrophoresis and imaging analysis are performed, and the brightness of the electrophoresis band corresponds to the amount of ssDNA eluted in each operation. Therefore, under the same PCR reaction system and amplification times, the brightness of the PCR product electrophoresis band is different due to the different template concentrations, and the progress of the screening is monitored according to the brightness of the band.

[0060] According to the monitoring results of the screening process, the enriched library is finally obtained after sequencing analysis and trimming optimization, and the nucleic acid aptamer Seq2-X capable of specifically recognizing maytansine is obtained. The 5' end of the nucleic acid aptamer Seq2-X is labeled with FAM, and is denoted as nucleic acid aptamer FAM-Seq2-X, and the DNA fragment is as follows:

[0061] 5'-FAM-CTCTCGGGACGACTTGTCATGTGTAGAAGGAGTGGGTTGAGATGTCGTCCC-3'.

[0062] In the present application, the nucleotide sequence of the nucleic acid aptamer Seq2-X is combined with biotin, digoxin, fluorescent substances, nanometer luminescent materials or enzyme labels, which can achieve the same or similar technical effects as those in Example 1. In the present embodiment, the fluorescent substance is a FAM fluorescent group, but other fluorescent groups can also produce the same technical effects, such as a Cy5 fluorescent group.

[0063] The nanometer luminescent material can be a quantum dot or an up-conversion nanoparticle. The enzyme label can be horseradish peroxidase or sucrose.

[0064] In the present application, under the premise that Seq2-X maintains the overall structure, the nucleotide sequence of the nucleic acid aptamer Seq2-X at a certain position is phosphorylated, methylated, aminated, sulfhydrylated or isotopically labeled, which can achieve the same or similar technical effects as those in Example 1. Amination is to label an amino group -NH2 at the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq2-X; sulfhydrylation is to label a sulfhydryl group -SH at the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq2-X.

[0065] In the present invention, the nucleic acid aptamer having a DNA sequence of any one of the following four sequences can achieve the same or similar technical effects as in Example 1:

[0066] (1) The homology with the nucleotide sequence of the nucleic acid aptamer Seq2-X is greater than 60%;

[0067] (2) a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer Seq2-X;

[0068] (3) an RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer Seq2-X;

[0069] (4) is a derivative of Seq2-X, wherein the derivative is a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer Seq2-X, or is a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer.

[0070] Comparative Example 1

[0071] The nucleotide sequence of the control chain is: Control: 5'-ATTGCGAAACGGAAGTCGCTTCTACACTTCCGATTCGCAAT-3'.

[0072] Experiment 1: Use the Mfold service to simulate the secondary structure of the nucleic acid aptamer sequence in Example 1.

[0073] The schematic diagram of the structure after the secondary structure simulation of the nucleic acid aptamer sequence of Example 1 using the Mfold service is shown in Figure 1 :from Figure 1 It can be seen that the Seq2-X sequence structure mainly includes a large loop, a small loop and two stem regions.

[0074] Experiment 2: Use a multifunctional microplate reader to investigate the optimal concentration ratio of the nucleic acid aptamer FAM-Seq2-X and the quenching probe Quencher.

[0075] Draw a fluorescence quenching saturation curve to determine the dissociation constant K when the complementary hybridization reaches equilibrium. d1, wherein the abscissa is a series of different concentrations of quencher, and the ordinate is fluorescence intensity. The specific process is as follows: the FAM-Seq2-X and quencher synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. are centrifuged at a speed of 10000 r for 5 min, and a certain volume of screening buffer is added to prepare a solution with a concentration of 10 μM, which is ready for use. The final concentration of FAM-Seq2-X is 50 nM, which is mixed with a series of different final concentrations of quencher (0 μM, 41 μM, 66 μM, 83 μM, 124 μM, 166 μM, 415 μM, 830 μM) and added to the screening buffer. The above solution is slowly annealed and hybridized in a PCR instrument (the hybridization program is the same as above). Then, a multifunctional enzyme marker is used for fluorescence test (operation is carried out in a dark environment), and the test conditions are as follows: Ex = 488 nm, Em = 520 nm, Ex Slit = 10.0 nm, and Em Slit = 10.0 nm. The fluorescence quenching saturation curve of the hybridization of the aptamer and the quencher is drawn to determine the dissociation constant K d 1, three parallel samples are set for each concentration in the experiment.

[0076] Figure 2 The concentration optimization diagram of the incubation of the aptamer FAM-Seq2-X and the quencher is shown in the figure, wherein the abscissa is a series of different concentrations of quencher, and the ordinate is fluorescence intensity. As can be seen from the figure, when the concentration of the aptamer FAM-Seq2-X is fixed at 50 nM, the fluorescence intensity of the system gradually decreases and then remains unchanged with the continuous increase of the concentration of the quencher. When the concentration of the quencher is about 28.2 nM, the fluorescence of FAM-Seq2-X is quenched by half. According to the following formula: The K d 1 calculated by the formula is about 3.2 nM.

[0077] Experiment three: the fluorescence recovery saturation curve after the incubation of different concentrations of maytansine under the optimal quenching ratio of FAM-Seq2-X and quencher is investigated by using a multifunctional enzyme marker.

[0078] The fluorescence recovery saturation curve is drawn to determine the dissociation constant K d2, where the horizontal axis is the target maytansine with gradually increasing concentrations, and the vertical axis is the fluorescence intensity. The specific process is: at the optimal quenching ratio of nucleic acid aptamer to quenching probe (50nM: 250nM), a series of concentrations of target maytansine (the target final concentration is 0μM, 0.1μM, 0.5μM, 1μM, 2μM, 3μM, 5μM, 7.5μM, 10μM, 20μM, 50μM and 100μM) are added to the nucleic acid aptamer-quenching probe complex solution, and incubated in an oscillating constant temperature metal bath at 25°C and in the dark for 1 hour. Three parallel samples are set for each concentration in the experiment. Subsequently, fluorescence testing is performed using a multifunctional microplate reader (operated in a dark environment) under the following test conditions: Ex = 488nm, Em = 520nm, Ex Slit = 10.0nm, Em Slit = 10.0nm. Then, the fluorescence recovery saturation curve of the target binding to the aptamer-quencher probe complex sensor was drawn to determine the dissociation constant K when the target induced the aptamer-quencher probe complex to dissociate and reach equilibrium. d 2.

[0079] Figure 3 The fluorescence recovery saturation curves after incubation with different concentrations of maytansine at the optimal quenching ratio of FAM-Seq2-X to Quencher in Example 1 of the present invention are shown, wherein the abscissa represents the target maytansine with gradually increasing concentrations, and the ordinate represents the fluorescence intensity. Figure 3 The results show that as the concentration of maytansine continues to increase, the fluorescence intensity of the system gradually recovers and then remains basically unchanged. When the concentration of maytansine is about 1.8 μM, the quenched fluorescence is restored to half. According to the following formula: The calculated K d 2 is approximately 0.13.

[0080] K d 1 and K d 2. Compare the equilibrium dissociation constant K between maytansine and nucleic acid aptamer d (K d =K d 1 / K d 2), the equilibrium dissociation constant K obtained from this d As shown in Table 1 below.

[0081] Table 1 Equilibrium dissociation constant results

[0082] Sequence name K d (nM) Seq2-X 24.6

[0083] Figure 3 and equilibrium dissociation constant K d The calculation results show that the nucleic acid aptamer Seq2-X of this embodiment has a strong binding ability to maytansine, and the equilibrium dissociation constant is at the nanomolar level.

[0084] Experiment four: chain displacement fluorescence method to investigate the specificity of FAM-Seq2-X.

[0085] Take 15 μL of 10 μM FAM-Seq2-X and 75 μL of 10 μM quencher probe (the nucleotide sequence of the quencher probe is: 5'-GTCGTCCCGAGAG-BHQ-1-3') dissolved in a total volume of 3000 μL of screening buffer, mix well and divide into 9 groups, each group of 100 μL, prepare three parallel samples. Then add 100 μL of blank sample (containing 2.5% DMSO screening buffer), 100 μL of 3 μM target maytansinol and 100 μL of 100 μM of other 7 kinds of small molecule compounds (cisplatin, 5-fluorouracil, tamoxifen, (S)-10-hydroxy camptothecin, docetaxel, sebufos, thiamethoxam) to the 9 groups of samples. After adding the sample, incubate the 9 groups of samples in a 25℃ and light-proof constant temperature metal bath for 1h. After incubation, measure the fluorescence intensity of the solution (Ex=488nm, Em=520nm, Ex Slit=10.0nm, Em Slit=10.0nm) with a multifunctional enzyme marker.

[0086] Figure 4 The specificity of the nucleic acid aptamer FAM-Seq2-X in Example 1 of the application is investigated. From the detection results of the nucleic acid aptamer FAM-Seq2-X in Example 1 of the application, it can be seen that the nucleic acid aptamer FAM-Seq2-X of the embodiment can specifically recognize maytansinol, and has no recognition for other small molecule compounds. Figure 4

[0087] Example 2

[0088] An application of the nucleic acid aptamer Seq2-X of Example 1 in improving the solubility of maytansinol.

[0089] The specific application method includes the following steps:

[0090] (1) Take an excess of maytansinol powder and dissolve it in the screening buffer, and ultrasonic it under the condition of 100W for 1h to prepare a saturated maytansinol suspension.

[0091] (2) Denature the nucleic acid aptamer Seq2-X at 95℃ for 5min, and keep it on ice at 4℃ for more than 10min for standby.

[0092] (3) Add the nucleic acid aptamer to the maytansinol suspension to make the final concentration of DNA in the solution 3 μM, mix the solution well, and then incubate it in a 25℃ constant temperature metal bath under oscillation for 0, 1, 2, 4, 6, 8, 12 and 24h respectively.

[0093] ​(4) After the incubation is completed, all samples are placed in a benchtop low-temperature refrigerated centrifuge and centrifuged at 10,000 x g for 10 min, and the supernatant is collected. The ultraviolet absorption of the sample supernatant from 500 nm to 200 nm is measured by ultraviolet spectrophotometry, and the absorbance values at 233 nm and 260 nm are recorded. Each sample is prepared in triplicate.

[0094] Experiment Five: Investigation of the effect of different incubation times of maytansine and aptamer on improving the solubility of maytansine.

[0095] Due to the poor solubility of maytansine, a long time is required to reach solubility equilibrium. Therefore, we first optimize the incubation time of maytansine and aptamer. According to the method of Example 2, the absorbance values of maytansine at 233 nm and 260 nm under different incubation times are recorded. According to the following equation set, the solubility (C x ) of maytansine is calculated.

[0096] A 233 = 2.98 x 10 4 C x + 1.77 x 10 5 C y

[0097] A 260 = 1.50 x 10 4 C x + 3.67 x 10 5 Cy

[0098] Figure 5 Results of the effect of aptamer Seq2-X on improving the solubility of maytansine under different incubation times. Figure 5 The results show that when the incubation time of aptamer Seq2-X at a final concentration of 3 μM with maytansine is extended from 1 h to 2 h, the solubility of maytansine is significantly improved, but as the incubation time continues to increase, the solubility of maytansine does not further improve, and basically reaches saturation. Considering that the solubility equilibrium of maytansine is a dynamic process, it takes a long time to reach equilibrium. Therefore, in the subsequent experiments, we directly incubate maytansine with aptamer in an oscillating constant-temperature metal bath at 25°C overnight for 24 h.

[0099] Experiment Six: Investigation of the effect of different concentrations of aptamer on improving the solubility of maytansine.

[0100] The effect of gradient concentrations of aptamer (0 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM) on improving the solubility of maytansine is investigated in the manner of Example 2.

[0101] Figure 6The results of the figure show that the solubility of maytansine gradually increases with the increasing concentration of the aptamer from 0.5 μM to 20 μM. When the concentration of the aptamer reaches 10 μM, the solubilization ability of the hydrophilic aptamer approaches a plateau, and when the concentration of the aptamer continues to increase to 20 μM, the solubility of maytansine increases from the initial 17 μM to 61 μM.

[0102] Example 3

[0103] An application of the aptamer Seq2-X of Example 1 in widening the therapeutic window of maytansine.

[0104] The specific application method is as follows:

[0105] (1) Maytansine is dissolved in a screening buffer containing 0.5% DMSO, and is ready for use after ultrasonic dissolution.

[0106] (2) The aptamer Seq2-X and the control strand Control are denatured at 95°C for 5 min and kept on ice at 4°C for more than 10 min, and are ready for use.

[0107] (3) The human tongue squamous cell drug-resistant cell Tca8113 / BLM is used as a model, and when the maytansine administration concentration is 400 nM, a series of complexes with different concentrations of the aptamer Seq2-X (0 nM, 10 nM, 100 nM, 200 nM, 400 nM, 1000 nM, 2000 nM, 5000 nM) are added to the above-mentioned 400 nM maytansine solution to investigate the killing effect of the complexes on the Tca8113 / BLM cells.

[0108] (4) When the maytansine administration concentration is 1000 nM, a series of complexes with different concentrations of the aptamer Seq2-X (0 nM, 10 nM, 100 nM, 200 nM, 400 nM, 1000 nM, 2000 nM, 5000 nM) are added to the above-mentioned 1000 nM maytansine solution to investigate the killing effect of the complexes on the Tca8113 / BLM cells.

[0109] (5) When the maytansine administration concentration is 2000 nM, a series of complexes with different concentrations of the aptamer Seq2-X (0 nM, 10 nM, 100 nM, 200 nM, 400 nM, 1000 nM, 2000 nM, 5000 nM) are added to the above-mentioned 2000 nM maytansine solution to investigate the killing effect of the complexes on the Tca8113 / BLM cells.

[0110] (6) The control strand was processed in the same manner as in steps 3-5 above.

[0111] Experiment 7: Investigate the effect of nucleic acid aptamer Seq2-X in broadening the therapeutic window of maytansine.

[0112] The specific experimental process is to draw the inhibition curve of maytansine alone on human tongue squamous cell carcinoma resistant cells Tca8113 / BLM, determine the optimal drug incubation time and the drug half inhibitory concentration IC 50 , select the IC 50 Three drug concentrations (400 nM, 1000 nM, and 2000 nM) with significant changes in the slope of the curve near the α-value were examined. At each fixed drug concentration, the effect of the aptamer Seq2-X on the cytotoxicity of maytansine was investigated when the drug concentration was varied from insufficient to excessive. The probe Control was used as the control strand, and five replicates were prepared for each sample group.

[0113] Figure 7 Results of an investigation into the use of the nucleic acid aptamer Seq2-X to broaden the therapeutic window of maytansine. Figure 7 The results of the investigation show that when the concentration of maytansine is 400nM, 1000nM and 2000nM respectively, as the concentration of the added nucleic acid aptamer gradually increases (from 0nM to 5000nM), the killing effect of the nucleic acid aptamer-maytansine complex on drug-resistant cells becomes more and more obvious. However, the addition of a control chain to form a control chain-maytansine complex of different concentrations does not change the efficacy of maytansine. This shows that nucleic acid aptamers increase the content of maytansine drugs entering cells and can significantly increase the cytotoxicity of low-concentration drugs. The nucleic acid aptamer-maytansine complex formed at the same time may also affect the process of drug-resistant cells pumping out drugs. In summary, nucleic acid aptamers can enhance the efficacy of maytansine, overcome the drug resistance of tumor cells to a certain extent, and ultimately broaden the therapeutic window of maytansine.

[0114] Example 4

[0115] An application of the nucleic acid aptamer Seq2-X of Example 1 in improving the stability of maytansine.

[0116] The specific application method is:

[0117] (1) Maytansine was dissolved in a screening buffer containing 0.5% DMSO, and ultrasonically dissolved for later use.

[0118] (2) Denature the nucleic acid aptamer Seq2-X at 95°C for 5 min and keep it on ice at 4°C for more than 10 min until ready for use.

[0119] (3) With MCF-7 cells as a model, 2nM maytansine, 2nM maytansine + 1nM Seq2-X, 2nM maytansine + 2nM Seq2-X and 2nM maytansine + 100nM Seq2-X were respectively placed at room temperature in the dark for 1 day, 4 days, 7 days and freshly prepared and then added to the above-mentioned cells for incubation for 72h, and then the survival rate of the cells was measured.

[0120] (4) With SCC4 cells as a model, 132nM maytansine, 132nM maytansine + 10nM Seq2-X, 132nM maytansine + 132nM Seq2-X and 132nM maytansine + 1000nM Seq2-X were respectively placed at room temperature in the dark for 1 day, 4 days, 7 days and freshly prepared and then added to the above-mentioned cells for incubation for 72h, and then the survival rate of the cells was measured.

[0121] (5) With Tca8113 / BLM cells as a model, 1000nM maytansine, 1000nM maytansine + 100nM Seq2-X, 1000nM maytansine + 1000nM Seq2-X and 1000nM maytansine + 2000nM Seq2-X were respectively placed at room temperature in the dark for 1 day, 4 days, 7 days and freshly prepared and then added to the above-mentioned cells for incubation for 72h, and then the survival rate of the cells was measured.

[0122] Experiment Eight: Effect of aptamer Seq2-X on improving the stability of maytansine.

[0123] With three different cells (MCF-7, SCC4, Tca8113 / BLM) as a model, the concentration near the IC 50 value obtained by fitting the inhibition curve was selected for investigation. The specific experimental method is as follows:

[0124] (1) Experimental method with MCF-7 cells as a model:

[0125] First, four groups of samples were prepared:

[0126] First group (free maytansine): 2nM maytansine.

[0127] Second group (maytansine-lack of aptamer): 2nM maytansine + 1nM Seq2-X.

[0128] Third group (maytansine-equal proportion of aptamer): 2nM maytansine + 2nM Seq2-X.

[0129] Fourth group (maytansine-excess aptamer): 2nM maytansine + 100nM Seq2-X.

[0130] The four groups of samples were respectively placed at room temperature in the dark for 0 days (freshly prepared), 1 day, 4 days, and 7 days, and were respectively recorded as Day 0, Day 1, Day 4, and Day 7. Then the four groups of samples stored in vitro for different days were respectively added to cells for incubation for 72 h, and after the incubation was completed, the survival rate of the cells was measured.

[0131] (2) Experimental method using SCC4 cells as a model:

[0132] First, four groups of samples were prepared:

[0133] The first group (free maytansine): 132 nM maytansine.

[0134] The second group (maytansine-insufficient amount of aptamer): 132 nM maytansine + 10 nM Seq2-X.

[0135] The third group (maytansine-equal proportion of aptamer): 132 nM maytansine + 132 nM Seq2-X.

[0136] The fourth group (maytansine-excessive amount of aptamer): 132 nM maytansine + 1000 nM Seq2-X.

[0137] The four groups of samples were respectively placed at room temperature in the dark for 0 days (freshly prepared), 1 day, 4 days, and 7 days, and were respectively recorded as Day 0, Day 1, Day 4, and Day 7. Then the four groups of samples stored in vitro for different days were respectively added to cells for incubation for 72 h, and after the incubation was completed, the survival rate of the cells was measured.

[0138] (3) Experimental method using Tca8113 / BLM cells as a model:

[0139] First, four groups of samples were prepared:

[0140] The first group (free maytansine): 1000 nM maytansine.

[0141] The second group (maytansine-insufficient amount of aptamer): 1000 nM maytansine + 100 nM Seq2-X.

[0142] The third group (maytansine-equal proportion of aptamer): 1000 nM maytansine + 1000 nM Seq2-X.

[0143] The fourth group (maytansine-excessive amount of aptamer): 1000 nM maytansine + 2000 nM Seq2-X.

[0144] The four groups of samples are respectively recorded as Day0, Day1, Day4 and Day7. Then the four groups of samples stored in vitro for different days are respectively added to the cells for incubation for 72 hours. After the incubation, the survival rate of the cells is measured.

[0145] Figure 8 The results of the investigation on the improvement of maytansine stability by the aptamer Seq2-X. The results show that the efficacy of free maytansine drug is reduced after being placed for 1 day, 4 days and 7 days, the killing effect of the drug placed for 7 days on MCF-7, SCC4 and Tca8113 / BLM cells is very weak, indicating that the efficacy of free maytansine drug is significantly reduced after being stored in vitro for more than 4 days. However, the maytansine-aptamer complex still has a killing effect on the three kinds of cells after being stored in vitro for 7 days, indicating that the aptamer can improve the stability of the drug.

[0146] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiment, it is not intended to limit the present application. Any skilled person in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.

Claims

1. A nucleic acid aptamer of a maytansinoid, characterized in that, The nucleic acid aptamer is Seq2-X, and a DNA sequence of the Seq2-X is shown as SEQ ID NO.

1.

2. The nucleic acid aptamer of claim 1, wherein, The nucleic acid aptamer is a nucleic acid aptamer combined with biotin, digoxin, a fluorescent substance, a nano luminescent material or an enzyme label on the DNA sequence of the Seq2-X.

3. The nucleic acid aptamer of claim 2, wherein, The fluorescent substance is a FAM fluorescent group or a Cy5 fluorescent group; the nano luminescent material is a quantum dot or an upconversion nanoparticle; and the enzyme label is horseradish peroxidase or sucrose.

4. The nucleic acid aptamer of claim 1, wherein, The nucleic acid aptamer is a nucleic acid aptamer in which a certain position on a nucleotide sequence of the nucleic acid aptamer Seq2-X is phosphorylated, methylated, aminated, sulfhydrylated or isotopically labeled, under the premise that an overall structure of the Seq2-X remains unchanged.

5. The nucleic acid aptamer of claim 4, wherein The amination is labeling of an amino group -NH2 at a 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq2-X; and the sulfhydrylation is labeling of a sulfhydryl group -SH at the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer Seq2-X.

6. Use of the nucleic acid aptamer according to any one of claims 1-5 in improving solubility of maytansine.

7. Use of the nucleic acid aptamer according to any one of claims 1-5 in preparing a drug for widening a therapeutic window of maytansine.

8. Use of the nucleic acid aptamer according to any one of claims 1-5 in improving stability of maytansine.

9. Use of the nucleic acid aptamer according to any one of claims 1-5 in preparing a drug carrier for targeted delivery of maytansine.

Images