Hydrophobic polypeptide grafted with oligonucleotide, nano assembly, preparation method and application

Abstract

The invention provides an oligonucleotide-grafted hydrophobic polypeptide, a nano assembly, a preparation method and application. The preparation method comprises the following steps: covalently linking a hydrophobic polypeptide FI and a 5'end of a hydrophilic oligonucleotide single chain (DNA) to prepare an FI-DNA amphiphilic molecule; the reaction system is subjected to purification characterization through gel electrophoresis, high performance liquid chromatography and time-of-flight mass spectrometry, and the morphology size of the nano assembly is characterized by using characterization means such as dynamic light scattering and a transmission electron microscope. The FI-DNA amphiphilic molecules can be self-assembled in a specific solvent to form nanofibers, the morphology of the FI-DNA amphiphilic molecules is converted into nano-micelles through introduction of host molecule cucurbituril, and the nano-micelle assembly is expected to be applied to drug delivery and achieve the effect of drug sustained release.

Description

Technical Field

[0001] The invention relates to the field of organic molecule synthesis, and in particular to the field of hierarchical self-assembly of DNA nanomaterials. Background Art

[0002] DNA nanotechnology has made remarkable progress since the 1990s, from the initial simple modular assembly to the diversified origami structure. Its unique molecular recognition performance and structural characteristics have made it show broad application prospects in many fields. Inspired by the hierarchical nanostructures presented in nature, based on the sequence programmability, good biocompatibility and addressability of DNA, DNA is widely used to construct nanomaterials with precise structures and specific functions, showing broad application prospects in the fields of materials science and biomedicine. In addition to using the precise base complementarity of the original DNA to construct exquisite, complex and controllable DNA assemblies, DNA amphiphilic assembly is also an indispensable method for constructing DNA nanostructures. Hydrophobic functional units are introduced into DNA to form amphiphilic hybrids, which can form nanostructures with designable size and function under the drive of amphiphilic action.

[0003] Host-guest interactions can significantly affect the hierarchical self-assembly process of molecules. Hierarchical self-assembly is a multi-level process. In it, molecules form ordered structures at different levels through non-covalent interactions. In this interaction, the combination of host molecules (usually macrocyclic molecules) and guest molecules (small molecules or ions) will lead to changes in the interaction forces in the system. Specifically, multiple interactions such as hydrogen bonds, π-π stacking, electrostatic interactions, and hydrophilic-hydrophobic interactions jointly participate, which determines the complexity and controllability of the self-assembly process. This self-assembly mechanism gives the material the ability to dynamically regulate, so that it undergoes reversible or irreversible structural transformations in response to external stimuli, thereby producing a series of functionalized nanoassemblies, which are widely used in drug delivery, fluorescence imaging, sensing detection, catalysis and other fields. Summary of the invention

[0004] DNA molecules have the advantages of precise base complementary pairing, sequence programmability, and clear secondary structure, and are widely used to construct various static micro-nano assembly structures. Peptides, as a kind of biological macromolecule, have become a widely used module for constructing supramolecular structures due to their structural diversity, ease of synthesis and modification, etc. Combining the addressability of DNA with the chemical diversity of peptides can achieve the integration and optimization of the characteristics of the two functional molecules and prepare nanostructures with controllable size and adjustable functions.

[0005] The present invention covalently links the hydrophobic dipeptide with the hydrophilic DNA, combines the functionality of the dipeptide molecule, and the phenylalanine at the end of the hydrophobic region can recognize cucurbituril, the molar ratio of the two is 1:2, with cucurbituril as the main body and amphiphilic FI-DNA molecules as the object for recognition, thereby generating a variety of hierarchical self-assembled nanomaterials. Due to the good biocompatibility of peptides and DNA, the material is expected to be used in medical fields such as drug delivery.

[0006] One of the purposes of the present invention is to construct an amphiphilic dipeptide-DNA molecule to achieve functional integration of two important functional macromolecules, DNA and polypeptide.

[0007] The second purpose of the present invention is to construct a nanofiber with good biocompatibility through hydrophilic-hydrophobic interaction, and to achieve morphological transformation into nanomicelles through host-guest interaction.

[0008] The third purpose of the present invention is to enable the nano-assembly to be efficiently taken up by cells and to test the sustained release effect of drugs.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is:

[0010] The preparation of FI-DNA nanoassemblies is mainly divided into two parts. The first part is the synthesis of dipeptide derivatives and dipeptide-DNA amphiphilic molecules, and the second part is the formation of nanoassemblies and host-guest mediated morphology regulation.

[0011] The dipeptide molecule is prepared by Fmoc solid phase synthesis method, and then undergoes amide condensation with 6-amino-1-hexanol to obtain a dipeptide derivative, which is then subjected to solid phase synthesis with DNA to obtain a dipeptide-DNA amphiphilic molecule.

[0012] The FI-DNA nanoassembly is prepared by the following method: a certain amount of dipeptide-DNA amphiphilic molecule, namely FI-DNA, in the above step is dissolved in a buffer solution, maintained at 90°C for 30 minutes, and then naturally cooled to room temperature and allowed to stand for 12 hours. By adding 0.5 equivalents of cucurbituril CB[8] solution and performing annealing again, the morphology can be controlled to obtain nanomicelles.

[0013] The nano micelles can be used as drug carriers. Hydrophobic drugs can be encapsulated in the hydrophobic cavity of the nano micelles. After the nanoassembly is taken up by cells, under the action of DNA enzymes, the nanoassembly gradually disintegrates and releases the drugs, achieving the effect of sustained drug release. Compared with the prior art, the present invention has the following outstanding effects:

[0014] 1. The present invention provides a synthesis method based on the covalent connection of a hydrophobic polypeptide to an oligonucleotide chain, wherein a hydroxyl group is modified at the C-terminus of a non-natural polypeptide, that is, a dipeptide with an Fmoc protecting group is synthesized, and an alkyl alcohol carbon chain with a specific length and properties is introduced, and then it is grafted to the 5' end of DNA by using a phosphoramidite chemistry method. When the DNA is aminolyzed from the CPG, the Fmoc protecting group of the non-natural polypeptide is also detached, thereby obtaining a natural dipeptide-DNA amphiphilic molecule; whereas the existing synthesis method first removes the protecting group on the polypeptide, and then obtains a polypeptide-DNA molecule by liquid phase synthesis on the polypeptide from which the protecting group has been removed; the method of the present invention omits the process of removing the Fmoc protecting group separately in the solid phase synthesis of the polypeptide, and instead removes the Fmoc protecting group on the amino group of the polypeptide while realizing the aminolysis of the DNA after grafting the DNA, thereby simplifying the synthesis steps.

[0015] 2. The amphiphilic FI-DNA molecules obtained by coupling endogenous macromolecular DNA with dipeptide derivatives are used as building units. The amphiphilic FI-DNA molecules are self-assembled in a buffer to form nanofibers. Compared with general polymer fibers, the amphiphilic nanoassemblies prepared by the present invention are qualitatively arranged on the periphery by specific DNA sequences. DNA can be further used to construct hierarchical self-assembled nanomaterials through complementary base pairing, and has excellent biocompatibility, non-toxicity, and other characteristics.

[0016] 3. The fourth-generation macrocyclic host cucurbituril can perform host-guest recognition with phenylalanine in the amphiphilic FI-DNA molecule prepared by the present invention at a molar ratio of 1:2. Cucurbituril, as the host molecule, has a cavity that can accommodate phenylalanine molecules and forms a stable host-guest complex with phenylalanine through interactions such as hydrophobic effects. The introduction of cucurbituril can change the stacking model of molecules, thereby achieving the effect of regulating the intermolecular stacking forces and thus realizing the change in the morphology of the assembly. This change is expected to be applied to drug delivery. This simple and widely applicable morphology control method can be used in the application of hierarchical self-assembly of various materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the H NMR spectrum of Fmoc-FI-C6-OH;

[0018] Figure 2 It is the NMR carbon spectrum of Fmoc-FI-C6-OH;

[0019] Figure 3 The electrophoresis results of the target product FI-DNA are shown in Figure 1. Lane 1 is the DNA raw material, and lane 2 is the FI-DNA product.

[0020] Figure 4 HPLC characterization of FI-DNA before and after purification;

[0021] Figure 5 is the FI-DNA time-of-flight mass spectrum;

[0022] Figure 6 For FI-DNA in H 2 Transmission electron microscopy (TEM) image of the assembly in O;

[0023] Figure 7 For FI-DNA in V DMSO :V H2O = Transmission electron microscopy (TEM) image of the assembly in a mixed solution of 1:20;

[0024] Figure 8 Transmission electron microscopy (TEM) images of FI-DNA assembled in PBS buffer;

[0025] Fig. 9 Dynamic light scattering characterization of the effect of CB[8] on the morphology of FI-DNA assemblies;

[0026] Fig.10 Transmission electron microscopy (TEM) images and assembly stacking model showing the effect of CB[8] on the morphology of FI-DNA assemblies;

[0027] Fig.11 is a flow chart of the solid phase synthesis of dipeptide Fmoc-FI in Example 1; DETAILED DESCRIPTION

[0028] The invention is further described below through examples in combination with the accompanying drawings. The examples are only used to further explain the technical solution of the present invention and cannot be considered as limiting the scope of protection of the present invention. Non-essential improvements or adjustments made by technicians in this field based on the contents of the above invention all fall within the scope of protection of the present invention.

[0029] Example 1

[0030] In this example, the target product FI-DNA was prepared by the following method, and the specific steps are as follows:

[0031] Step 1. Solid phase synthesis of dipeptide Fmoc-FI, the process is as follows Fig.11 As shown:

[0032] Specific experimental steps:

[0033] (A1) The carboxyl group of the first amino acid with a protective group is coupled through the chlorine atom of CTC to form an ester bond, thereby connecting the amino acid to the resin to form an amino acid-resin complex: 2 mL of N,N-dimethylformamide (DMF) is added to 500 mg of CTC resin (2-chlorotrityl chloride resin), transferred into a horn tube, and placed on a shaker for activation for 30 minutes; the waste liquid is blown out with an ear bulb, and the first amino acid with a protective group: 424.1 mg of N-(9-fluorenylmethoxycarbonyl)-L-isoleucine (Fmoc-L-isoleucine) is added, mixed with 2 mL of DMF containing 315 μL of N,N-diisopropylethylamine (DIPEA), and reacted for 8 hours; the resin is washed three times with DMF to wash away the excess amino acid. 1 mL of methanol is added for 30 minutes to cap the unreacted chlorine active sites on the resin, and the resin is washed three times with DMF.

[0034] (A2) Removal of 9-fluorenylmethoxycarbonyl protecting group (Fmoc) and detection: After the first amino acid coupling is completed, the Fmoc protecting group is removed with 20% piperidine / DMF solution to expose the amino group required for the next first amino acid coupling. After the resin is washed with DMF three times, the Kaiser test results in a blue color, which indicates the presence of the amino group, indicating that the deprotection is complete.

[0035] (A3) The amino group of the first amino acid is coupled to the carboxyl group of the second amino acid with a protective group: 387.4 mg of the second amino acid with a protective group: N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-L-phenylalanine) is added, along with 202.8 mg of 1-hydroxybenzotriazole (HOBT), 570 mg of 2-(7-aza- Benzotriazole )-N,N,N',N'- Tetramethyluronium hexafluorophosphate A mixed solution of HATU and 265 μL DIPEA in DMF was reacted for 8 h. After the reaction, the excess amino acids were washed away with DMF and the Kaiser test was performed again to show yellow, indicating that the coupling of phenylalanine was completed.

[0036] (A4) Cleavage of the carboxyl group of the first amino acid from the CTC resin to form a free dipeptide: The resin was cleaved several dozen times with a 2.5% trifluoroacetic acid / dichloromethane (TFA / DCM) solution, the mixed solution was collected, the solvent was removed, and ice ether was added for precipitation to obtain Fmoc-FI dipeptide, i.e., isoleucylphenylalanine dipeptide with the N-terminus protected by an Fmoc protecting group, with a yield of 82%.

[0037] Step 2. Synthesis of Fmoc-FI-C6-OH, the reaction equation is as follows:

[0038]

[0039] Specific experimental steps:

[0040] (B1) 250 mg of Fmoc-FI dipeptide was placed in a round-bottom flask, 25 mL of DCM was added, and the mixture was sonicated until dissolved. Then 89 mg of 6-amino-1-hexanol was added and sonicated until dissolved. Then 284 mg of peptide coupling reagent HATU and 130.6 μL of DIPEA were added, and the mixture was stirred at room temperature for 48 h.

[0041] (B2) After the reaction, the solvent DCM was removed by rotary evaporation, 50 mL of acetonitrile was added to dissolve the excess unreacted HATU and 6-amino-1-hexanol, and a white solid was obtained by suction filtration, and then rinsed twice with acetonitrile. The white solid was Fmoc-FI-C6-OH, and the yield was 85%.

[0042] Step 3. Solid Phase Synthesis of FI-DNA

[0043] The following DNA sequences are from Huzhou Hippo Biotechnology Co., Ltd.

[0044] Specific experimental steps:

[0045] (C1) Weigh 120 mg of Fmoc-FI-C6-OH obtained in step (B2) into a 10 mL round-bottom flask, add 3 mL of anhydrous and oxygen-free dichloromethane (DCM) under argon atmosphere (to prevent interference from oxygen and moisture), then add 204.48 μL of N,N-diisopropylethylamine (DIPEA), and stir at room temperature for 5 min to ensure that the reagents are fully mixed.

[0046] (C2) Take 140.88 μL of 2-cyanoethyl N, N-diisopropyl chlorophosphoramidite and add it below the liquid surface to prevent air from entering and causing oxidation of the product. Continue stirring at room temperature for 40 minutes. Use TLC to monitor the progress of the reaction to ensure that the reaction is complete. 2-cyanoethyl N, N-diisopropyl chlorophosphoramidite undergoes a substitution reaction with the hydroxyl group of Fmoc-FI-C6-OH to form a phosphoramidite ester, which is used to connect to the DNA chain. After the reaction is completed, use 5 mL of saturated NaHCO 3 The reaction was quenched with saturated NaHCO 3 The organic layer was extracted with anhydrous NaCl solution twice, water once, and saturated NaCl solution twice. 2 SO 4 After drying, the solvent was removed by rotary evaporation under argon atmosphere to obtain a white solid.

[0047] (C3) Add 2 mL of anhydrous and oxygen-free DCM to the white solid, sonicate for 1 min to dissolve it, take 1.5 mL and add it to a pear-shaped bottle containing 1 μmol CPG-DNA-OH (5'-HO-ACG CGC CCA-CPG, purchased from Huzhou Hippo Biotechnology Co., Ltd.) and 13.017 mg of ethylthiotetrazole, and react for 3 h under argon atmosphere. The hydroxyl group of the 5' end DNA reacts with the phosphoramidite described in (C2) to undergo FI-DNA coupling reaction. After the reaction is completed, the solid is washed with dichloromethane and acetonitrile in turn to remove unreacted reagents and impurities. Then, 1 mL of oxidizing reagent (I 2 |THF|H 2 O|Py) oxidation for 1 min to oxidize the phosphoramidite to a phosphodiester bond. The excess oxidant was washed away with acetonitrile.

[0048] (C4) Add 1 mL of aqueous ammonia and react at 55° C. for 3 h. Transfer the liquid to another 1.5 mL centrifuge tube, place it in a concentrator to remove the aqueous ammonia, and purify it using high performance liquid chromatography (HPLC) to obtain a pure FI-DNA product.

[0049] The products obtained in the above steps (C2) and (C3) were characterized and analyzed, and the results were as follows:

[0050] (1) Using a nuclear magnetic resonance spectrometer to characterize the product obtained in step (B2), i.e., a white solid, such as Figure 1 , Figure 2 shown.

[0051] (2) Prepare a 20% denaturing polyacrylamide gel to characterize the product obtained in step (C4). The electrophoresis condition is 150V for 1h. The results are as follows: Figure 3 As shown, the migration rate of the FI-DNA band is significantly slower than that of the raw DNA band, which indicates the successful coupling of the hydrophobic dipeptide to DNA.

[0052] (3) The product obtained in step (C3) was characterized by HPLC, and the eluent used in HPLC was 0.1 M TEAA and anhydrous acetonitrile. Figure 4 , collecting the product at a retention time of 21.3 min.

[0053] (4) The purified product of step (C4) is characterized by time-of-flight mass spectrometry, and the proton peak of the target product can be obtained. Figure 5 The ordinate of the figure is the percentage of ion signal intensity, and the highest peak of the time-of-flight mass spectrum represents the content of the target product FI-DNA.

[0054] Example 2

[0055] The target product FI-DNA nanofiber is prepared by the following method, and the specific preparation steps are as follows:

[0056] The FI-DNA molecules purified in step (C4) of Example 1 were dissolved in H 2 O.V DMSO :V H2O =1:20 mixed solution and PBS buffer, then, the prepared solution was kept at 90°C for 30 minutes, naturally cooled to room temperature, and after standing for 12 hours, the FI-DNA nanoassembly was obtained.

[0057] like Figures 6 to 8 As shown in Figure 2, the nanoassemblies prepared above were characterized by transmission electron microscopy (TEM). 2 In V O, TEM showed that FI-DNA formed filamentous nanoassemblies. Compared with the assembly in pure water, the introduction of the organic solvent DMSO made the hydrophobic part more stretchable in the solution, allowing the amphiphilic molecules to assemble in an orderly manner. DMSO :V H2O =1:20, TEM showed that FI-DNA formed nanofibers with an average diameter of 15nm. In a solution with high salt concentration, salt ions stabilize the charge on the DNA phosphate backbone and weaken the electrostatic repulsion between molecules, leading to the formation of a more ordered assembly. In PBS buffer, FI-DNA formed nanofibers with the most regular morphology and an average diameter of 16nm.

[0058] Example 3

[0059] In Example 2, it was confirmed that the most regular nanofibers can be obtained in PBS buffer. Under this condition, this example achieves morphology control of the FI-DNA assembly by the following method.

[0060] (1) 25 μL of a 100 μM cucurbituril CB[8] aqueous solution was added to the nanoassembly prepared in Example 2. After thorough mixing, the prepared solution was kept at 90°C for 30 min, cooled naturally to room temperature, and allowed to stand for 12 h. The solution was then characterized by dynamic light scattering and transmission electron microscopy (TEM). Fig. 9 and Fig.10 As shown, dynamic light scattering showed that the size of the assembly was significantly reduced. CB[8] was mixed with FI-DNA at a molar ratio of 1:2. After CB[8] recognized the phenylalanine at the end of the hydrophobic dipeptide at a molar ratio of 1:2, CB[8] changed the molecular stacking model, making the molecules more densely stacked, and obtained thermodynamically stable nanomicelles with an average diameter of 19 nm. The morphology did not change after standing for another 3 days.

[0061] (2) Drug loading and drug release scheme of the nanoassemblies in Examples 2 and 3:

[0062] After the FI-DNA@CB[8] solution and the hydrophobic drug molecules (Dox for example) are mixed evenly, Dox is encapsulated in the formed nanoassembly after annealing. Then, the targeted nucleic acid aptamer is added to the assembly solution and incubated at room temperature for 12 hours. The nucleic acid aptamer can be loaded on the periphery of the assembly through base complementary pairing. When entering the cell, the nucleic acid aptamer increases the cancer cell's uptake of the nanoassembly. After entering the cell, the DNA enzyme in the cell destroys the structure of the assembly, realizing the gradual release of Dox, achieving the effect of targeted delivery of chemotherapy drugs to kill cancer cells.

Claims

1. A hydrophobic polypeptide grafted with an oligonucleotide, characterized in that: The hydrophobic polypeptide of the grafted oligonucleotide comprises: a first amino acid connected to the carboxyl group of phenylalanine through a peptide chain, an alkyl alcohol connected to the carboxyl group of the first amino acid through a peptide bond, and an oligonucleotide single chain connected to the hydroxyl group of the alkyl alcohol through phosphoramidite.

2. The hydrophobic polypeptide grafted with oligonucleotide according to claim 1, characterized in that The first amino acid includes isoleucine, the carbon number of the alkyl alcohol is 5 to 16, and the structural formula of the hydrophobic polypeptide of the grafted oligonucleotide is as follows: Its molecular structure is as follows, wherein n=3-14.

3. The hydrophobic polypeptide grafted with oligonucleotide according to claim 2, characterized in that The number of bases of the single-stranded oligonucleotide is 9, and the sequence of the single-stranded oligonucleotide is HO-D9-CPG:5'-HO-ACG CGC CCA-CPG.

4. A nanoassembly, characterized in that: The hydrophobic polypeptide grafted with oligonucleotide according to any one of claims 1 to 3 is dissolved in water or a mixed solution of DMSO and water in a volume ratio of 1:

20.

5. A nanoassembly, characterized in that: The hydrophobic polypeptide grafted with oligonucleotide according to any one of claims 1 to 3 is dissolved in PBS buffer to form nanofibers with an average diameter of 16 nm.

6. A nanoassembly, characterized in that: The aqueous solution of cucurbituril and the nanoassembly according to claim 5 are mixed at a molar ratio of cucurbituril to phenylalanine of 1:2, and the nanomicelles with an average diameter of 19 nm are self-assembled.

7. A method for preparing a hydrophobic polypeptide grafted with an oligonucleotide according to any one of claims 1 to 3, characterized in that: The following steps are included: (1) coupling and fixing the carboxyl group of the first amino acid with an Fmoc protecting group to a resin by a solid phase synthesis method, removing the Fmoc protecting group after the coupling of the first amino acid is completed, coupling the carboxyl group of phenylalanine with an Fmoc protecting group to the amino group of the first amino acid, and after the coupling of the two amino acids is completed, cleaving the resin to obtain an Fmoc-protected hydrophobic polypeptide, i.e., an Fmoc-polypeptide, wherein the above amino acids are all hydrophobic amino acids; (2) subjecting the Fmoc-polypeptide obtained in step (1) to an amide condensation reaction with an alkyl alcohol to obtain an Fmoc-protected polypeptide derivative; (3) The Fmoc-protected polypeptide derivative obtained in step (2) is reacted with a phosphochlorine reagent to generate an intermediate product, which is then subjected to a coupling substitution reaction with the 5'-terminal hydroxyl group of the oligonucleotide single chain loaded on the CPG bead, and the CPG bead is removed by aminolysis, and the Fmoc protecting group of the polypeptide is removed at the same time, and then the hydrophobic polypeptide grafted with the oligonucleotide is obtained after purification.

8. The preparation method according to claim 7, characterized in that: In the step (1), the first amino acid is selected from one of isoleucine, leucine and valine; in the step (2), the number of carbon atoms in the alkyl alcohol is 5 to 16, and the product obtained in the step (2) is Fmoc-FI-Cn-OH; the number of bases in the oligonucleotide single chain is 9, and the sequence of the oligonucleotide single chain is HO-D9-CPG: 5'-HO-ACG CGC CCA-CPG; the hydrophobic polypeptide of the grafted oligonucleotide is FI-DNA.

9. The preparation method according to claim 7, characterized in that: In the step (2), the molar ratio of the Fmoc-polypeptide to the alkyl alcohol is 2:3, and the alkyl alcohol is one of 6-amino-1-hexanol and 5-amino-1-hexanol.

10. The preparation method according to claim 7, characterized in that: The resin in step (1) is CTC resin; the Fmoc protecting group is removed by cleavage with 2.5% trifluoroacetic acid / dichloromethane solution, and after removing the solvent, the Fmoc-polypeptide, namely Fmoc-FI, is obtained by precipitation with icy ether.

11. Use of the nanoassembly according to any one of claims 3 to 6 in the preparation of sustained-release drugs, characterized in that: The drug comprises a nucleic acid aptamer and a hydrophobic drug molecule. The nucleic acid aptamer and the oligonucleotide single strand in the nanoassembly are loaded outside the nanoassembly through base complementary pairing, and the hydrophobic drug molecule is encapsulated in the nanoassembly.

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