A class of peptide ligands that can specifically bind to and / or significantly promote the assembly of Holliday linkers and their applications

Abstract

This invention discloses a class of polypeptide ligands that can specifically bind to and / or significantly promote the assembly of Holliday linkers and their applications, belonging to the field of biomedical technology. The general formula for the amino acid sequence of the polypeptide ligand is: AX₄BQX₃CXn, where A and B are each independently selected from any one of phenylalanine, tryptophan, tyrosine, lysine, and arginine; C is selected from any one of arginine and lysine; X represents any amino acid; and n is an integer ranging from 0 to 4. The advantages of this invention are: the polypeptide provided by this invention has a high affinity for Holliday linkers (HJs), a strong ability to promote HJ formation, and exhibits certain antitumor activity against human liver cancer cells A549.

Description

Technical Field

[0001] This invention relates to polypeptide ligands and their applications, specifically to a class of polypeptide ligands that can specifically bind to and / or significantly promote the assembly of Holliday linkers and their applications, belonging to the field of biomedical technology. Background Technology

[0002] The Holliday junction (HJ) is a high-order nucleic acid structure formed by the complementary pairing of four DNA strands. This theoretical model was first proposed by Robin Holliday in 1964 to explain the molecular mechanism of homologous recombination during meiosis. With ongoing research, the structure and function of the HJ have been extended to various DNA metabolic processes. For example, during cell division, HJ structures can form when replication forks are blocked, and these structures further participate in DNA damage repair; during homologous recombination repair, damaged DNA strands form HJ structures under the mediation of relevant repair proteins, thus ensuring the accuracy of the repair process; furthermore, terminal repetitive sequences on chromosomes can also form HJ structures, maintaining genome stability through telomere elongation and replacement mechanisms. Homologous recombination technology, developed based on the HJ theoretical model, has been widely applied in molecular biology, gene editing, and nanocarrier construction.

[0003] Despite the significant research value of hemispheres (HJs) in genetics, cell biology, and nucleic acid chemistry, they still face numerous challenges in chemical targeting, identification of interacting proteins, and research on intracellular regulatory mechanisms. First, HJ structures can branch and migrate along the DNA strand, and their formation is independent of specific base sequences, making precise localization difficult using conventional techniques such as gene editing and in situ hybridization. Second, under physiological conditions, DNA typically tends to form thermodynamically more stable double-stranded structures rather than quadruple-stranded HJ structures, resulting in lower abundance of HJs in the genome and greater difficulty in extraction and purification. Third, the highly flexible hydrophobic cavity at the center of the HJ structure increases the difficulty of ligand design and screening. Furthermore, existing chemical ligands targeting HJs generally suffer from insufficient selectivity, and their physicochemical properties limit their ability to achieve specific recognition of HJs in vitro and in vivo.

[0004] Therefore, significant technical bottlenecks remain in the targeting, interaction protein discovery, and regulatory network analysis of Helicobacter pylori (HJ), with the key issue being the lack of chemical tool molecules possessing both high selectivity and excellent physicochemical properties. Developing novel ligands capable of specifically recognizing HJs and exhibiting good application performance is of great significance for advancing basic research and biomedical applications related to HJs. Summary of the Invention

[0005] The purpose of this invention is to provide a class of polypeptide ligands that can specifically bind to and / or significantly promote the assembly of Holliday linkers, and the application of such polypeptide ligands in the biomedical field.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A class of polypeptide ligands that can specifically bind to and / or significantly promote the assembly of Holliday linkers, wherein the general formula of the amino acid sequence of the polypeptide ligand is: AX4BQX3CX n In this context, A and B are each independently selected from any one of phenylalanine, tryptophan, tyrosine, lysine, and arginine; C is selected from any one of arginine and lysine; X represents any amino acid; and n is an integer ranging from 0 to 4.

[0007] Preferably, the polypeptide ligand is: HJBP-3: FACRRWQWRMKKLGK; HJBP-4: FKARRWQWRMKKLGK; HJBP-5: FKCARWQWRMKKLGK; HJBP-6: FKCRAWQWRMKKLGK; HJBP-9:FKCRRWQARMKKLGK; HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-21: FFCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-23: FKCFRWQWRMKKLGK; HJBP-24: FKCRFWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-26: FKCRRFQWRMKKLGK; HJBP-27: FKCRRYQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-29: FKCRRKQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; HJBP-36: FKCRRWQWFMKKLGK; HJBP-37: FKCRRWQWRRKKLGK; HJBP-38: FKCRRWQWRMRKLGK; HJBP-42: FKCRRWQWRMKKLGF; HJBP-43: FKCRRWQRRMKRRGK; Alternatively, HJBP-44: FKRRRWQRRMKRRRK.

[0008] More preferably, the polypeptide ligand is: HJBP-3: FACRRWQWRMKKLGK; HJBP-5: FKCARWQWRMKKLGK; HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-21: FFCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-23: FKCFRWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; Alternatively, HJBP-37: FKCRRWQWRRKKLGK; The above-mentioned polypeptide ligands have an equilibrium dissociation constant of less than 0.3 μM for Holliday linkers, and can specifically bind to Holliday linkers.

[0009] More preferably, the polypeptide ligand is: HJBP-6: FKCRAWQWRMKKLGK; HJBP-9:FKCRRWQARMKKLGK; HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-24: FKCRFWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-27: FKCRRYQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-29: FKCRRKQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; HJBP-37: FKCRRWQWRRKKLGK; HJBP-38: FKCRRWQWRMRKLGK; HJBP-42: FKCRRWQWRMKKLGF; HJBP-43: FKCRRWQRRMKRRGK; Alternatively, HJBP-44: FKRRRWQRRMKRRRK; The above-mentioned polypeptide ligands promote the formation of Holliday linkers at a half-maximal effective concentration of less than 9.0 μM, and can significantly promote Holliday linker assembly.

[0010] More preferably, the polypeptide ligand is: HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; Alternatively, HJBP-37: FKCRRWQWRRKKLGK; The above-mentioned polypeptide ligands have an equilibrium dissociation constant for Holliday linkers of less than 0.3 μM and a half-maximal effective concentration for promoting Holliday linker formation of less than 9.0 μM. They can both specifically bind to Holliday linkers and significantly promote Holliday linker assembly.

[0011] The aforementioned application of polypeptide ligands that can specifically bind to and / or significantly promote the assembly of Holliday linkers in the preparation of anti-hepatocellular carcinoma drugs, wherein the polypeptide ligands are: HJBP-3: FACRRWQWRMKKLGK; HJBP-4: FKARRWQWRMKKLGK; HJBP-5: FKCARWQWRMKKLGK; HJBP-6: FKCRAWQWRMKKLGK; HJBP-9:FKCRRWQARMKKLGK; HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-21: FFCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-23: FKCFRWQWRMKKLGK; HJBP-24: FKCRFWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-26: FKCRRFQWRMKKLGK; HJBP-27: FKCRRYQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-29: FKCRRKQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; HJBP-36: FKCRRWQWFMKKLGK; HJBP-37: FKCRRWQWRRKKLGK; HJBP-38: FKCRRWQWRMRKLGK; HJBP-42: FKCRRWQWRMKKLGF; HJBP-43: FKCRRWQRRMKRRGK; Alternatively, HJBP-44: FKRRRWQRRMKRRRK.

[0012] The advantages of this invention are: (1) The amino acid sequence provided by the present invention conforms to AX4BQX3CX n The characteristic peptides have a high affinity for HJ. K D The concentration range is 0.1 μM-1.7 μM. Even under high-concentration competitive DNA conditions, it can still specifically recognize HJ, but has no significant effect on other nucleic acid structures; however, the amino acid sequence does not conform to AX4BQX3CX. n The characteristic peptides have a low affinity for HJ; (2) The amino acid sequence provided by the present invention conforms to AX4BQX3CX. n The characteristic peptides have a strong ability to promote HJ formation, with 75.8% of the peptides being EC. 50 Below 9 μM; and the amino acid sequence does not conform to AX4BQX3CX n The characteristic peptides have a weak ability to promote HJ formation, with 64.7% of the peptides being EC. 50 Above 14 μM.

[0013] (3) The amino acid sequence provided by the present invention conforms to AX4BQX3CX n The characteristic peptides exhibited certain antitumor activity against human liver cancer cells A549, with a cell proliferation inhibition rate of 29.6%-45.6% after 48 hours of treatment; however, the amino acid sequence did not conform to AX4BQX3CX. n The cell proliferation inhibition rate after 48 hours of treatment with the characteristic peptide was only 0.2%-11.2%. Attached Figure Description

[0014] Figure 1 This is the original spectrum of the biomembrane interference experiment from HJBP-1 peptide to HJBP-9 peptide; Figure 2 This is the original spectrum of the biomembrane interference experiment of HJBP-10 peptide to HJBP-18 peptide; Figure 3 This is the original spectrum of the biomembrane interference experiment from HJBP-19 peptide to HJBP-27 peptide; Figure 4 This is the original spectrum of the biomembrane interference experiment from HJBP-28 peptide to HJBP-36 peptide; Figure 5 This is the original spectrum of the biomembrane interference experiment from HJBP-37 peptide to HJBP-45 peptide; Figure 6 This is the original spectrum of the biomembrane interference experiment from HJBP-46 peptide to HJBP-50 peptide; Figure 7 It is AX4BQX3CX n Structure-activity relationship diagram of motif binding HJ; Figure 8 This is the original pattern of the gel migration retardation experiment from HJBP-1 peptide to HJBP-12 peptide; Figure 9 This is the original pattern of the gel migration retardation experiment of HJBP-13 peptide to HJBP-24 peptide; Figure 10 This is the original pattern of the gel migration retardation experiment of HJBP-25 peptide to HJBP-36 peptide; Figure 11 This is the original pattern of the gel migration retardation experiment of HJBP-37 peptide to HJBP-48 peptide; Figure 12 This is the original spectrum of the gel migration retardation experiment of HJBP-49 peptide and HJBP-50 peptide; Figure 13 This is a gel scanning image showing the effect of HJBP-1 peptide promoting the binding of FAM fluorescently labeled HJ under competitive DNA conditions. Figure 14 This is a gel scanning image showing the results of FAM fluorescently labeled HJBP-1 peptide promoting HJ binding under competitive DNA conditions. Figure 15 This is a schematic diagram of the binding mode between the core amino acids of the HJBP series polypeptides and HJ. Figure 16 This is a graph showing the calculated inhibition rates of HJBP-1 to HJBP-50 peptides on the proliferation of human liver cancer cells A549 at a concentration of 12.5 μM. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0017] I. Amino acid sequence of a polypeptide The polypeptide provided by this invention has the general formula of its amino acid sequence: AX4BQX3CX n In this context, A and B are each independently selected from any one of phenylalanine (F), tryptophan (W), tyrosine (Y), lysine (K), and arginine (R); C is selected from any one of arginine (R) and lysine (K); X represents any amino acid; and n is an integer ranging from 0 to 4.

[0018] II. Screening for peptide ligands that specifically bind to and promote the assembly of Holliday linkers using biomembrane interference techniques. This experiment used four partially complementary single-stranded DNA strands (HJ1, HJ2, HJ3, and HJ4) as substrates, with FAM fluorescent labeling applied to the 5' end of single-stranded HJ1 (FAM-HJ1). The HJ structure is assembled from HJ1, HJ2, HJ3, and HJ4, and is located in Mg... 2+ It exists stably under certain conditions.

[0019] The nucleotide sequences (5'→3') of HJ1, HJ2, HJ3, and HJ4 are as follows: HJ1:GGCAGTTGACGTCATGCTAGGC (SEQ ID NO: 51); HJ2: GCTAGCATGATACTGCTACCG (SEQ ID NO: 52); HJ3: CGGTAGCAGTACCGTTGGTGGC (SEQ ID NO: 53); HJ4: GCCACCAACGGCGTCAACTGCC (SEQ ID NO: 54).

[0020] The peptides used in this experiment include those conforming to AX4BQX3CX. n Polypeptides that do not conform to the motif also include those that do not conform to AX4BQX3CX. n The motifs of the polypeptides are detailed in Tables 1-1 to 1-4.

[0021] Table 1-1 Conforms to AX4BQX3CX n Amino acid sequence of a motif polypeptide (Part 1)

[0022] Table 1-2 Conforms to AX4BQX3CX n Amino acid sequences of motif-based polypeptides (Part 2)

[0023] Table 1-3 Not conforming to AX4BQX3CX n Amino acid sequence of a motif polypeptide (Part 1)

[0024] Table 1-4 Not conforming to AX4BQX3CX n Amino acid sequences of motif-based polypeptides (Part 2)

[0025] The specific steps of the biological membrane interference experiment are as follows: (1) The streptavidin-modified BLI sensor was immersed in a solution containing 50 μg / mL biotin-labeled HJ (annealed in 2.0 mM MgCl2 solution), fixed at 37 °C for 300 s, and washed with deionized water after the response value reached 0.6 nm.

[0026] (2) Equilibrate the BLI sensor in pre-cooled PBS buffer (pH=7.4) for 180s to obtain a stable baseline.

[0027] (3) The BLI sensor was transferred into a micropore containing a polypeptide (0 nM-1000 nM) and the binding and dissociation reactions were carried out at a constant temperature. The changes in the response signal of the binding phase were monitored in real time.

[0028] (4) Use the instrument’s matching dynamic analysis software to calculate the equilibrium dissociation constant by steady-state fitting.

[0029] The original spectra of the above 50 polypeptides in the biomembrane interference experiment are shown below. Figures 1 to 6 .

[0030] Calculations show that the equilibrium dissociation constants of the above 50 polypeptides with respect to HJ ( K D )as follows: Table 2-1 Conforms to AX4BQX3CX n motif peptides for HJ K D Calculation results

[0031] Table 2-2 Not conforming to AX4BQX3CX n motif peptides for HJ K D Calculation results

[0032] Note: ND indicates that affinity cannot be detected.

[0033] The results showed that the amino acid sequence conformed to AX4BQX3CX. nThe characteristic peptides generally bind effectively to HJ, while peptides that do not conform to this characteristic have significantly weaker affinity for HJ. For example, some peptides HJBP-2, HJBP-7, HJBP-8, HJBP-30, HJBP-31, HJBP-32, HJBP-33, HJBP-34, HJBP-39, HJBP-40, HJBP-41, HJBP-45, HJBP-46, HJBP-48, HJBP-49, and HJBP-50 show a significantly decreased affinity for HJ.

[0034] AX4BQX3CX n The structure-activity relationship of motif binding HJ is shown in Figure 7 Detailed analysis revealed that phenylalanine at position 1, tryptophan at position 6, glutamine at position 7, and lysine at position 11 play crucial roles in peptide binding to nucleic acids. Specifically, phenylalanine at position 1 can be replaced by tryptophan or tyrosine, both containing an aromatic ring, or by lysine or arginine, whose R group contains a terminal amino group; the active structure of tryptophan at position 6 is similar to that of phenylalanine at position 1; glutamine at position 7 is critical, and replacing it with other amino acids reduces peptide activity; lysine at position 11 can be replaced by arginine. Furthermore, individual amino acid analysis showed that HJBP-47 of the 11-peptide maintains the basic binding of the peptide to HJ, while the deletion of 2-3 consecutive amino acids (HJBP-45, HJBP-46) completely eliminates the peptide's activity, indirectly verifying the binding of AX4BQX3CX. n The universality of motifs for peptide binding to HJ.

[0035] III. Investigating the Influence of Peptides on HJ Assembly Using Gel Migration Retention Assay The HJ structure is assembled from HJ1, HJ2, HJ3, and HJ4. Its molecular weight is higher than that of single-stranded, double-stranded, and replication fork DNAs. It migrates slowly in non-denaturing polyacrylamide gels. The formation of HJs can be determined by observing the degree of migration retardation in the gel. The specific method is as follows: (1) Mix FAM-HJ1, HJ2, HJ3 and HJ4 in equimolar ratio and dissolve them in 1×TB buffer; (2) Different concentrations of peptides (0 μM, 0.3 μM, 0.6 μM, 1.3 μM, 2.5 μM, 5.0 μM, 10 μM and 20 μM) were incubated with HJ (final concentration 1.25 μM) in a 20 μL system at 37 °C in the dark. (3) After incubation for 1 hour, add 4 μL of 6× DNA loading buffer to terminate the reaction; (4) Electrophoretic separation was performed using 12% non-denaturing polyacrylamide gel at a constant voltage of 120V until the bromophenol blue reached 1 / 2 of the gel. (5) Use a gel imaging system to acquire images.

[0036] The imaging results of the gel migration retardation experiment of the above 50 peptides are shown in [the image]. Figures 8 to 12 The half-maximal effective concentration (EC50) of the peptide in promoting HJ formation was calculated through three independent experiments. 50 ).

[0037] Calculations show that the above 50 polypeptides promote the formation of HJ in EC. 50 as follows: Table 3-1 Conforms to AX4BQX3CX n motif polypeptides form ECs with HJ 50 Calculation Results (I)

[0038] Table 3-2 Conforms to AX4BQX3CX n motif polypeptides form ECs with HJ 50 Calculation Results (II)

[0039] Table 3-3 Not conforming to AX4BQX3CX n motif polypeptides form ECs with HJ 50 Calculation results

[0040] The results showed that the amino acid sequence conformed to AX4BQX3CX. n Characteristic peptides significantly promote HJ assembly, while peptides that do not conform to these characteristics have poor or no assembly ability of HJ. EC 50 and K D The significant positive correlation, consistent with structure-activity relationship analysis, indicates that the amino acid sequence conforms to AX4BQX3CX. n The characteristic polypeptide can both bind to HJ and promote the assembly and formation of HJ structures.

[0041] IV. Study on the effect of peptides on HJ formation under different concentrations of competitive DNA. Select amino acid sequences that conform to AX4BQX3CX n The characteristic peptide HJBP-15 was used as a representative peptide to study the effect of the peptide on HJ formation under different concentrations of competitive DNA.

[0042] (1) FAM-labeled HJ group: FAM-labeled HJ (1.25 μM, about 0.3 μg / mL) and unlabeled competitive DNA (single-stranded / double-stranded) at different concentrations (0.05 mg / mL-50 mg / mL) were added to a 20 μL reaction system, followed by the addition of peptide HJBP-15.

[0043] (2) FAM-labeled peptide group: Unlabeled HJ (1.25 μM, about 0.3 μg / mL) and unlabeled competitive DNA (single-stranded / double-stranded) at different concentrations (0.05 mg / mL-50 mg / mL) were added to a 20 μL reaction system, followed by the addition of FAM-labeled peptide HJBP-15.

[0044] (3) Incubate the reaction system at 37°C for 30 min, load the sample onto a 12% non-denaturing polyacrylamide gel, and perform electrophoresis separation in 1×TBE buffer at 4°C and 100V constant voltage until the bromophenol blue indicator migrates to about 2 / 3 of the bottom of the gel.

[0045] (4) Use a fluorescence imaging system (excitation wavelength 488nm, emission wavelength 520nm) to scan the gel, analyze the position and intensity of the fluorescence signal bands in each lane, and determine the effect of the peptide on HJ formation.

[0046] The gel scanning results of the FAM-labeled HJ group are shown in [the table]. Figure 13 The results showed that under low concentration competitive DNA conditions, the peptide could effectively promote HJ assembly, but under high concentration competitive DNA conditions (>10 mg / mL), the high concentration of competitive DNA could promote HJ assembly, and the peptide's function in promoting HJ assembly was masked.

[0047] Gel scanning results of the FAM-labeled polypeptide group are shown in […]. Figure 14 The results showed that as the concentration of competitive DNA increased, the FAM fluorescent band always appeared at the position of the HJ structure, indicating that the peptide could still specifically capture the HJ structure even under high concentrations of competitive DNA.

[0048] V. Study the structure-activity relationship and binding mode between peptides and HJ. The structure-activity relationship and binding mode of the peptides and HJ were studied by selecting the core amino acids of the HJBP series peptides and comparing them with the structure of HJ, as detailed below: (1) Molecular docking and long-term (≥100 ns) dynamic simulations were performed using GROMACS and AMBER to analyze the interactions between peptide amino acid residues and HJ in the simulated trajectory (such as hydrogen bonding, electrostatic interactions or π-π stacking) and preliminarily identify key sites that may interact with the peptide.

[0049] (2) The key action sites identified by the simulation results may be located in the central active cavity of the HJ structure, which is composed of nucleotides at positions 9-14 of the HJ1 to HJ4 chains. To further elucidate the molecular mechanism of peptide binding to HJ, baseless deoxynucleotides (lacking hydrogen bonds and base stacking ability) were introduced at the above sites to explore possible binding modes.

[0050] (3) Biotin-labeled HJ mutants were immobilized on the surface of the sensor chip by biomembrane interference method. HJBP-1 peptide was used as an analyte and flowed through the chip at a gradient concentration. The affinity between the peptide and each nucleic acid structure was calculated by steady-state fitting.

[0051] A schematic diagram illustrating the binding mode between the core amino acids of the HJBP series polypeptides and the HJ structure is shown below. Figure 15 .

[0052] By comparing the affinity of the peptides with various HJ mutants, it was found that the deletion of the nucleotide at position 12 (BD) in the HJ1 and HJ3 chains significantly reduced the binding affinity of the peptides to HJ to the point of being undetectable. Given that the nucleotide at position 10 in the above chains does not participate in complementary strand pairing and is exposed alone during HJ formation, it is speculated that this nucleotide may have a stacking effect with the phenylalanine at position 1 of the peptide. In addition, the deletion of the nucleotide at position 14 in the HJ2 and HJ4 chains did not reduce the binding of the peptides to HJ. The docking results showed that the hydroxyl group on the phosphate backbone at this position may have formed a hydrogen bond with the glutamine at position 7 of the peptide (key binding residues are marked in red).

[0053] VI. Antitumor Activity of Peptides (1) Human liver cancer cells A549 were seeded at 5000 cells / well in 96-well plates and cultured in DMEM medium containing 10% fetal bovine serum for 24 h in a cell culture incubator at 37°C and 5% CO2 saturated humidity.

[0054] (2) Peptide treatment group, solvent control group and blank group were set up respectively. In the peptide treatment group, the peptide to be tested was added to a final concentration of 12.5 μM and three replicates were set up. After administration, the mixture was cultured for 48 h. The solvent control group was added with an equal amount of DMSO. The blank group contained only culture medium.

[0055] (3) Add 20 μL of MTT solution (5.0 mg / mL) to each well of each group, incubate for 4 h, discard the supernatant, add 150 μL of DMSO to each well to dissolve formazan, and shake slowly for 10 min. Measure the absorbance at 490 nm using a microplate reader, and calculate the cell proliferation inhibition rate according to the following formula:

[0056] The calculated inhibition rates of each test peptide on the proliferation of human hepatocellular carcinoma A549 cells at a concentration of 12.5 μM are shown in the figure below. Figure 16 .

[0057] The results showed that the amino acid sequence conformed to AX4BQX3CX. n The characteristic peptides generally exhibit significant inhibitory effects on tumor proliferation, with a cell proliferation inhibition rate of approximately 29.6%-45.6% after 48 hours of treatment; however, the amino acid sequence does not conform to AX4BQX3CX. nThe characteristic peptides (such as HJBP-2, HJBP-7, HJBP-8, HJBP-12, HJBP-30, HJBP-31, HJBP-32, HJBP-33, HJBP-34, HJBP-39, HJBP-40, HJBP-41, HJBP-45, HJBP-46, HJBP-48, HJBP-49, and HJBP-50) showed significantly weaker antitumor effects, with cell proliferation inhibition rates of approximately 0.2%–11.2% after 48 hours of treatment.

[0058] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A class of polypeptide ligands that specifically bind to and / or significantly promote the assembly of Holliday linkers, characterized in that, The general formula for the amino acid sequence of the polypeptide ligand is: AX4BQX3CX n In this context, A and B are each independently selected from any one of phenylalanine, tryptophan, tyrosine, lysine, and arginine; C is selected from any one of arginine and lysine; X represents any amino acid; and n is an integer ranging from 0 to 4.

2. The polypeptide ligand according to claim 1, which specifically binds to and / or significantly promotes the assembly of Holliday linkers, is characterized in that, The polypeptide ligand is: HJBP-3: FACRRWQWRMKKLGK; HJBP-4: FKARRWQWRMKKLGK; HJBP-5: FKCARWQWRMKKLGK; HJBP-6: FKCRAWQWRMKKLGK; HJBP-9:FKCRRWQARMKKLGK; HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-21: FFCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-23: FKCFRWQWRMKKLGK; HJBP-24: FKCRFWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-26: FKCRRFQWRMKKLGK; HJBP-27: FKCRRYQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-29: FKCRRKQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; HJBP-36: FKCRRWQWFMKKLGK; HJBP-37: FKCRRWQWRRKKLGK; HJBP-38: FKCRRWQWRMRKLGK; HJBP-42: FKCRRWQWRMKKLGF; HJBP-43: FKCRRWQRRMKRRGK; Alternatively, HJBP-44: FKRRRWQRRMKRRRK.

3. The polypeptide ligand according to claim 2, which can specifically bind to and / or significantly promote the assembly of Holliday linkers, is characterized in that, The polypeptide ligand is: HJBP-3: FACRRWQWRMKKLGK; HJBP-5: FKCARWQWRMKKLGK; HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-21: FFCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-23: FKCFRWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; Alternatively, HJBP-37: FKCRRWQWRRKKLGK; The above-mentioned polypeptide ligands have an equilibrium dissociation constant of less than 0.3 μM for Holliday linkers, and can specifically bind to Holliday linkers.

4. The polypeptide ligand according to claim 2, which can specifically bind to and / or significantly promote the assembly of Holliday linkers, is characterized in that, The polypeptide ligand is: HJBP-6: FKCRAWQWRMKKLGK; HJBP-9:FKCRRWQARMKKLGK; HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-24: FKCRFWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-27: FKCRRYQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-29: FKCRRKQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; HJBP-37: FKCRRWQWRRKKLGK; HJBP-38: FKCRRWQWRMRKLGK; HJBP-42: FKCRRWQWRMKKLGF; HJBP-43: FKCRRWQRRMKRRGK; Alternatively, HJBP-44: FKRRRWQRRMKRRRK; The above-mentioned polypeptide ligands promote the formation of Holliday linkers at a half-maximal effective concentration of less than 9.0 μM, and can significantly promote Holliday linker assembly.

5. The polypeptide ligand according to claim 2, characterized in that, The polypeptide ligand is: HJBP-10: FKCRRWQWAMKKLGK; HJBP-11:FKCRRWQWRAKKLGK; HJBP-13: FKCRRWQWRMKALGK; HJBP-14: FKCRRWQWRMKKAGK; HJBP-15: FKCRRWQWRMKKLAK; HJBP-16: FKCRRWQWRMKKLGA; HJBP-17: YKCRRWQWRMKKLGK; HJBP-18: WKCRRWQWRMKKLGK; HJBP-19: RKCRRWQWRMKKLGK; HJBP-20: KKCRRWQWRMKKLGK; HJBP-22: FKRRRWQWRMKKLGK; HJBP-25: FKCRKWQWRMKKLGK; HJBP-28: FKCRRRQWRMKKLGK; HJBP-35: FKCRRWQRRMKKLGK; Alternatively, HJBP-37: FKCRRWQWRRKKLGK; The above-mentioned polypeptide ligands have an equilibrium dissociation constant for Holliday linkers of less than 0.3 μM and a half-maximal effective concentration for promoting Holliday linker formation of less than 9.0 μM. They can both specifically bind to Holliday linkers and significantly promote Holliday linker assembly.

6. The use of the polypeptide ligands of claim 2 that can specifically bind to and / or significantly promote the assembly of Holliday linkers in the preparation of anti-hepatocellular carcinoma drugs.

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