DNA nano-material-based regulator for promoting interaction between lysosome and mitochondria and preparation method thereof

Abstract

The invention discloses a regulator for promoting interaction between lysosome and mitochondria based on a DNA nano-material and a preparation method of the regulator, the regulator is of a DNA nano-structure and is formed by connecting a DNA aptamer targeted by a targeted lysosome membrane protein CD63, connecting DNA and a triphenylphosphine molecule with targeted mitochondria, and a B3 sequence of an ultraviolet light cutting site and a disulfide bond is designed at a position complementary to the DNA aptamer to close the DNA aptamer. The regulator provided by the invention has good targeting property on lysosome and mitochondria, has good controllability and biocompatibility, is easy to biodegrade, and does not influence the expression of proteins on the surfaces of the lysosome and the mitochondria.

Description

Technical Field

[0001] The present invention belongs to the technical field of organelle interaction regulators, and in particular relates to a regulator based on DNA nanomaterials for promoting the interaction between lysosomes and mitochondria, and a preparation method thereof. Background Art

[0002] Both mitochondria and lysosomes are dynamic regulators that maintain cell homeostasis. Mitochondria provide power to cells by producing ATP through intracellular oxidative phosphorylation. Mitochondrial dysfunction can cause cell aging, metabolic disorders, and a variety of neurodegenerative diseases. Lysosomes participate in the recycling of intracellular substances and provide cells with energy and nutrients. Lysosomes contain more than 60 hydrolases that can nitrate almost all types of biopolymers, including proteins, carbohydrates, nucleic acids, and lipids. Mitochondria-lysosome interactions regulate a variety of cellular life activities, such as cell metabolism. Under physiological and pathological conditions, mitochondria and lysosomes often establish some short-term contacts, which are also called mitochondria-lysosome contacts (MLCs), allowing them to exchange lipids, proteins, ions, and other molecules, and trigger mitochondrial fission through lysosomes. In addition, mitophagy is also a key biological process that requires the participation of mitochondria and lysosomes. As a type of autophagy, mitophagy is an important mechanism for controlling mitochondrial quality to eliminate damaged or dysfunctional mitochondria to maintain cell survival. Generally speaking, the occurrence of mitophagy increases the adaptability of cancer to mitochondrial targeted drugs. However, recent studies have shown that excessive or long-term mitophagy can lead to cancer cell death ([1] Zhang H, Xuan X, Wang Y, et al. In situautophagy regulation in synergy with phototherapy for breast cancer treatment [J]. Acta Pharmaceutica Sinica B, 2024, 14 (5): 2317-2332. [2] Ji M, Sun L, Zhang M, et al. RN0D, agalactoglucan from Panax notoginseng flower induces cancer cell death via PINK1 / Parkin mitophagy [J]. Carbohydrate Polymers, 2024, 332: 121889.). Therefore, it is of great significance to develop new regulators of lysosome-mitochondrial interactions.

[0003] DNA nanostructures have excellent programmability, addressability, and near-atomic structural accuracy. Compared with DNA oligonucleotides, DNA nanostructures show better stability in physiological environments containing nucleases. These advantages, coupled with the inherent biocompatibility and degradability of DNA materials, make DNA nanostructures superior to other materials in in vivo applications. With the continuous deepening of tumor cell research, it is known that mutations in key genes and proteins in cells usually occur at the subcellular level. More and more studies are focusing on subcellular organelles as targets for anti-tumor drugs. Nanomedicines can be modified with some targeting units so that they can be precisely located in subcellular organelles, thereby enhancing their ability to destroy tumor tissues and reducing the dosage of nanomedicines, thereby reducing side effects and improving therapeutic efficacy ([3] Gough N R. Emerging roles for organelles in cellular regulation [J]. Science Signaling, 2016, 9 (457): eg11-eg11. [4] Zhen W, An S, Wang S, et al. Precise subcellular organelle targeting for boosting endogenous-stimuli-mediated tumor therapy [J]. Advanced Materials, 2021, 33 (51): 2101572.). Therefore, DNA nanomaterials can provide a suitable platform for organelle regulation. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a regulator based on DNA nanomaterials for promoting the interaction between lysosomes and mitochondria and a preparation method thereof.

[0005] In the first aspect, an embodiment of the present invention provides a regulator based on DNA nanomaterials for promoting the interaction between lysosomes and mitochondria. The regulator is a DNA nanostructure, which is formed by connecting a DNA aptamer targeting the lysosomal membrane protein CD63, a connecting DNA, and a triphenylphosphine molecule targeting mitochondria, and a B3 sequence with an ultraviolet cleavage site and a disulfide bond is designed at a position complementary to the DNA aptamer to block the DNA aptamer.

[0006] Furthermore, the DNA aptamer is formed by connecting a fixed sequence with a repeated base sequence.

[0007] Furthermore, the DNA aptamer has the following fixed sequence:

[0008] CACCCCACCTCGCTCCCGTGACACTAATGCTAGTTCTA.

[0009] Furthermore, the number of repeated bases is 20, 40, 60, or 80.

[0010] Furthermore, the number of repeated bases is preferably 60.

[0011] Furthermore, the B3 sequence is:

[0012] TAGCATTAGTGIPClinkerTCACGGGAGCGSH-SHAGGTGGGGTG.

[0013] Furthermore, the labeling site of the triphenylphosphine molecule is connected to the 3' end of the DNA.

[0014] In a second aspect, an embodiment of the present invention provides a method for preparing a regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials, the method comprising:

[0015] Step S1, designing and synthesizing a DNA aptamer having a photo-cleavage site and a disulfide bond and a DNA backbone for connecting DNA;

[0016] Step S2, mixing propargyl triphenylphosphine with DNA labeled with an azide group, adding dimethyl sulfoxide and ascorbic acid solution, and then adding copper sulfate solution, reacting fully at room temperature, purifying to obtain a DNA backbone labeled with a TPP molecule targeting mitochondria, and ultrafiltration to obtain a purified dual-targeting chimera;

[0017] Step S3, reacting the dual-targeting chimera with the complementary sequence B3 at 95°C for 5 minutes and then continuously cooling to 25°C at a rate of 1°C every 30 seconds to form a complementary double chain, thereby obtaining a regulator.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The regulator of the present invention has good targeting to lysosomes and mitochondria, good controllability and biocompatibility, can combine large and small molecules to regulate the activities between the two organelles, is easily biodegradable, and will not affect the expression of lysosome and mitochondrial surface proteins. It is of great significance in the clinical application and development in the field of cell biology research and precision diagnosis and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0021] Figure 1 Schematic diagram of the preparation of regulators and the process of inducing the interaction between lysosomes and mitochondria;

[0022] Figure 2 Co-localization analysis of the degree of contact between lysosomes and mitochondria induced by the regulators containing base-linked DNA of different lengths prepared in Examples 1 to 3;

[0023] Figure 3 Transmission electron micrograph of the regulator-induced interaction between lysosomes and mitochondria. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] It should be noted that, in the absence of conflict, the features in the following embodiments and implementations may be combined with each other.

[0026] like Figure 1 As shown, an embodiment of the present invention provides a regulator based on DNA nanomaterials for promoting the interaction between lysosomes and mitochondria, wherein the regulator is a DNA nanostructure, which is formed by connecting a DNA aptamer targeting the lysosomal membrane protein CD63, a connecting DNA, and a triphenylphosphine molecule targeting mitochondria, and a B3 sequence with an ultraviolet cleavage site and a disulfide bond is designed at a position complementary to the DNA aptamer to block the DNA aptamer.

[0027] Furthermore, the DNA aptamer is formed by connecting a fixed sequence with a repeated base sequence. The fixed sequence is as follows:

[0028] CACCCCACCTCGCTCCCGTGACACTAATGCTAGTTCTA.

[0029] Furthermore, the number of repeated bases is 20, 40, 60, 80, preferably 60.

[0030] Furthermore, the B3 sequence is:

[0031] TAGCATTAGTGIPClinkerTCACGGGAGCGSH-SHAGGTGGGGTG.

[0032] Furthermore, the labeling site of the triphenylphosphine molecule is connected to the 3' end of the DNA.

[0033] like Figure 1 As shown, the present invention also provides a method for preparing a regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials, the method comprising:

[0034] Step S1, designing and synthesizing a DNA aptamer having a photo-cleavage site and a disulfide bond and a DNA backbone for connecting DNA;

[0035] Step S2, mixing propargyl triphenylphosphine with DNA labeled with an azide group, adding dimethyl sulfoxide and ascorbic acid solution, and then adding copper sulfate solution, at room temperature for more than 24 hours, purifying with a silica gel column to obtain a DNA backbone labeled with a TPP molecule targeting mitochondria, and ultrafiltration using a 10kD ultrafiltration tube for more than 3 times to obtain a purified dual-targeting chimera;

[0036] Step S3, reacting the dual-targeting chimera with the complementary sequence B3 at 95°C for 5 minutes and then continuously cooling to 25°C at a rate of 1°C every 30 seconds to form a complementary double chain, thereby obtaining a regulator.

[0037] The present invention is further described in detail below with reference to specific embodiments and accompanying drawings.

[0038] Example 1

[0039] Schematic diagram of the preparation of lysosomal regulators based on DNA nanomaterials and the induction of mitochondrial aggregation and fusion Figure 1 As shown, the specific synthesis steps are as follows:

[0040] (1) Preparation of dual-targeting aptamers: Design and synthesize a DNA backbone with a linker sequence, the number of bases in the linker sequence = 60, 5 μL, 5 mM propargyl triphenylphosphine and 200 μL, 100 μM DNA backbone labeled with an azide group are mixed, and then 250 μL DMSO and 40 μL, 50 mM ascorbic acid are added. 50 μL, 10 mM copper sulfate solution is added to the above mixed solution and reacted at room temperature for 24 hours and then purified by silica gel column to obtain the linker DNA: CACCCCACCTCGCTCCCGTGACACTAATGCTATTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT. After the reaction, a 10 kD ultrafiltration tube is used for ultrafiltration 3 times to obtain a purified single-stranded DNA dual-targeting aptamer.

[0041] (3) The linked DNA dual-targeting aptamer obtained in step (1) was mixed with 200 μL of 20 μM complementary DNA B3 sequence, reacted at 95°C for 5 min, and then continuously cooled to 25°C at a rate of 1°C every 30 s to form a complementary double strand, thereby obtaining a regulator that promotes the interaction between lysosomes and mitochondria.

[0042] Example 2

[0043] This embodiment is basically the same as embodiment 1, the only difference being that the number of bases selected for the connecting DNA is 80, and the connecting DNA obtained is:

[0044] CACCCCACCTCGCTCCCGTGACACTAATGCTATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0045] Example 3

[0046] This embodiment is basically the same as embodiment 1, the only difference being that the number of bases selected for the connecting DNA is 40, and the connecting DNA obtained is:

[0047] CACCCCACCTCGCTCCCGTGACACTAATGCTATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0048] Example 4

[0049] This embodiment is basically the same as embodiment 1, the only difference being that the number of bases selected for the connecting DNA is 20, and the connecting DNA obtained is:

[0050] from Figure 2 The confocal experiment showed that when the number of linker sequence bases was 60, the co-localization effect of mitochondria and lysosomes was best.

[0051] Example 5

[0052] The line regulator prepared in Example 1 will first target the mitochondria. Under the simultaneous action of ultraviolet light and glutathione, the regulator will further target the lysosomes to achieve the interaction between mitochondria and lysosomes. We took transmission electron microscopy photos of the regulator.

[0053] 500 ng / ul of the regulator was transfected into cancer cells and incubated at 37°C for 6 hours, then exposed to UV light for 10 minutes. After further incubation for 12 hours, the cells were fixed with 2.5% pentanediol, sectioned, and imaged using a transmission electron microscope.

[0054] from Figure 3 It can be seen that compared with the control group samples (without adding any regulators), in the samples containing regulators, the state of mitochondria changed and the autophagy phenomenon increased, indicating that the regulator can promote the contact between lysosomes and mitochondria and cause mitochondrial autophagy.

[0055] Those skilled in the art will readily appreciate other embodiments of the present application after considering the description and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application. The description and examples are intended to be exemplary only.

[0056] It should be understood that the present application is not limited to the exact construction that has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof.

Claims

1. A regulator based on DNA nanomaterials for promoting the interaction between lysosomes and mitochondria, characterized in that: The regulator is a DNA nanostructure, which is formed by connecting a DNA aptamer targeting lysosomal membrane protein CD63, a connecting DNA, and a triphenylphosphine molecule targeting mitochondria, and a B3 sequence with an ultraviolet cleavage site and a disulfide bond is designed at a position complementary to the DNA aptamer to close the DNA aptamer.

2. The regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials according to claim 1, characterized in that: The DNA aptamer is formed by connecting a fixed sequence with a repeated base sequence.

3. The regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials according to claim 2, characterized in that: The DNA aptamer has the following fixed sequence: CACCCCACCTCGCTCCCGTGACACTAATGCTAGTTCTA.

4. The regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials according to claim 2, characterized in that: The number of repeated bases is 20, 40, 60, and 80.

5. The regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials according to claim 4, characterized in that: The number of repeating bases is preferably 60.

6. The regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials according to claim 1, characterized in that: The B3 sequence is: TAGCATTAGTGIPClinkerTCACGGGAGCGSH-SHAGGTGGGGTG.

7. The regulator for promoting the interaction between lysosomes and mitochondria based on DNA nanomaterials according to claim 1, characterized in that: The labeling site of the triphenylphosphine molecule is the 3' end of the attached DNA.

8. A method for preparing a regulator for promoting interaction between lysosomes and mitochondria based on DNA nanomaterials according to any one of claims 1 to 7, characterized in that: The method comprises: Step S1, designing and synthesizing a DNA aptamer having a photo-cleavage site and a disulfide bond and a DNA backbone for connecting DNA; Step S2, mixing propargyl triphenylphosphine with DNA labeled with an azide group, adding dimethyl sulfoxide and ascorbic acid solution, and then adding copper sulfate solution, reacting fully at room temperature, purifying to obtain a DNA backbone labeled with a TPP molecule targeting mitochondria, and ultrafiltration to obtain a purified dual-targeting chimera; Step S3, reacting the dual-targeting chimera with the complementary sequence B3 at 95°C for 5 minutes and then continuously cooling to 25°C at a rate of 1°C every 30 seconds to form a complementary double chain, thereby obtaining a regulator.

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