A tnd-tim interaction switch aptamer and application, kit, and pharmaceutical composition thereof

Abstract

This invention belongs to the field of molecular biology, specifically relating to a TND-TIM interaction switch nucleic acid aptamer and its applications, kits, and pharmaceutical compositions. The nucleic acid aptamer of this invention can specifically recognize multiple transcriptional regulatory factors, including TND, solving the technical challenge of traditional small molecules intervening in TND-TIM interactions. The aptamer has a simple structure, is easy to chemically modify and scale up, and possesses high affinity and specificity. It can be widely used for protein-protein interaction analysis, molecular mechanism research, imaging tracing, and the construction of PPI intervention systems for various TND-TIM transcriptional regulatory factors. Validated through cell and animal research models, it can serve as a reliable research tool for elucidating the TND-TIM protein interaction and transcriptional regulation mechanisms, and as a potential pharmaceutical active ingredient for tumor treatment / inhibition of disease progression.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a TND-TIM interacting switch nucleic acid aptamer and its applications, reagent kits, and pharmaceutical compositions. Background Technology

[0002] Precise regulation of gene expression is central to life processes, with RNA polymerase II (RNAPII)-mediated transcription being regulated by multi-level, dynamic protein complexes. Recent studies have revealed that a class of protein interaction modules, composed of the TFIIS N-terminal domain (TND) and transcription interaction motifs (TIMs), plays a crucial role in the transcriptional regulatory network, becoming a new focus for understanding transcriptional mechanisms and related disease mechanisms.

[0003] The TND domain is an evolutionarily conserved five-helix bundle fold structure widely found in various important transcriptional regulators, including elongation factor ELOA, the phosphatase regulatory subunit PNUTS, the chromatin remodeling-related protein HRP2, and the transcription elongation factor TFIIS itself. The core function of this domain is to provide a molecular recognition interface for the TIM motif. TIM, typically located in an intrinsically disordered region, is a short peptide sequence that specifically recognizes and binds to TND, forming a dynamic, reversible protein-protein interaction pair. This interaction mode represents an important molecular "docking" mechanism in transcriptional regulation.

[0004] Studies have shown that the N-terminal region (MYC Box 0, MB0) of the proto-oncoprotein MYC contains a typical TIM sequence, which can directly participate in regulating the transcriptional elongation process of RNAPII by binding to a specific TND domain, thereby affecting the expression of a large number of genes and driving cell proliferation and tumorigenesis. This suggests that the TND-TIM interaction interface is a key node in regulating MYC function and even the broader transcriptional program, and has important value for basic research and clinical applications.

[0005] However, the functional study and development of intervention methods targeting the TND-TIM interaction interface face significant technical bottlenecks. This interaction interface is primarily formed by the surface interaction between α-helices and linear short peptides, lacking typical hydrophobic pockets or deep trenches suitable for small molecule binding. This structural feature makes traditional structure-based small molecule drug design methods ineffective, hindering the development of high-affinity, high-specificity small molecule inhibitors or modulators. This deficiency severely restricts scientists from precisely manipulating this interaction in living cells or in vivo environments, elucidating its downstream biological effects, and exploring its potential as a therapeutic target for diseases.

[0006] To address this challenge, the search for novel molecular tools capable of specifically recognizing and intervening in such "difficult-to-target" protein interfaces is imperative. Nucleic acid aptamers, single-stranded DNA or RNA molecules obtained through in vitro screening technology (SELEX), exhibit unique advantages in this regard. Aptamers can fold themselves into complex three-dimensional structures, binding to specific epitopes of target proteins with high affinity and specificity, including flat protein surfaces, in a manner similar to antibodies. Compared to antibodies, aptamers are characterized by small molecular weight, ease of chemical synthesis and modification, minimal batch-to-batch variability, high stability, and low immunogenicity. Compared to small molecules, aptamers possess higher target specificity and affinity potential, particularly adept at targeting protein-protein interaction interfaces.

[0007] Therefore, developing nucleic acid aptamers that can act as switches for TND-TIM interactions will not only fill the gap in molecular tools in this field and deepen our understanding of the transcriptional regulatory mechanisms of RNAPII, but may also open up new avenues for the development of innovative therapies that target the transcriptional functions of oncoproteins such as MYC. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a TND-TIM interaction switch nucleic acid aptamer that can specifically recognize the TND domain of transcription factors and block their interaction with the TIM module.

[0009] A second objective of this invention is to provide applications for TND-TIM interaction switch nucleic acid aptamers, such as specific probes and competitive inhibitors, to trace the TND-TIM interaction state and elucidate the specific mechanisms of action of different TND-containing proteins (such as ELOA, PNUTS, etc.) in the transcriptional complex; they can also serve as lead molecules to lay the foundation for feasibility studies in the subsequent development of therapeutic strategies based on oligonucleotides or small molecule mimics; and they can also serve as active ingredients for the preparation of drugs for the prevention / treatment of tumors.

[0010] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0011] A TND-TIM interaction switch nucleic acid aptamer, wherein the nucleic acid aptamer is a single-stranded DNA containing the nucleotide sequence shown in SEQ ID NO.1, specifically 5'-GGAGGGCTTGAGCA-3'; or a nucleotide sequence having at least 30% homology with SEQ ID NO.1 and specifically binding to TND domain proteins;

[0012] Alternatively, the nucleic acid aptamer may be a single-stranded RNA containing the nucleotide sequence shown in SEQ ID NO.2, specifically 5'-GGAGGGCUUGAGCA-3', or a nucleotide sequence that has at least 30% homology with SEQ ID NO.2 and specifically binds to TND domain proteins.

[0013] In a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] Based on the inventive concept of this invention, in order to further improve the application performance of nucleic acid aptamers, the nucleotide sequence of the above-mentioned nucleic acid aptamers is modified and the modified nucleic acid aptamers bind to TND domain proteins. The modification includes at least one of phosphorylation, 2'-O-methyl modification, amination, carboxylation, aldehyde modification, thiophosphate modification, oxygen substitution with selenium, and isotopization.

[0015] This invention utilizes the aforementioned nucleic acid aptamer to specifically bind to the N-terminal domain of transcriptional regulatory factors, thereby blocking the interaction between the aptamer and its corresponding transcriptional interaction motif or similar peptides; the dissociation constant Kd of the nucleic acid aptamer binding to the protein TND domain is < 10 μM; preferably, the dissociation constant Kd is < 5 μM; more preferably, the dissociation constant Kd is < 2 μM; and even more preferably, the dissociation constant Kd is < 1 μM.

[0016] In a specific embodiment of the present invention, a substance for labeling, detection, diagnosis or treatment is provided to be attached to the nucleotide sequence of the above-mentioned nucleic acid aptamer, and the nucleic acid aptamer conjugate after attaching the substance specifically binds to the TND domain protein; the substance is selected from at least one of fluorescent markers, quenching groups, azide groups, alkynyl groups, radioactive substances, therapeutic substances, biotin, digoxigenin, maleimide, nanomaterials, polyethylene glycol, small peptides, siRNA and enzyme labeling.

[0017] In specific embodiments of the present invention, the transcriptional regulatory factors are selected from ELOA, HRP2, TFIIS, IWS1, PNUTS, and MED26. The above-mentioned nucleic acid aptamers can serve as TND-TIM interaction switch molecules for these transcriptional regulatory factors, constructing an in vitro analysis system for TND-TIM protein interaction; at the cellular level, they can be used to study the regulatory role of TND-TIM protein interaction and related transcriptional pathway changes; aptamer derivatives can be used as research tools to conduct research on protein recognition, interaction analysis, signaling pathway studies, and imaging tracing; and they can be used as research validation tools in animal models to analyze the biological functions of the TND-TIM module. Specifically, as a specific probe, it can detect and trace the TND domain and the TND-TIM interaction state in vitro or intracellularly; as a competitive inhibitor, it can specifically block the binding of the TIM peptide to TND, thereby studying the biological consequences of the lack of this interaction at the molecular, cellular, and even animal levels; as a functional research tool, it can help elucidate the specific mechanisms of action of different TND-containing proteins in the transcriptional complex; as a lead molecule, it can lay the foundation for subsequent development of therapeutic strategies based on oligonucleotides or small molecule mimics. As a pharmaceutical active ingredient, it is used in drug preparation and is expected to prevent / treat tumors or inhibit tumor progression / development. In specific embodiments of this invention, its potential value in preventing / treating / inhibiting the progression of colorectal cancer and breast cancer has been preliminarily verified.

[0018] Based on the above applications, the present invention also provides a TND-TIM interaction or regulation performance characterization kit, comprising the above-mentioned nucleic acid aptamer conjugate, auxiliary reagents, and / or reporter-signal detection system and / or solid-phase support carrier; wherein the reporter-signal detection system and / or solid-phase support carrier are detection systems that support fluorescence polarization method, surface plasmon resonance (SPR) method or electrophoretic mobility analysis.

[0019] In a specific embodiment of the present invention, the reporter-signal detection system of the above-mentioned kit is based on a fluorescence polarization detection system, and the kit further includes a fluorescently labeled ligand, wherein the fluorescently labeled ligand is a transcriptional interaction motif of the transcriptional regulatory factor to be tested or a similar peptide.

[0020] As an example, the present invention provides a method for characterizing the TND domain of the transcriptional regulatory factor TFIIS and the MB0 region of the MYC protein or its functionally similar peptide PPI, comprising the following steps:

[0021] (1) Incubate the TFIIS TND domain protein to be tested with the fluorescently labeled MYC protein MB0 region or its functionally similar peptide;

[0022] (2) Add the above nucleic acid aptamers to the reaction system;

[0023] (3) Determine the blocking effect of the aptamer on protein-protein interactions.

[0024] Beneficial effects of this invention:

[0025] This invention provides a nucleic acid aptamer that specifically recognizes multiple transcriptional regulatory factors, including TND, solving the technical challenge of traditional small molecules intervening in TND–TIM interactions. The aptamer has a simple structure, is easy to chemically modify and scale up, and possesses high affinity and specificity. It can be widely used for protein-protein interaction analysis, molecular mechanism research, imaging tracing, and the construction of PPI intervention systems for various TND-TIM transcriptional regulatory factors. Validated through cell and animal research models, it can serve as a reliable research tool for elucidating the TND–TIM protein interaction and transcriptional regulation mechanisms, and as a potential pharmaceutical active ingredient for tumor treatment / inhibition of disease progression. Attached Figure Description

[0026] Figure 1 This is a schematic diagram showing the results of the binding affinity test between the nucleic acid aptamer and the ELOA protein.

[0027] Figure 2 This is a schematic diagram showing the results of affinity assays for binding nucleic acid aptamers to HRP2, TFIIS, IWS1, PNUTS, MED26, and LEDGF proteins.

[0028] Figure 3 This is a schematic diagram showing the results of affinity assays for binding nucleic acid aptamers to proteins without the TND domain.

[0029] Figure 4 This is a schematic diagram showing the detection results of the blocking effect of nucleic acid aptamers on the ELOA–MYC MB0 interaction;

[0030] Figure 5 This is a schematic diagram showing the detection results of the blocking effect of nucleic acid aptamers on the interaction between other TND proteins and the MYC MB0 peptide;

[0031] Figure 6 This is a schematic diagram showing the detection results of the blocking effect of nucleic acid aptamers on the interaction of TND protein with other TIM peptides (such as LEO1 and PAF1);

[0032] Figure 7 A comparison of the stability of unmodified and phosphate-thiolated nucleic acid aptamers in serum;

[0033] Figure 8 Schematic diagram of cellular uptake of unmodified and phosphate-thiolated nucleic acid aptamers;

[0034] Figure 9A schematic diagram showing the binding of the phosphate-thiocyanate backbone-modified nucleic acid aptamer to ELOA TND and the inhibition of ELOA–MYC MB0 interaction;

[0035] Figure 10 A graph showing the regulation of MYC transcriptional levels after nucleic acid aptamer treatment;

[0036] Figure 11 This is a schematic diagram illustrating the activation of the tumor cell apoptosis pathway after treatment with nucleic acid aptamers.

[0037] Figure 12 Figure showing the ability of nucleic acid aptamers to induce tumor cell apoptosis.

[0038] Figure 13 This diagram shows the functional validation of the TND–TIM interaction of nucleic acid aptamers in a mouse xenograft model. Detailed Implementation

[0039] To facilitate a better understanding of the technical solutions of this invention by those skilled in the art, the invention is further described below with reference to embodiments. Unless otherwise specified, experimental methods are performed according to conventional methods or operating conditions recommended by relevant reagent manufacturers; unless otherwise specified, reagents and materials are purchased from conventional commercial suppliers. Analogs or equivalent substitutes for the methods and materials are also applicable to this invention. The following embodiments are merely illustrative of the invention and are not intended to limit its scope.

[0040] 1. Expression and purification of proteins containing the TND domain

[0041] The recombinant plasmid pET28-TND, containing the coding sequences for the TND domains (PNUTS, HPR2, MED26, IWS1, ELOA, LEDGF, TFIIS), was transformed into E. coli BL21(DE3) competent cells. Expression was induced overnight at 18 °C with 0.5 mM MIPTG. After cell collection, the cells were lysed by sonication, and the supernatant was collected by centrifugation and purified by Ni-NTA affinity chromatography. The purification steps included: (1) equilibration with 1×DPBS buffer (containing 0.05 mM EDTA and 5 mM imidazole); (2) washing with 1×DPBS buffer (containing 40 mM imidazole and 2 M NaCl); and (3) elution of the target protein with 1×DPBS buffer (containing 300 mM imidazole and 5% glycerol). The eluted product was subjected to TEV protease to remove the His tag, and then collected by Ni-NTA chromatography again to obtain high-purity TND protein for subsequent screening experiments.

[0042] 2. The MYC MB0 peptide was synthesized by Shanghai Chutai Biotechnology; all nucleic acid sequences were synthesized by Suzhou Genewise Biotechnology Co., Ltd.; all cell lines were purchased from the ATCC cell bank.

[0043] Example 1:

[0044] This embodiment provides a nucleic acid aptamer, which is a single-stranded DNA, with the sequence GGAGGGCTTGAGCA (as shown in SEQ ID NO.1), denoted as TTiApt.

[0045] Example 2

[0046] This embodiment is used to verify the function of the nucleic acid aptamer described in Example 1:

[0047] 1. Affinity test:

[0048] Detection method: The function of the aptamer in Example 1 was verified by fluorescence polarization experiment. Its affinity with ELOA, HRP2, TFIIS, IWS1, PNUTS, MED26 and LEDGF was detected. The specific detection method was as follows: the target protein stock solution and the aptamer stock solution described in Example 1 with fluorescent label at the 3' end were prepared. The target protein stock solution was serially diluted with buffer solution to dilute the aptamer stock solution to the working solution concentration. The target protein dilution solution and the aptamer working solution were mixed and incubated. The change of mP value during the binding of aptamer and target protein was detected, and its dissociation constant was calculated to characterize its binding affinity.

[0049] Test results: such as Figure 1 The results show the binding affinity detection results between the aptamer and ELOA TND, which are concentration-dependent, with a dissociation constant of 0.266 ± 0.020 μM.

[0050] like Figure 2 The results show the affinity assays of the aptamer for binding to TND proteins such as HRP2, TFIIS, IWS1, PNUTS, MED26, and LEDGF. The aptamer showed high affinity for HRP2 and TFIIS (Kd < 1 μM), and detectable binding ability for IWS1, PNUTS, and MED26 (Kd < 10 μM), while no obvious binding signal was detected for LEDGF.

[0051] This indicates that the aptamer provided by the present invention can recognize and bind to the TND domains of various transcriptional regulatory factors, but also exhibits a certain degree of selectivity.

[0052] 2. Specificity assessment:

[0053] To verify the specificity of aptamer binding, the fluorescently labeled aptamers were subjected to binding affinity assays with three non-TND proteins (pepsin, streptavidin, and recombinant protein A) using the method described above. The results are as follows: Figure 3 As shown, no obvious binding signal was detected, indicating that the nucleic acid aptamer provided by the present invention can specifically recognize the TND domain of transcription regulatory factors.

[0054] (3) Characterization of the TND–TIM interaction switching function of the nucleic acid aptamer of the present invention:

[0055] Characterization method: includes the following steps:

[0056] (1) Incubate the protein containing the TND domain to be tested with the fluorescently labeled transcription interaction motif peptide;

[0057] (2) Add the nucleic acid aptamer provided in Example 1 above to the reaction system;

[0058] (3) The blocking effect of the aptamer on TND-TIM interaction was determined by fluorescence polarization method.

[0059] This method was used to characterize the blocking effects of the nucleic acid aptamers of the present invention on the interaction of HRP2, IWS1, PNUTS, TFIIS, MED26 and FAM-MYC MB0; the blocking effects of ELOA, HRP2, PNUTS, TFIIS and FAM-LEO1 TIM peptide; and the blocking effects of ELOA, HRP2, PNUTS, TFIIS and FAM-PAF1 TIM peptide.

[0060] Test results: such as Figure 4 The results show that the nucleic acid aptamer of the present invention can effectively block the binding of ELOA TND to FAM-MYC MB0, with a half-maximal inhibitory concentration (IC50) of 100%. 50 The value was 0.284 ± 0.042 μM.

[0061] like Figure 5 The results show that the nucleic acid aptamer of this invention can effectively inhibit the binding of proteins containing the TND domain, such as HRP2, IWS1, PNUTS, TFIIS, and MED26, to MYC MB0, with varying blocking efficiencies among different proteins. This indicates that the nucleic acid aptamer of this invention can serve as a competitive molecular tool for studying various TND–TIM interactions.

[0062] like Figure 6The results show that the nucleic acid aptamers of this invention can effectively inhibit the binding of proteins containing the TND domain, such as ELOA, HRP2, PNUTS, and TFIIS, to the LEO1 or PAF1 TIM peptide. This indicates that the nucleic acid aptamers of this invention can not only act on TND–MYC MB0 interactions, but are also applicable to various TND–TIM interaction systems, demonstrating their broad applicability as a molecular tool.

[0063] Example 3

[0064] Performance characterization, stability improvement, and functional verification of nucleic acid aptamers

[0065] This embodiment provides a nucleic acid aptamer, which is a single-stranded DNA with the sequence GGAGGGCTTGAGCA (as shown in SEQ ID NO.1), and is modified with total phosphorus thioester (PS), that is, the non-bridging oxygen atoms of the phosphate backbone of 14 nucleotides are replaced by sulfur atoms, denoted as TTiApt-PS.

[0066] 1. Serum stability assessment

[0067] The nucleic acid aptamers provided in Example 1 and Example 3 were incubated in 20% FBS at 37°C, and the degradation of the nucleic acid aptamers was detected at different time points. The results are as follows: Figure 7 As shown, the thiomodified nucleic acid aptamer of Example 3 remained intact after 96 hours of incubation in 20% FBS, while the unmodified nucleic acid aptamer provided in Example 1 was essentially degraded within 12 hours, suggesting that PS modification significantly prolongs the stability of the aptamer in the serum environment.

[0068] 2. Cellular uptake and subcellular localization

[0069] FAM-labeled TTiApt-PS and TTiApt were co-incubated with HEK293T cells for 48 hours, and cell uptake and distribution were detected using laser confocal microscopy. Figure 8 The results show that TTiApt-PS can enter cells and accumulate in the nucleus, while unmodified TTiApt mainly remains in the extracellular space or on the cell membrane surface, suggesting that PS modification helps improve the cellular uptake capacity of aptamers.

[0070] 3. Combining specificity and interaction inhibition

[0071] Fluorescence polarization experiments were used to detect the binding performance of the thiomodified aptamer TTiApt-PS provided in Example 3 and the unmodified aptamer TTiApt provided in Example 1 to the ELOA TND domain. Figure 9Results A showed that the binding affinity of TTiApt-PS to the ELOA TND domain was (Kd = 0.126 ± 0.007 μM), which was significantly better than that of unmodified TTiApt (Kd = 0.266 ± 0.020 μM). Figure 9 A).

[0072] Further investigation was conducted to determine its blocking / inhibiting effect on the interaction between ELOA and TND–MYC MB0, such as... Figure 9 Results B show that the IC of TTiApt-PS 50 The concentration was 0.074 ± 0.036 μM, which was significantly better than that of unmodified TTiApt (IC). 50 = 0.284 ± 0.042μM)( Figure 9 B).

[0073] Example 4: Transcriptional Regulation of Nucleic Acid Aptamers and Validation at the Cellular / Animal Level

[0074] 1. Transcriptional Regulation Analysis

[0075] After treating HCT116 cells with the thiomodified aptamer TTiApt-PS provided in Example 3 for 48 hours, transcriptome analysis was performed. Figure 10 The results showed a decrease in MYC gene transcription levels, which was confirmed by qRT-PCR (Figures 10A and 10B). Figure 11 The gene set enrichment analysis results indicated that apoptosis-related pathways were activated, suggesting that TTiApt-PS treatment may affect MYC-dependent transcriptional activity and activate apoptosis-related signaling pathways. This demonstrates that the nucleic acid aptamer of this invention can serve as a competitive inhibitor, acting as a TND-TIM interaction switch, for constructing research models of transcriptional regulatory mechanisms / pathways.

[0076] 2. Cell function verification

[0077] The thiolated aptamer TTiApt-PS provided in Example 3 was applied to colorectal cancer cell lines (HCT116, LOVO, HT29) and non-tumor cells, respectively. The results showed that in tumor cells, TTiApt-PS treatment induced a dose-dependent increase in apoptosis, while the control aptamer Random-PS had a smaller effect; no significant toxicity was observed in non-tumor cells (Figure 12A). Further Annexin V–FITC / PI double staining flow cytometry analysis in HCT116 cells confirmed that TTiApt-PS could induce apoptosis (Figure 12B). This indicates that the aptamer of this invention can serve as a lead molecule, laying the foundation for subsequent development of therapeutic strategies based on oligonucleotides or small molecule mimics.

[0078] 3. Animal-level verification

[0079] A tumor model was established by subcutaneously inoculating EMT6 cells into BALB / c mice. After tumor formation, the mice were randomly divided into groups and treated with PBS, Random-PS, or TTiApt-PS, respectively. The results showed that the tumor volume growth in the TTiApt-PS treatment group was significantly slower than that in the control group (Figure 13), suggesting that this aptamer has potential tumor-suppressing activity in vivo. This indicates that the nucleic acid aptamer of the present invention has the potential as a pharmaceutical active ingredient for the prevention / treatment / inhibition of breast cancer progression.

[0080] Conclusion: The experimental results of the above embodiments demonstrate that the nucleic acid aptamer provided by this invention can specifically bind to the TND domain and block its interaction with TIM motifs (including MYCMB0, LEO1, and PAF1). It can serve as a TND-TIM interaction switch molecule (specific probe, competitive inhibitor, etc.) for studying protein-protein interaction regulatory mechanisms. In cell experiments, the nucleic acid aptamer can induce apoptosis in tumor cells; in mouse models, tumor growth in the treated group was significantly slowed, indicating that the nucleic acid aptamer of this invention can be used as a molecular tool for studying TND–TIM interaction regulation and provides a basis for exploring related disease mechanisms, drug development, and bioassay applications.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A TND-TIM interaction switch nucleic acid aptamer, characterized in that, The nucleic acid aptamer is a single-stranded DNA containing the nucleotide sequence shown in SEQ ID NO.1, specifically 5'-GGAGGGCTTGAGCA-3'; or a nucleotide sequence that has at least 30% homology with SEQ ID NO.1 and specifically binds to the TND domain protein; Alternatively, the nucleic acid aptamer may be a single-stranded RNA containing the nucleotide sequence shown in SEQ ID NO.2, specifically 5'-GGAGGGCUUGAGCA-3', or a nucleotide sequence that has at least 30% homology with SEQ ID NO.2 and specifically binds to TND domain proteins.

2. The TND-TIM interaction switch nucleic acid aptamer as described in claim 1, characterized in that, The nucleotide sequence of the nucleic acid aptamer is modified and the modified nucleic acid aptamer binds to the TND domain protein. The modification includes at least one of phosphorylation, 2'-O-methylation, amination, carboxylation, aldehydeation, thiolation, thiophosphate modification, oxygen substitution with selenium, and isotopization.

3. The TND-TIM interaction switch nucleic acid aptamer as described in claim 1, characterized in that, The nucleic acid aptamer specifically binds to the N-terminal domain of the transcription regulatory factor, blocking the interaction between the aptamer and its corresponding transcriptional interaction motif or similar peptide; the dissociation constant Kd of the nucleic acid aptamer binding to the TND domain of the protein is < 10 μM; the transcription regulatory factor is selected from ELOA, HRP2, TFIIS, IWS1, PNUTS, and MED26.

4. The TND-TIM interaction switch nucleic acid aptamer as described in claim 3, characterized in that, The nucleic acid aptamer specifically binds to the N-terminal domain of the transcriptional regulatory factor, blocking the interaction between the aptamer and its corresponding transcriptional interaction motif or similar peptide; the dissociation constant Kd of the nucleic acid aptamer binding to the TND domain of the protein is < 5 μM.

5. The TND-TIM interaction switch nucleic acid aptamer as described in claim 4, characterized in that, The dissociation constant Kd of the nucleic acid aptamer binding to the TND domain of the protein is <2 μM.

6. A nucleic acid aptamer conjugate, characterized in that, The nucleic acid aptamer conjugate is a conjugate of a nucleic acid aptamer according to any one of claims 1-5 with other substances for labeling, detection, diagnosis or treatment attached to the nucleotide sequence of the nucleic acid aptamer, and the conjugate of the nucleic acid aptamer with the attached substances specifically binds to the TND domain protein. The substance is selected from at least one of fluorescent markers, quenching groups, azide groups, alkynyl groups, radioactive substances, therapeutic substances, biotin, digoxin, maleimide, nanomaterials, polyethylene glycol, small peptides, siRNA, and enzyme labeling.

7. The application of the nucleic acid aptamer conjugate as described in claim 6, characterized in that, As specific probes, competitive inhibitors, lead molecules, and / or active pharmaceutical ingredients, they are used for the construction of models of the regulatory and / or molecular interaction mechanisms of transcriptional regulatory factors, the preparation of detection kits, and / or drug preparation.

8. A TND-TIM interaction or regulation performance characterization kit, characterized in that, Includes the nucleic acid aptamer conjugate as described in claim 6, auxiliary reagents, and / or reporter signal detection systems and / or solid-phase support carriers; The report-signal detection system and / or solid support are detection systems that support fluorescence polarization method, surface plasmon resonance (SPR) method or electrophoretic mobility analysis.

9. The TND-TIM interaction or regulation performance characterization kit as described in claim 8, characterized in that, The report-signal detection system is based on a fluorescence polarization detection system. The kit also includes fluorescently labeled ligands, which are transcriptional interaction motifs of the transcriptional regulatory factor to be tested or similar peptides.

10. A pharmaceutical composition, characterized in that, Its active pharmaceutical ingredient is the nucleic acid aptamer conjugate as described in claim 6.

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