Protein-degrading DNA materials, their preparation methods and applications

Abstract

This invention discloses a protein-degrading DNA material, its preparation method, and its applications. The protein-degrading DNA material includes a degradation initiation module, a protein targeting module, and a response module. The degradation initiation module is an E3 ubiquitin ligase targeting module, and the response module is a telomerase response module. The preparation method includes the following steps: dissolving the ssDNA strand used in the degradation initiation module in a solvent and annealing it to obtain the degradation initiation module; dissolving the ssDNA strand used in the protein targeting module in a solvent and annealing it to obtain the protein targeting module; dissolving the ssDNA strand used in the response module in a solvent and annealing it to obtain the response module; and mixing the degradation initiation module, the protein targeting module, and the response module to obtain the protein-degrading DNA material. The protein-degrading DNA material of this invention possesses tumor microenvironment triggering characteristics, can precisely target tumor cells, and has a high efficiency in degrading pathogenic proteins.

Description

Technical Field

[0001] This invention belongs to the field of nanobiotechnology, specifically relating to a protein-degradable DNA material, its preparation method, and its application. Background Technology

[0002] Current protein degradation technologies are mainly divided into chemically synthesized small molecule degradative agents (such as PROTAC) and degradation systems based on biomacromolecules. In the field of DNA nanotechnology, existing technologies typically employ the following preparation schemes: 1) Multi-stranded DNA origami / complex nanoassembly scheme: Hundreds of short single-stranded DNA strands are self-assembled with a long scaffold through thermal denaturation followed by programmed degradation temperature to construct large DNA nanostructures. Subsequently, protein ligands or degradation tags are coupled to the surface of the structure through chemical modification or physical adsorption.

[0003] 2) Traditional chemical cross-linking scheme: The DNA sequence with the target function (such as nucleic acid aptamer) is directly linked to the artificially synthesized degradation inducing molecule through chemical reaction (such as Click chemistry, amino-carboxyl condensation, etc.), and the target product is obtained by purification by chromatographic column.

[0004] Existing methods for preparing protein-degraded DNA nanomaterials typically suffer from drawbacks such as complex and costly preparation processes, difficult purification, and low yields, which require further improvement. Summary of the Invention

[0005] The purpose of this invention is to provide a protein-degrading DNA material, its preparation method and application. It is simple to prepare, has tumor microenvironment triggering characteristics, can accurately target tumor cells, and has a high efficiency in degrading pathogenic proteins.

[0006] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0007] A protein-degrading DNA material, comprising a degradation initiation module, a protein targeting module, and a response module, wherein the degradation initiation module is an E3 ubiquitin ligase targeting module, and the response module is a telomerase response module.

[0008] In one or more embodiments of the present invention, the degradation initiation module is assembled from ssDNA1, ssDNA2, and ssDNA3 connected with E3 ubiquitin ligase targeting polypeptides, wherein the ssDNA1 sequence is shown in SEQ ID NO.1, the ssDNA2 sequence is shown in SEQ ID NO.2, and the ssDNA3 sequence is shown in SEQ ID NO.3.

[0009] In one or more embodiments of the present invention, the E3 ubiquitin ligase targeting polypeptide is LA-(Hyp)-YI-(L-Pra).

[0010] In one or more embodiments of the present invention, the protein targeting module is a TRF protein targeting module; and / or,

[0011] The protein targeting module is assembled from ssDNA4, ssDNA5, and ssDNA6, wherein the ssDNA4 sequence is shown in SEQ ID NO.4, the ssDNA5 sequence is shown in SEQ ID NO.5, and the ssDNA6 sequence is shown in SEQ ID NO.6.

[0012] In one or more embodiments of the present invention, the response module is assembled from ssDNA7, ssDNA8, ssDNA9 and ssDNA-X, wherein the ssDNA7 sequence is shown in SEQ ID NO.7, the ssDNA8 sequence is shown in SEQ ID NO.8 and the ssDNA9 sequence is shown in SEQ ID NO.9;

[0013] Among them, ssDNA-X satisfies any of the following sets:

[0014] The La1 and Lb1 groups, wherein the La1 sequence is shown in SEQ ID NO.10 and the Lb1 sequence is shown in SEQ ID NO.11;

[0015] The La2 and Lb2 groups, wherein the La2 sequence is shown in SEQ ID NO.12 and the Lb2 sequence is shown in SEQ ID NO.13;

[0016] The La3 and Lb3 groups, wherein the La3 sequence is shown in SEQ ID NO.14 and the Lb3 sequence is shown in SEQ ID NO.15.

[0017] In one or more embodiments of the present invention, the molar ratio of the degradation initiation module, the protein targeting module, and the response module is 1:1:2 to 1:1:5.

[0018] Another specific embodiment of the present invention provides the following technical solution:

[0019] A method for preparing a protein-degradable DNA material, the method comprising the following steps:

[0020] The ssDNA strand used in the degradation initiation module was dissolved in a solvent and then annealed to obtain the degradation initiation module.

[0021] The ssDNA strand used in the protein targeting module is dissolved in a solvent and then annealed to obtain the protein targeting module.

[0022] The ssDNA strand used in the response module is dissolved in a solvent and then annealed to obtain the response module.

[0023] The degradation initiation module, protein targeting module, and response module are mixed to obtain protein-degraded DNA material.

[0024] In one or more embodiments of the present invention, the concentration of any one of the ssDNA strands used in the degradation initiation module, the protein targeting module, and the response module in the solvent is 10µM-30µM; and / or,

[0025] The solvent is 1× TAE Mg 2+ Buffer solution, wherein Mg 2+ The concentration is 10mM-20mM.

[0026] In one or more embodiments of the present invention, the annealing procedure in any one of the preparation steps of the degradation initiation module, protein targeting module, and response module is as follows: 95°C for 2 min; 65°C for 2 min; 60°C for 5 min; 60°C for 30 s, 40 cycles; 20°C for 30 s; and 10°C for heat treatment.

[0027] Another specific embodiment of the present invention provides the following technical solution:

[0028] Application of a protein-degraded DNA material or a protein-degraded DNA material prepared by a method thereof in tumor treatment and anti-tumor drug preparation.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The preparation process is extremely simplified, significantly improving production efficiency and product yield: Traditional DNA nanostructures (such as DNA origami) typically require hundreds of auxiliary strands and annealing times of tens of hours. This invention employs a modular "one-pot" assembly method based on a core framework, where the three assembly units can complete self-assembly within hours.

[0031] 2. Precise temporal and spatial control of degradation activity is achieved, reducing off-target toxicity: Existing materials are mostly in a continuously activated state, easily causing degradation in non-lesion tissues. This invention integrates telomerase response sequences during the preparation process, giving the material tumor microenvironment-triggered characteristics.

[0032] 3. It achieves precise quantification and spatial configuration at the molecular level, thereby improving degradation efficiency: Unlike the randomness of ligand ratios and structural heterogeneity caused by chemical cross-linking methods, this invention utilizes the programmability of the DNA double helix to strictly match the effective metric ratio of the targeting aptamer and the degradation unit.

[0033] 4. High scalability and compatibility: The preparation framework of this invention has a high degree of modularity. By simply changing the nucleic acid aptamer sequence in the ssDNA chain, it can be quickly transformed into a degradation platform for other pathogenic proteins. This "plug and play" characteristic makes this preparation method highly valuable and competitive in the field of new drug development that targets a wide variety of diseases. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram and gel electrophoresis image of the ssDNA1 ligation with the E3 ubiquitin ligase targeting polypeptide in Example 1 of the present invention.

[0036] Figure 2 Y in Embodiment 2 of the present invention E3 - Gel electrophoresis image of DNA;

[0037] Figure 3 Y in Embodiment 3 of the present invention TRF - Gel electrophoresis image of DNA;

[0038] Figure 4 This is a gel electrophoresis image of TL-DNA in Example 4 of the present invention;

[0039] Figure 5 The fluorescence emission spectra of each group of samples in Example 5 of this invention are in the wavelength range of 550-700 nm.

[0040] Figure 6 This is a quantitative statistical analysis of the fluorescence intensity at the characteristic emission peak (~580nm) of TAMRA in Example 5 of the present invention;

[0041] Figures 7-9 These are gel electrophoresis images of each group of samples in Example 7 of the present invention;

[0042] Figure 10 This is the result of Western blotting of proteins in Example 8 of the present invention. Detailed Implementation

[0043] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0044] Existing methods for preparing protein-degrading DNA nanomaterials suffer from the following drawbacks: Complex and costly preparation processes: 1) Assembly of complex structures such as DNA origami often requires a large number of auxiliary strands, involving cumbersome sequence design and extremely time-consuming annealing procedures (usually several hours or even days), hindering large-scale preparation and industrial production. 2) Difficult purification and low yield: Complex DNA nanoassemblies are prone to generating byproducts or incompletely assembled single strands during preparation, requiring time-consuming and inefficient purification via PAGE gel electrophoresis or HPLC, resulting in extremely low final product yields. 3) Difficulty in precisely controlling spatial orientation and proportions: Traditional chemical cross-linking methods often struggle to precisely control the grafting positions and proportions of degradation functional units on the DNA strand, leading to significant heterogeneity at the molecular level in the prepared nanomaterials, affecting the uniformity and stability of the material structure. 4) Strong dependence on the assembly environment: Existing technologies often require specific high-salt-concentration buffer systems for assembly, limiting the flexibility of the preparation process, and the material structure is easily disintegrated once the environment changes.

[0045] A specific embodiment of the present invention provides a protein-degrading DNA material, which includes a degradation initiation module, a protein targeting module, and a response module, wherein the degradation initiation module is an E3 ubiquitin ligase targeting module, and the response module is a telomerase response module.

[0046] Specifically, the degradation initiation module (Y E3 The ssDNA is assembled from ssDNA1, ssDNA2, and ssDNA3, which are linked with an E3 ubiquitin ligase targeting polypeptide. The ssDNA1 sequence is shown in SEQ ID NO.1, the ssDNA2 sequence is shown in SEQ ID NO.2, and the ssDNA3 sequence is shown in SEQ ID NO.3. The E3 ubiquitin ligase targeting polypeptide is LA-(Hyp)-YI-(L-Pra).

[0047] The protein targeting module is the TRF protein targeting module (Y TRFssDNA), which is assembled from ssDNA4, ssDNA5, and ssDNA6. The ssDNA4 sequence is shown in SEQ ID NO.4, the ssDNA5 sequence is shown in SEQ ID NO.5, and the ssDNA6 sequence is shown in SEQ ID NO.6.

[0048] The response module (TL-DNA) is a telomerase response module, which is assembled from ssDNA7, ssDNA8, ssDNA9 and ssDNA-X. The ssDNA7 sequence is shown in SEQ ID NO.7, the ssDNA8 sequence is shown in SEQ ID NO.8, and the ssDNA9 sequence is shown in SEQ ID NO.9.

[0049] Among them, ssDNA-X satisfies any of the following groups: La1 and Lb1 group, where the La1 sequence is shown in SEQ ID NO.10 and the Lb1 sequence is shown in SEQ ID NO.11; La2 and Lb2 group, where the La2 sequence is shown in SEQ ID NO.12 and the Lb2 sequence is shown in SEQ ID NO.13; La3 and Lb3 group, where the La3 sequence is shown in SEQ ID NO.14 and the Lb3 sequence is shown in SEQ ID NO.15.

[0050] The protein-degrading DNA material of this invention is a mixed system of a degradation initiation module, a protein targeting module, and a response module, with a molar ratio of 1:1:2 to 1:1:5, specifically 1:1:2, 1:1:3, 1:1:4, and 1:1:5. After delivery into telomerase-overexpressing tumor cells, this mixed system… E3 -DNA and Y TRF -DNA is pre-bound to E3 ubiquitin ligase and TRF1 and TRF2 respectively; TL-DNA is activated by telomerase in the cell and releases L-DNA, which then mediates the in-situ assembly of the three into a Y / L-shaped DNA nanoassembly. This assembly reduces the spatial distance between E3 and TRF1 / 2, induces TRF1 / 2 ubiquitination and degradation by the proteasome, and achieves targeted protein degradation.

[0051] Another specific embodiment of the present invention provides a method for preparing protein-degraded DNA material, which specifically includes the following steps:

[0052] Step 1: Prepare raw materials.

[0053] Specifically, ssDNA1, ssDNA2, and ssDNA3, which are linked to E3 ubiquitin ligase targeting peptides, are dissolved in 1×TAEMg. 2+ Buffer solution, to obtain the first solution.

[0054] Dissolve ssDNA4, ssDNA5, and ssDNA6 in 1×TAE Mg 2+ Buffer solution, to obtain the second solution.

[0055] Dissolve ssDNA8, ssDNA9, and ssDNA-X in 1×TAE Mg 2+ Buffer solution, to obtain the third solution.

[0056] The buffer solution used contains 10mM-20mM Mg 2+ .

[0057] The concentrations of ssDNA1, ssDNA2, ssDNA3, ssDNA4, ssDNA5, ssDNA6, ssDNA7, ssDNA8, ssDNA9, and ssDNA-X in the first, second, and third solutions were all 10µM-30µM. When ssDNA-X was in the La1 and Lb1 group, the concentrations of La1 and Lb1 were both 10µM-30µM. Similarly, when ssDNA-X was in the La2 and Lb2 group or the La3 and Lb3 group, the concentrations of La2, Lb2, La3, and Lb3 were all 10µM-30µM.

[0058] Step 2, program self-assembly.

[0059] Specifically, the first, second, and third solutions were placed in a PCR instrument and subjected to an annealing program. The annealing program was as follows: 95°C for 2 min; 65°C for 2 min; 60°C for 5 min; 60°C for 30 s, for 40 cycles; 20°C for 30 s; and incubation at 10°C. Through the annealing program, a stable degradation initiation module (Y) was formed based on the base pairing principle. E3 -DNA), protein targeting module (Y TRF -DNA), response module (TL-DNA).

[0060] Step 3, mix.

[0061] Specifically, the degradation initiation module (Y) E3 -DNA), protein targeting module (Y TRF The protein-degradable DNA material is obtained by mixing the DNA-DNA and the response module (TL-DNA) in a molar ratio of 1:1:2 to 1:1:5.

[0062] Another specific embodiment of the present invention provides the application of a protein-degraded DNA material or a protein-degraded DNA material prepared by the above-described method for preparing protein-degraded DNA material in tumor treatment and anti-tumor drug preparation.

[0063] The present invention will be further described in detail below with reference to specific embodiments.

[0064] The reagents used in this invention are available from the following sources. Unless otherwise specified, all reagents used in this invention can be obtained commercially.

[0065] Table 1. Reagent Sources

[0066]

[0067] The DNA sequence used in this invention is shown in Table 2.

[0068] Table 2 DNA Sequences

[0069]

[0070] In Table 1, the modification of ssDNA1 was carried out by solid-phase synthesis of the modified chain, which was purified by HPLC with a purity of >95%.

[0071] Example 1

[0072] In this embodiment, ssDNA1 linked with an E3 ubiquitin ligase-targeting peptide was prepared. The DNA-peptide was precisely coupled by using a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) on an alkyne-modified functional single-stranded DNA. The resulting DNA was purified by 12% non-denaturing polyacrylamide gel electrophoresis (PAGE). The specific procedures are as follows:

[0073] (1) Click reaction

[0074] Prepare 10×click working solution. The composition of 10×click working solution is as follows: CuSO4 10mM, BTTP 1mM, sodium ascorbate 100mM. The system is then brought to the final volume with PBS, specifically CuSO4 1μL, BTTP 11μL, sodium ascorbate 2μL, and PBS 6μL.

[0075] The reaction system consisted of 1 μL of 10×click working solution, 1 μL of Ya-N3-DNA (i.e., ssDNA1), 5 μL of Pep-E3 (LA-(Hyp)-YI-(L-Pra), purchased from Qiangyao Biotechnology), and 3 μL of MQ (ultrapure water). The reaction system was gently shaken at room temperature for 30 min to obtain the click reaction product.

[0076] (2) Gel purification: The coupling products were purified by 12% non-denaturing polyacrylamide gel electrophoresis (PAGE).

[0077] 1) Sample preparation

[0078] The click reaction products were separated by electrophoresis.

[0079] 2) Electrophoretic separation

[0080] Electrophoresis was performed using a 12% non-denaturing polyacrylamide gel at 120V until the target bands were fully separated.

[0081] 3) Rubber cutting and recycling

[0082] After electrophoresis, place the gel under a UV lamp (or use a gel imaging system) to identify the target conjugate band based on the molecular weight marker (Ladder) and control bands (such as ssDNA1).

[0083] Use a gel cutter or sterile blade to cut off the target band, removing as much excess gel as possible, and transfer it to a clean centrifuge tube.

[0084] 4) Dissolution of gel blocks and DNA recovery

[0085] Add an equal volume of PC solution to the gel block (if the gel weight is 0.1g, its volume can be considered as 100µl, then add 100µl of PC solution). Use a gel cutter to cut a 1% agarose gel; the weight of a single block is approximately 0.06g. The actual weight of the gel block depends on the gel concentration and thickness. Incubate in a 50°C water bath for about 10 minutes, gently turning the centrifuge tube up and down continuously to ensure the gel block is fully dissolved (if the gel block is too large, it can be cut into smaller pieces beforehand). If the solution color is not yellow, it can be adjusted to yellow using 3M sodium acetate (pH 5.0).

[0086] 5) Adsorption column purification

[0087] Add the dissolved solution to the pretreated adsorption column CB2 with equilibration buffer BL, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column CB2 into the collection tube. Add 600 μL of wash buffer PW to the adsorption column CB2 (please check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column CB2 into the collection tube. Centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube. Place the adsorption column CB2 into the collection tube, centrifuge at 12,000 rpm for 2 min, and remove as much wash liquid as possible. Let the adsorption column air dry completely at room temperature for 2-5 min.

[0088] 6) Elution and Collection

[0089] Place the adsorption column in a new centrifuge tube, and add 40 μL of elution buffer EB (pH 7.0-8.5) dropwise to the center of the membrane. Incubate at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min and collect the eluent. To improve the recovery rate, the eluent can be added back to the adsorption column and the centrifugation can be repeated once.

[0090] 7) Product preservation

[0091] The purified DNA-peptide conjugate can be stored at -20°C for subsequent characterization or functional experiments.

[0092] like Figure 1 As shown, covalent coupling of Ya-N3-DNA and Pep-E3 was successfully achieved by CuAAC click chemistry, resulting in ssDNA1 linked with an E3 ubiquitin ligase targeting polypeptide.

[0093] Example 2

[0094] In this embodiment, the E3 ubiquitin ligase targeting module is synthesized, as detailed below:

[0095] The ssDNA1, ssDNA2, and ssDNA3 molecules linked with the E3 ubiquitin ligase targeting peptide prepared in Example 1 were dissolved in 1×TAE Mg 2+ Buffer solution (containing 20mM Mg) 2+ In the study, the concentrations of ssDNA1, ssDNA2, and ssDNA3, which are linked to E3 ubiquitin ligase targeting peptides, were all 10 µM.

[0096] Place the mixed solution in a PCR instrument and perform the annealing procedure:

[0097] Step 1: Pre-denaturation at 95℃ for 2 minutes;

[0098] Step 2: Denaturation at 65℃ for 2 minutes;

[0099] Step 3: Denaturation at 60℃ for 5 minutes;

[0100] Step 4: Anneal at 60℃ for 30 seconds, repeat 40 times;

[0101] Step 5: Extend for 20 seconds at 20°C;

[0102] Step 6: Keep warm at 10℃.

[0103] like Figure 2 As shown, Y E3 - The DNA bands are clear and positioned higher than ssDNA1, ssDNA2, and ssDNA3, indicating that ssDNA1, ssDNA2, and ssDNA3 successfully formed a Y-shaped structure after annealing. E3 -DNA.

[0104] Example 3

[0105] In this embodiment, the protein targeting module is synthesized, as detailed below:

[0106] Dissolve ssDNA4, ssDNA5, and ssDNA6 in 1×TAE Mg2+ Buffer solution (containing 20mM Mg) 2+ In the sample, the concentrations of ssDNA4, ssDNA5, and ssDNA6 were all 10 µM.

[0107] Place the mixed solution in a PCR instrument and perform the annealing procedure:

[0108] Step 1: Pre-denaturation at 95℃ for 2 minutes;

[0109] Step 2: Denaturation at 65℃ for 2 minutes;

[0110] Step 3: Denaturation at 60℃ for 5 minutes;

[0111] Step 4: Anneal at 60℃ for 30 seconds, repeat 40 times;

[0112] Step 5: Extend for 20 seconds at 20°C;

[0113] Step 6: Keep warm at 10℃.

[0114] like Figure 3 As shown, Figure 3 ssDNA(4+5) is synthesized using ssDNA4 and ssDNA5 according to the method in this embodiment; ssDNA(4+6) is synthesized using ssDNA4 and ssDNA6 according to the method in this embodiment; and ssDNA(5+6) is synthesized using ssDNA5 and ssDNA6 according to the method in this embodiment. As can be seen from the figure, Y... TRF - The DNA band is clear and at the highest position, indicating that ssDNA4, ssDNA5, and ssDNA6 have successfully assembled into a Y-shaped structure. TRF -DNA.

[0115] Example 4

[0116] In this embodiment, the telomerase response module is synthesized, as detailed below:

[0117] Dissolve ssDNA7, ssDNA8, ssDNA9, La2, and Lb2 in 1×TAE Mg 2+ Buffer solution (containing 20mM Mg) 2+ In the sample, the concentrations of ssDNA7, ssDNA8, ssDNA9, La2, and Lb2 were all 10 µM.

[0118] Place the mixed solution in a PCR instrument and perform the annealing procedure:

[0119] Step 1: Pre-denaturation at 95℃ for 2 minutes;

[0120] Step 2: Denaturation at 65℃ for 2 minutes;

[0121] Step 3: Denaturation at 60℃ for 5 minutes;

[0122] Step 4: Anneal at 60℃ for 30 seconds, repeat 40 times;

[0123] Step 5: Extend for 20 seconds at 20°C;

[0124] Step 6: Keep warm at 10℃.

[0125] like Figure 4 As shown, Figure 4 In the figure, La refers to La2, Lb refers to Lb2, I-pet refers to ssDNA8, I-apt refers to ssDNA9, and Ts refers to ssDNA7. As can be seen from the figure, the TL band, which is the target product TL-DNA, is clear and without tails, indicating that ssDNA7, ssDNA8, ssDNA9, La2, and Lb2 have been successfully assembled into TL-DNA.

[0126] Example 5

[0127] This embodiment verifies the responsiveness via in vitro FRET, specifically as follows:

[0128] Following the method described in Example 4, L-DNA was synthesized using La2 and Lb2 and modified with the fluorescent group TAMRA.

[0129] In this embodiment, the TL-DNA from Example 4 was used for the experiment. The experimental procedure for verifying the TL-DNA response by in vitro FRET was as follows: Each group of samples was mixed with different telomerases in telomerase reaction buffer (20mM Tris-HCl, pH 8.3, 1.5mM MgCl2, 63mM KCl, 0.005% Tween 20, 1mM EGTA, 0.1mg / mL BSA). The sample volume was 0.5μM, and the enzyme volume was 1x10. 6 Telomerase extracted from cells was reacted at 37°C for 60 minutes, and the fluorescence intensity emitted at the corresponding excitation wavelength of the fluorescent group was monitored using a microplate reader. By comparing the fluorescence intensity of each group at the TAMRA emission wavelength, it was found that only when active telomerase was present did the probe structure open, resulting in a significant recovery of fluorescence, thus verifying the specificity and effectiveness of the response.

[0130] The samples in each group and the corresponding telomerases used are as follows:

[0131] Group i: The sample is TL-DNA modified with TAMRA-BHQ2 fluorescence quenching pair. No telomerase is added to this group.

[0132] Group ii: The sample was TL-DNA modified with TAMRA-BHQ2 fluorescence quenching pair, and the telomerase was heat-inactivated telomerase D-TE, which was inactivated by heating and served as a negative control.

[0133] Group iii: The sample was TL-DNA modified with TAMRA-BHQ2 fluorescence quencher, and the telomerase was N-TE, an extract of telomerase from normal cells. No significant fluorescence enhancement was expected.

[0134] Group IV: The sample was TL-DNA modified with TAMRA-BHQ2 fluorescence quenching pair. The telomerase was extracted from MCF-7 cells after pre-incubation with the telomerase inhibitor azidothymidine. It was used as the inhibitor treatment group AZT-TE to verify that the signal originated from telomerase activity.

[0135] Group v: TAMRA-modified L-DNA, serving as a positive control group, representing the maximum fluorescence signal after the probe is fully opened. This group does not contain telomerase.

[0136] Group vi: The sample was TL-DNA modified with TAMRA-BHQ2 fluorescence quenching pair, and the telomerase was telomerase extract (TE of cancer cells) from cancer cells (MCF-7 cells).

[0137] The fluorescence recovery of probes in different treatment groups was systematically evaluated using fluorescence spectroscopy and quantitative analysis. Figure 5 The fluorescence emission spectra of each group of samples in the wavelength range of 550-700 nm are shown. Figure 6 Quantitative statistics of fluorescence intensity at the characteristic emission peak (~580 nm) of TAMRA.

[0138] The control group (i), the heat-inactivated telomerase group (ii, D-TE), the normal cell telomerase extract group (iii, N-TE), and the telomerase inhibitor AZT pretreatment group (iv, AZT-TE) containing only the quenched probe TL-DNA all showed extremely low fluorescence signals, indicating that the TAMRA fluorescence of the probe was effectively quenched by BHQ2 under conditions without active telomerase, and the structure remained closed. The positive control group (v, L-DNA), i.e., TAMRA-labeled L-DNA, showed a significant fluorescence intensity, representing the maximum fluorescence signal after the probe was fully opened, providing a reference for the upper limit of the system's fluorescence response. The fluorescence intensity of the cancer cell telomerase extract group (vi, TL-DNA-TE) was comparable to that of the positive control group (v), achieving a significant recovery of fluorescence, confirming that active telomerase can specifically trigger the opening of the probe structure and relieve the fluorescence quenching effect.

[0139] The above results indicate that the DNA nanoprobe exhibits specific fluorescence recovery only in the presence of active telomerase, and the response signal can be effectively blocked by the telomerase inhibitor AZT. Meanwhile, telomerase extract from normal cells showed no significant activation effect, fully verifying the high specificity, high sensitivity, and response effectiveness of the probe for telomerase activity detection, and laying an experimental foundation for the precise response of telomerase in tumor cells in subsequent studies.

[0140] Example 6

[0141] The preparation method of the protein-degraded DNA material in this embodiment is as follows:

[0142] Take the annealed product Y from Example 2 E3 -DNA, annealing product Y from Example 3 TRF -DNA, the annealed product TL-DNA from Example 4, with Y E3 -DNA, Y TRF The molar ratio of TL-DNA to TL-DNA is 1:1:3. The resulting protein-degraded DNA material is denoted as Y. E3-TRF -TL.

[0143] Example 7

[0144] This embodiment describes the regulation and assembly of a DNA dynamic assembly material module.

[0145] Following the preparation method in Example 4, L-DNA-16 was synthesized using La1 and Lb1, L-DNA-32 was synthesized using La2 and Lb2, and L-DNA-64 was synthesized using La3 and Lb3.

[0146] Y in Example 2 E3 -DNA, Y in Example 3 TRF -DNA and L-DNA of different lengths, namely L-DNA-16, L-DNA-32, and L-DNA-64, were used to construct binary linker systems and multi-component assembly systems. The binary linker system included Y-DNA in a molar ratio of 1:3. E3 -DNA and L-DNA-16, Y in a molar ratio of 1:3 E3 -DNA and L-DNA-32, Y in a molar ratio of 1:3 E3 -DNA and L-DNA-64, Y in a molar ratio of 1:3 TRF -DNA and L-DNA-16, Y in a molar ratio of 1:3 TRF -DNA and L-DNA-32, Y in a molar ratio of 1:3 TRF -DNA and L-DNA-64. The multi-component assembly system includes: Y DNA in a molar ratio of 1:1:3. E3 -DNA, Y TRF-DNA and L-DNA-16, in a molar ratio of 1:1:3, Y E3 -DNA, Y TRF -DNA and L-DNA-32, in a molar ratio of 1:1:3, Y E3 -DNA, Y TRF -DNA and L-DNA-64. Each group used 1×TAE Mg 2+ Buffer solution (containing 20mM Mg) 2+ Dilute to 10 μM according to the ratio, mix well and place in a PCR instrument for annealing: pre-denaturation: 95℃ 2 min; denaturation: 65℃ 2 min; denaturation: 60℃ 5 min; annealing: 60℃ 30s 40 cycles; extension: 20℃ 30s; incubation: 10℃.

[0147] Prepare a 3% agarose gel: Weigh 0.9 g of agarose and add it to 30 mL of 1×TAE buffer. Heat to dissolve, then add the nucleic acid dye (Gel-Red (10000 x), Beyotime, 1.5 μL) and mix well. Pour the mixture into a gel mold and insert a comb. Let it stand at room temperature for 30 min until solidified. Place the gel in an electrophoresis tank, add 1×TAE buffer to cover the gel surface, take 5 μL of annealing product and mix it with 1 μL of 6×DNA loading buffer, then load the sample. At the same time, add 5 μL of 50 bp DNA ladder as a molecular weight marker. Electrophoresis at a constant voltage of 100 V for 40 min.

[0148] After electrophoresis, the gel was placed in a gel imaging system and the band migration was observed under ultraviolet light. The band differences between the ligation products and multi-component assembly products mediated by L-DNA of different lengths were compared to verify the integrity and efficiency of DNA network assembly.

[0149] like Figures 7-9 As shown, by changing the length of L-DNA, the contact between the target protein and E3 ubiquitin ligase in the DNA degradation network can be regulated, and L-DNA of different lengths can be assembled into DNA network structures.

[0150] Example 8

[0151] This embodiment evaluates the tumor protein-targeted degradation performance of DNA dynamic assembly materials.

[0152] Set up the following experimental groups:

[0153] PBS group: blank control, used for normalization analysis.

[0154] AZT-Y E3-TRF -TL group: Telomerase inhibition group, using both a telomerase inhibitor and the protein-degrading DNA material Y prepared in Example 6. E3-TRF -TL, unable to trigger Y-E3 With Y- TRF The assembly includes an E3 ubiquitin ligase targeting module and a TRF1 / TRF2 targeting module.

[0155] Y E3 -TL group: Referring to the preparation method in Example 6, the Y from Example 2 was used... E3 -DNA and TL-DNA from Example 4 were mixed at a molar ratio of 1:3 to obtain Y. E3 -TL can be assembled by telomerase, contains an E3 ubiquitin ligase targeting module, and does not contain a TRF1 / TRF2 targeting module.

[0156] YTL group: Following the preparation method in Example 2, Y-DNA-1 was synthesized using ssDNA1, ssDNA2, and ssDNA3. Following the preparation method in Example 3, Y-DNA-2 was synthesized using ssDNA4, ssDNA5-1, and ssDNA6. Following the preparation method in Example 6, Y-DNA-1, Y-DNA-2, and TL-DNA from Example 4 were mixed in a molar ratio of 1:1:3 to obtain YTL, which can be assembled by telomerase triggering and does not contain the E3 ubiquitin ligase targeting module or the TRF1 / TRF2 targeting module.

[0157] Y TRF -TL group: Referring to the preparation method in Example 6, the Y from Example 3 was used... TRF -DNA and TL-DNA from Example 4 were mixed at a molar ratio of 1:3 to obtain Y. TRF -TL can be assembled by telomerase, does not contain the E3 ubiquitin ligase targeting module, and contains the TRF1 / TRF2 targeting module.

[0158] Y E3-TRF -L group: L-DNA was synthesized using La2 and Lb2 according to the preparation method in Example 4. The Y2 from Example 2 was prepared using the preparation method in Example 6. E3 -DNA, Y in Example 3 TRF -DNA and L-DNA are mixed in a molar ratio of 1:1:3 to obtain Y. E3-TRF -L, positive control group, can be assembled without telomerase triggering, containing E3 ubiquitin ligase targeting module and TRF1 / TRF2 targeting module.

[0159] Y E3-TRF -TL group: Final material group, i.e., using the protein-degraded DNA material Y prepared in Example 6. E3-TRF -TL can be assembled by telomerase and contains an E3 ubiquitin ligase targeting module and a TRF1 / TRF2 targeting module.

[0160] The following experiment was conducted using the above materials:

[0161] (1) Protein extraction. Human breast cancer cells MCF-7 were extracted at a rate of 2×10⁻⁶. 5 Cells were seeded at a density of 1 μL per well in 6-well plates. For the inhibitor group, 1 μL of telomerase inhibitor Zidovudine was added during plate formation. After cell adhesion, the corresponding materials were added according to the above groupings, with a dosage of 200 nM for each group, and incubated for 72 h. After incubation, the culture medium was discarded, and the cells were washed three times with PBS. Cells were then digested with 500 μL of trypsin and the digestion was terminated with 800 μL of culture medium. The cell suspension from each well was collected into centrifuge tubes, centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. 300 μL of protein lysis buffer was added to the cell pellet, and the cells were repeatedly pipetted with a 1 mL pipette tip to ensure complete lysis. The lysate was heated in a 100°C metal bath for 10 min to denature the protein. The protein concentration was then measured using a micro-UV spectrophotometer. Samples were aliquoted and stored at -80°C for later use.

[0162] (2) Western Blot validation. Take 25 μg of protein sample from each group, add 5×SDS loading buffer, make up the system with deionized water, denature again at 100℃ for 10 min, and add to the well of pre-made protein polyacrylamide gel. Use the three-color pre-stained protein marker as the molecular weight standard (load 4.5 μL, 1 on the left and 2 on the right), use MOPS-SDS as electrophoresis buffer, and electrophoresis at 200V constant voltage for 35 min.

[0163] After electrophoresis, the sponge, filter paper, gel, and pre-activated PVDF membrane were assembled into a "sandwich" structure for transfer, following the sequence: "black clip (negative electrode) → sponge → filter paper → gel → PVDF membrane → filter paper → sponge → white clip (positive electrode). The membrane was then placed in 1× rapid transfer buffer and electrophoretically transferred at a constant current of 400 mA for 35 min. After transfer, the PVDF membrane was washed five times (6 min each time) with 1× TBST buffer, blocked in 5% BSA blocking buffer at room temperature for 4 h, and washed again with 1× TBST buffer.

[0164] Subsequently, the membrane was cut according to the protein molecular weight, and the target protein primary antibody working solution (TRF1 or TRF2 protein, primary antibody dilution ratio 1:1000) and the internal control protein primary antibody working solution (β-actin protein, primary antibody dilution ratio 1:10000) were added respectively, and incubated overnight at 4°C.

[0165] The next day, the primary antibody was recovered, washed thoroughly with 1× TBST, and HRP-labeled secondary antibody (goat anti-mouse or goat anti-rabbit IgG, with a dilution ratio of 1:10000) was added. The mixture was incubated on a shaker at room temperature for 1 hour, and finally washed 5 times with 1× TBST. ECL ultrasensitive luminescent solution was added, and the bands were collected and the protein expression level was quantitatively analyzed using a fully automated chemiluminescence image analysis system.

[0166] like Figure 10 As shown, regarding the expression of TRF1 and TRF2 proteins, Y E3-TRF The -TL group exhibited a significant inhibitory effect, indicating that DNA material assembly can downregulate the expression of target proteins.

[0167] In summary, the present invention has the following advantages:

[0168] 1. Simplification and integration of modular "one-pot" assembly architecture

[0169] Existing technologies typically employ complex DNA origami techniques involving hundreds of short chains, or stepwise chemical cross-linking methods (first synthesizing DNA aptamers, then coupling and degrading tags through chemical reactions), which are cumbersome and produce numerous byproducts.

[0170] Improvements of this invention: A modular assembly process based on a tri-fork / Y-shaped core backbone is proposed. By designing short DNA strands (ssDNAs) with specific complementary regions, the targeting unit, degradation initiation unit and response switch are synchronously self-assembled in the same annealing process.

[0171] Technical benefits: It greatly simplifies the preparation process, shortens the preparation cycle from several days to several hours, and because it is based entirely on complementary base pairing, the structural uniformity of the product is significantly better than that of the chemical cross-linking method.

[0172] 2. Structured integration of environmentally responsive switching sequences

[0173] Existing technology: Protein degradation materials are mostly in a continuously activated state, lacking the ability to recognize the tumor microenvironment, and are prone to off-target side effects.

[0174] The improvement of this invention lies in the fact that, during the preparation process, the telomerase recognition primer is directly used as a response switch for the self-assembled backbone. This sequence is not only structurally supportive but also a functional switch.

[0175] Technical effect: The prepared nanomaterials have "logic gate" control function. Only in specific environments with high telomerase expression (such as in tumor cells) will the material structure undergo depolymerization or conformational change, thereby releasing / activating degradation activity, achieving a high degree of integration between preparation process and functional response.

[0176] 3. Stoichiometric ratio control

[0177] Existing technology: Traditional chemical modification methods have difficulty in precisely controlling the physical distance between the target ligand and the degradation tag, as well as the 1:1 molecular ratio, resulting in unstable degradation efficiency.

[0178] The improvement of this invention is that it optimizes the kinetics of the formation of the ternary complex (target protein-DNA material-E3 enzyme), which significantly improves the rate and depth of protein degradation.

[0179] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this disclosure.

[0180] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A protein-degradable DNA material, characterized in that, The protein-degrading DNA material includes a degradation initiation module, a protein targeting module, and a response module, wherein the degradation initiation module is an E3 ubiquitin ligase targeting module, and the response module is a telomerase response module.

2. The protein-degrading DNA material according to claim 1, characterized in that, The degradation initiation module is assembled from ssDNA1, ssDNA2, and ssDNA3, which are linked to E3 ubiquitin ligase targeting peptides. The ssDNA1 sequence is shown in SEQ ID NO.1, the ssDNA2 sequence is shown in SEQ ID NO.2, and the ssDNA3 sequence is shown in SEQ ID NO.

3.

3. The protein-degrading DNA material according to claim 2, characterized in that, The E3 ubiquitin ligase targeting polypeptide is LA-(Hyp)-YI-(L-Pra).

4. The protein-degrading DNA material according to claim 1, characterized in that, The protein targeting module is a TRF protein targeting module; and / or, The protein targeting module is assembled from ssDNA4, ssDNA5, and ssDNA6, wherein the ssDNA4 sequence is shown in SEQ ID NO.4, the ssDNA5 sequence is shown in SEQ ID NO.5, and the ssDNA6 sequence is shown in SEQ ID NO.

6.

5. The protein-degrading DNA material according to claim 1, characterized in that, The response module is assembled from ssDNA7, ssDNA8, ssDNA9 and ssDNA-X, wherein the ssDNA7 sequence is shown in SEQ ID NO.7, the ssDNA8 sequence is shown in SEQ ID NO.8 and the ssDNA9 sequence is shown in SEQ ID NO.9; Among them, ssDNA-X satisfies any of the following sets: The La1 and Lb1 groups, wherein the La1 sequence is shown in SEQ ID NO.10 and the Lb1 sequence is shown in SEQ ID NO.11; The La2 and Lb2 groups, wherein the La2 sequence is shown in SEQ ID NO.12 and the Lb2 sequence is shown in SEQ ID NO.13; The La3 and Lb3 groups, wherein the La3 sequence is shown in SEQ ID NO.14 and the Lb3 sequence is shown in SEQ ID NO.

15.

6. The protein-degrading DNA material according to claim 1, characterized in that, The molar ratio of the degradation initiation module, protein targeting module, and response module is 1:1:2 to 1:1:

5.

7. A method for preparing the protein-degradable DNA material according to claim 1, characterized in that, The preparation method includes the following steps: The ssDNA strand used in the degradation initiation module was dissolved in a solvent and then annealed to obtain the degradation initiation module. The ssDNA strand used in the protein targeting module is dissolved in a solvent and then annealed to obtain the protein targeting module. The ssDNA strand used in the response module is dissolved in a solvent and then annealed to obtain the response module. The degradation initiation module, protein targeting module, and response module are mixed to obtain protein-degraded DNA material.

8. The method for preparing protein-degradable DNA material according to claim 7, characterized in that, The concentration of any one of the ssDNA strands used in the degradation initiation module, the protein targeting module, and the response module in the solvent is 10µM-30µM; and / or, The solvent is 1× TAE Mg 2+ Buffer solution, wherein Mg 2+ The concentration is 10mM-20mM.

9. The method for preparing protein-degradable DNA material according to claim 7, characterized in that, The annealing procedure in any one of the preparation steps of the degradation initiation module, protein targeting module, and response module is as follows: 95℃ for 2 min; 65℃ for 2 min; 60℃ for 5 min; 60℃ for 30 s, 40 cycles; 20℃ for 30 s; and 10℃ for heat preservation.

10. The application of the protein-degraded DNA material according to claim 1 or the protein-degraded DNA material prepared by the preparation method of the protein-degraded DNA material according to claim 7 in tumor treatment and anti-tumor drug preparation.

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