Synthesis method and application of biotinylated polypeptide conjugated drugs
Patent Information
- Application Number
- CN202510432170.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2026-10-16
AI Technical Summary
然而，补骨脂素水溶性较差、缺乏核定位能力、DNA亲和性低以及交联效率不足等方面的局限性制约了其在染色质特异性标记中的应用
[0021] This method provides a modification technique for drugs that are poorly soluble, have low cell entry efficiency, and are not targeted.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a biotinylated polypeptide drug-drug conjugate, comprising the polypeptide being a TAT membrane-penetrating peptide, the drug being psoralen, a method for synthesizing the conjugate, and its application in reversible nucleic acid labeling and in vivo chromatin-specific labeling. Background Technology
[0002] Psoralen is a compound derived from the traditional Chinese medicine psoralen. Its core structure is furanocoumarin. It exerts its pharmacological effects under ultraviolet radiation and has been widely used in photochemotherapy for psoriasis, vitiligo, and cutaneous T-cell lymphoma. 8-Methoxysarrin is commonly used for vitiligo patients, and amotoxa hydrochloride is commonly used to inactivate platelets and infectious pathogens in plasma.
[0003] Psoralen, as a photocrosslinking agent for nucleic acids, exhibits a 2+2 cycloaddition reaction between the carbon-carbon double bonds of its furan and pyrrolidone rings and the thymine bases of nucleic acids under 365 nm light illumination. It is also frequently used for nucleic acid labeling. When interacting with paired double-stranded nucleic acids, it tends to insert between complementary AT / TA bonds, producing monoaddition or diaddition products, known as interstrand crosslinking (ICL). Researchers have already used it to detect RNA secondary structures and interactions in vivo, developing a method called PARIS (Psoralen analysis of RNA interactions and structures), which led to the discovery of novel interactions between U16 and U6.
[0004] TAT transmembrane peptides are classic tool peptides in the field of drug delivery. They are derived from the trans-activator of transcription of human immunodeficiency virus type 1 (HIV-1). The core sequence is a short peptide YGRKKRRQRRR with 11 amino acids, rich in arginine and lysine, and carrying a strong positive charge. Its advantage lies in its ability to carry a variety of biomolecules into cells and its effectiveness against most cell types. Currently, using TAT transmembrane peptides as drug delivery carriers is a classic method to enhance cellular uptake and drug efficacy. It also has wide applications in the field of nanoparticle delivery. Studies have also shown that it has a certain ability to localize to the cell nucleus (Journal of the American Chemical Society 2012, 134(13)).
[0005] Biotin is a water-soluble B vitamin. Due to its high binding affinity to avidin, it has become a commonly used derivatizing group in protein and nucleic acid chemistry. After biotinylation, biological macromolecules can be coupled with biotin-antibody fluorescent dyes or streptomycin-avidin magnetic beads to achieve immunofluorescence and immunohistochemical imaging of live cells, or be specifically enriched for subsequent analysis.
[0006] In eukaryotes, DNA encoding genetic information is stored in the chromatin of the cell nucleus in a highly compressed manner, also known as the higher-order structure of the genome. This structure is characterized by DNA and histones arranged like beads on a string, constantly repositioning themselves to adjust gene expression. The DNA regions wrapped around histones are called chromatin "closed regions," where transcriptional activity is inhibited. The intermediate DNA regions connecting adjacent nucleosomes are called chromatin "open regions," which are actively expressed and generally contain transcription start sites, promoters, and other gene regulatory elements, typically exhibiting active gene transcription activity. Understanding the information from chromatin's open / closed regions is of significant scientific value for comprehending the impact of chromatin accessibility on disease development and progression.
[0007] Molecularly specific recognition of nucleic acid chemical labeling techniques is an important means of elucidating DNA / RNA structural information. For example, N3-Kethoxal can specifically label single-stranded DNA formed during whole-genome transcription and detect ssDNA produced during transcription or other processes. Studies have shown that psoralen can specifically add to negatively coiled DNA in open chromatin regions to form ICLs, demonstrating its potential application value in chromatin accessibility characterization. However, limitations such as poor water solubility, lack of nuclear localization ability, low DNA affinity, and insufficient cross-linking efficiency restrict its application in chromatin-specific labeling. Summary of the Invention
[0008] The purpose of this invention is to provide a simple and universal method for peptide-conjugated drugs, wherein the amino terminus of the peptide is modified with biotin, the conjugation method is a click reaction, and the drug is a poorly soluble drug.
[0009] The polypeptide of the present invention is characterized as a TAT membrane-penetrating peptide with the amino acid sequence YGRKKRRQRRR, rich in arginine and lysine, and carrying a strong positive charge.
[0010] The polypeptide of the present invention is characterized by being synthesized by solid-phase synthesis, with the addition of a non-natural amino acid with an azide side chain to the synthon, and biotin modified at the amino terminus.
[0011] The poorly soluble drug of the present invention is characterized as psoralen, which is synthesized from trimethasone and modified with cyclooctyne (DBCO).
[0012] The poorly soluble drug of the present invention is characterized by undergoing a 2+2 cycloaddition reaction with thymine of nucleic acids under 365nm light irradiation, and undergoing decrosslinking under 254nm light irradiation.
[0013] The coupling method described in this invention is characterized by a cycloalkyne-azide click reaction (SPAAC).
[0014] The biotinylated polypeptide conjugate of the present invention is characterized in that it can be used for photoreversible labeling of nucleic acids.
[0015] The biotinylated polypeptide conjugate of the present invention is characterized by good water solubility and strong affinity for DNA.
[0016] The biotinylated polypeptide conjugate of the present invention is characterized by high cellular uptake rate, nuclear colocalization function, and enrichment.
[0017] The biotinylated polypeptide conjugate of the present invention is characterized by its ability to be used for specific labeling of open region DNA in living chromatin.
[0018] The core design of this invention lies in the fact that the TAT membrane-penetrating peptide enables nuclear-targeted delivery of psoralen. The positive charge of its side chain groups significantly enhances the interaction between psoralen and negatively charged nucleic acids, thereby effectively enhancing the efficiency of photocrosslinking. Furthermore, biotin labeling can be used for in vivo imaging and enrichment of crosslinked DNA in open regions.
[0019] Preferably, the photoreversibility of the nucleic acid labeling of the present invention is characterized by decrosslinking at 254 nm under the protection of acridine orange.
[0020] Preferably, the chromatin open region marker of the present invention is characterized by being analyzeable by the Bal 31 nuclease crosslinking site retardation method. The present invention has the following beneficial effects
[0021] This method provides a modification technique for drugs that are poorly soluble, have low cell entry efficiency, and are not targeted.
[0022] The synthesis of peptide-conjugated drugs is simple and versatile, and can be used for the conjugation of various peptides with drugs.
[0023] Biotinylated peptide-conjugated drugs provide an efficient imaging and enrichment analysis method for exploring drug action sites.
[0024] The coupling method is carried out in an aqueous environment under mild conditions, without extreme high temperature or high pressure requirements, which meets the requirements of green chemistry.
[0025] Compared with conventional psoralen, TAT transmembrane peptides significantly enhance the efficiency of interstrand crosslinking with DNA under 365nm light irradiation.
[0026] Compared with conventional nucleic acid labeling methods, this method is photoreversible, achieving decrosslinking under 254nm light illumination. The singlet oxygen caused by 254nm can be removed by acridine orange, and the sequence information of the nucleic acid can be completely restored.
[0027] Compared with conventional chromatin open region labeling methods, this method can obtain cross-linked fragments of chromatin open regions in a short time using Bal 31 nuclease.
[0028] Compared with conventional chromatin accessibility analysis techniques, this method can obtain information on open regions of live chromatin and has better spatiotemporal controllability. Detailed Implementation
[0029] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention, and should not be construed as limiting the present invention.
[0030] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art. Attached Figure Description
[0031] Figure 1 The synthesis route diagram for Biotin-TAT-Lys(N3) in Example 1 is shown.
[0032] Figure 2 This is the synthesis route diagram of DBCO-Amotosalen in Example 1.
[0033] Figure 3 This is a structural diagram of the Biotin-TAT-Amotosalen in Example 1.
[0034] Figure 4 The image shows the 1H NMR spectrum of Biotin-TAT-Amotosalen in Example 1.
[0035] Figure 5 This is a modified PAGE gel image of Example 2.
[0036] Figure 6 This is a statistical graph of qPCR in Example 2.
[0037] Figure 7 This is the MALDI-TOF image of Example 2.
[0038] Figure 8 This is a modified PAGE gel image of Example 3.
[0039] Figure 9 This is a statistical graph of qPCR in Example 3.
[0040] Figure 10 This is a schematic diagram of Example 4.
[0041] Figure 11 This is an agarose gel image of Example 4.
[0042] Figure 12 This is the immunoblot image from Example 4.
[0043] Figure 13 This is a confocal microscope image of Example 5.
[0044] Figure 14 This is a flow cytometry diagram from Example 5.
[0045] Figure 15 This is a heat map of Example 6.
[0046] Example 1
[0047] First, the biotinylated polypeptide Biotin-TAT-Lys(N3) was obtained by solid-phase synthesis. The solid-phase synthesis steps ( Figure 1 The process involved: In a polypeptide synthesis tube, using 2-chlorotriphenylmethyl chloride resin as a carrier, side-chain protected amino acid synthons were gradually introduced. Fmoc-Lys(N3)-OH was added as a synthon, and the amino terminus was capped with carboxybiotin. The mixture was recrystallized with ice-cold ether, washed, and the resulting unprotected polypeptide was obtained. This unprotected polypeptide was dissolved in a deprotecting agent, reacted for 5 minutes, recrystallized with ice-cold ether, washed, and filtered to obtain a grayish-white powder. After purification by preparative liquid chromatography, the powder was lyophilized, combined, weighed, and stored at 4°C.
[0048] Amotosalen·HCl, which exhibits good water solubility and DNA affinity, was synthesized from trimethalsalen (CAS: 3902-71-4). Then, DBCO amide was condensed to form DBCO-Amotosalen. (Steps...) Figure 2The reaction was as follows: 1,3,5-trioxacyclopentane (0.35 g, 3.95 mmol) was dissolved in 22.5 mL of concentrated hydrochloric acid. Trimethylsalicyline (1 g, 4.5 mmol) was gradually added in 10 portions under vigorous stirring, and the mixture was stirred overnight at room temperature. After terminating the reaction with 60 mL of water, compound CMT was extracted with dichloromethane. The organic phase was washed three times with water and dried over sodium sulfate. Rotary evaporation yielded a white solid, namely compound CMT, with a yield of approximately 70%. Subsequently, compound CMT was converted to compound CF3-CMT via Williamson ether synthesis. After removing trifluoroacetamide, compound CF3-CMT was obtained. Compound CMT (0.75 g, 2.7 mmol) was mixed with N-(2-hydroxyethyl)trifluoroacetamide (1.5 g, 9.55 mmol), heated to 100 °C, and stirred for 4 hours. After cooling the mixture to room temperature, recrystallization from methanol yielded a grayish-white powder, namely compound CF3-CMT, with a yield of approximately 70%. Compound CF3-CMT was dissolved in methanol containing 0.5 M cesium carbonate and stirred at room temperature for 16 hours. The mixture was then rotary evaporated until no solvent evaporated, and separated by flash chromatography. The fraction containing dichloromethane:methanol = 9:1 (v / v) was collected, dissolved in ethanol, cooled on an ice bath, and then added to diethyl ether containing 1 M hydrochloric acid. Stirring was continued on ice for 4 hours, and the resulting white precipitate was Amotosalen·HCl, with a final yield of approximately 50%. Compound Amotosalen·HCl underwent a condensation reaction with dibenzocyclooctyne-N-hydroxysuccinimide in a triethylamine alkaline environment to give compound DBCO-Amotosalen.
[0049] Biotin-TAT-Lys(N3) was dissolved in water to a final concentration of 300 μM, and DBCO-Amotosalen was dissolved in DMSO to a final concentration of 3 mM. The two solutions were mixed at a ratio of H2O:DMSO = 9:1 (v / v), with an actual molar ratio of 1:1. The mixture was shaken at room temperature for 12 hours. The reaction was monitored by RP-TLC, and the Biotin-TAT-Amotosalen solution was purified by liquid chromatography to obtain the solution described in this method. Figure 3 ).
[0050] Analysis by high-resolution mass spectrometry and proton nuclear magnetic resonance (NMR) spectroscopy Figure 4 All of the above compounds showed good agreement with the theoretical values, indicating successful synthesis. The high-resolution mass spectrometry and hydrogen nuclear magnetic resonance (HMR) results for Biotin-TAT-Amotosalen are as follows: C 118 H 179 N 41 O 24 S 2+ The theoretical m / z value is 1293.6895, but we obtained 1293.6894 (err. 0.08ppm), corresponding to [M+2H].2+ C 118 H 180 N 40 O 24 S 3+ The theoretical value of m / z is 862.7954, but we obtained 862.7963 (err. 1.04ppm), corresponding to [M+3H]. 3+ C 118 H 181 N 40 O 24 S 4+ The theoretical m / z value is 647.3484, but we obtained 647.3485 (err. 0.15ppm), corresponding to [M+4H]. 4+ C 105 H 175 N 40 O 24 S 5+ The theoretical value of m / z is 518.0802, yielding 518.0802 (err. 0ppm), corresponding to [M+5H]. 5+ . 1 H NMR (300MHz, DMSO-d6, ppm) δ: 11.99 (br, 1H), 9.26 (s, 1H), 7.95-8.69 (m, 21H), 6.43-7.28 (m, 36H), 7.16 (J = 8.1Hz, 2H), 7.00 (J = 8.4 Hz,2H),4.6(s,2H),3.73-4.41(m,18H),3.27(m,4H),3.07-3.27(m,20H),2.42-2.50(m,5H),2.00-2.12(m,4H),1.23-1.74(m,61H).
[0051] Example 2
[0052] Preferably, FAM-ACTATAGGGTTGTCTCTGGTCCTTACTTAATTGCTAC and its complementary oligonucleotide sequence are selected and annealed to a final concentration of 200 nM to form double-stranded DNA. This DNA is then mixed with Amotosalen·HCl and Biotin-TAT-Amotosalen in buffer systems, respectively, and incubated on ice for 10 minutes. Afterward, it is placed in a 365 nm UV crosslinker (UVP, CL-1000), maintaining a 4 cm distance between the lamp and the mixture. Photocrosslinking is performed for 1, 3, 5, 10, and 20 minutes at a power of approximately 1 J·cm⁻¹. -2 Products collected at different time points were placed in equal volumes of 95% formamide loading buffer and analyzed by electrophoresis on a 20% denaturing polyacrylamide sugar gel (200 mV, 1.5 h), and observed in a fluorescence imager. Figure 5 The results of the denaturing PAGE gel show that the ICL product gradually increases with time. The cross-linking products of Biotin-TAT-Amotosalen and DNA exhibit a diffuse state in the PAGE gel image. This may be due to the large positive charge and molecular weight of the TAT peptide, which alters the electrophoretic behavior of the DNA interstrand cross-linking products as the number of cross-links increases. Band grayscale analysis revealed that the cross-linking efficiency of Biotin-TAT-Amotosalen and DNA reaches over 60% within 10 minutes, demonstrating higher efficiency compared to Amotosalen·HCl at the same concentration. This is related to the abundant positive charge on the lysine and arginine side chains of the TAT peptide.
[0053] Preferably, a 216bp DNA segment encoding a GFP gene is amplified by PCR and used as a substrate template. The template is placed in 100μL of reaction buffer to a final concentration of 0.6ng / μL. After incubation with Amotosalen·HCl and Biotin-TAT-Amotosalen, respectively, and then irradiated with 365nm light, 10μL of the template is taken out at 1, 3, 5, 10, and 20 minutes and used as a template for qPCR quantitative analysis under standard conditions. Figure 6 The graph shows the statistical results of qPCR. Since interstrand crosslinks inhibit DNA replication, the Ct value is positively correlated with the crosslinking ratio. The Ct value increases with increasing 365nm illumination time, and Biotin-TAT-Amotosalen exhibits comparable crosslinking efficiency compared to Amotosalen·HCl.
[0054] Preferably, TGTGCAGCTCCTCCACGCG and its complementary oligonucleotide sequence are selected, and double-stranded DNA is formed by annealing and hybridization at a final concentration of 200 nM. The DNA is then incubated with different concentrations of Biotin-TAT-Amotosalen in reaction buffer and irradiated with 365 nm light. The reaction solution is desalted by a Bio-Spin 6 column and analyzed by MALDI-TOF MS. Figure 7 The MALDI-TOF image shows that after cross-linking, the mass spectrum peak increased from 12234 to 14820, indicating that double-stranded DNA and Biotin-TAT-Amotosalen formed a covalent cross-linked product with BTA through the addition reaction of psoralen under light irradiation.
[0055] Example 3
[0056] Preferably, FAM-ACTATAGGGTTGTCTCTGGTCCTTACTTAATTGCTAC and its complementary oligonucleotide sequence are selected and annealed to a final concentration of 200 nM to form double-stranded DNA. This DNA is then mixed with Biotin-TAT-Amotosalen in a buffer system, incubated on ice for 10 minutes, and then placed in a 365 nm UV crosslinker (UVP, CL-1000). The distance between the lamp and the mixture is maintained at 4 cm. The power is turned on for photocrosslinking for 20 minutes at a power of approximately 1 J·cm⁻¹. -2 The lamp was then changed to 254nm, and a high concentration of acridine orange was added to the product. The power was turned on to decrosslink for 1, 5, 10, and 20 minutes. The products at different times were placed in an equal volume of 95% formamide loading buffer and electrophoretically analyzed on a 20% denaturing polyacrylamide gel (200mV, 1.5h). The results were observed in a fluorescence imager. Figure 8 The results from denaturing PAGE gels showed that as the interstrand-crosslinked DNA was exposed to light at 254 nm for an extended period, the diffuse bands of the ICL shifted downwards and decreased until they completely overlapped with the single-stranded DNA, demonstrating the photoreversibility of the crosslinking process.
[0057] Preferably, a 216bp DNA segment encoding a GFP gene is amplified by PCR and used as a substrate template. This template is placed in 100μL of reaction buffer to a final concentration of 0.6ng / μL. After incubation with Amotosalen·HCl and Biotin-TAT-Amotosalen, respectively, the mixture is irradiated at 365nm for 20 minutes. The irradiation is then changed to 254nm, and a high concentration of acridine orange is added to the product. The mixture is then decrosslinked for 1, 5, 10, and 20 minutes. 10μL of the product from each time point is collected as a template and subjected to qPCR quantitative analysis under standard conditions. Figure 9 The graph shows the statistics of qPCR. Due to the high light energy of UVC, a large amount of singlet oxygen will be generated, which will reduce the activity of nucleic acids. To offset this loss, acridine orange was introduced as a singlet oxygen scavenger. Finally, the proportion of DNA that can recover its initial activity under 254nm light and acridine orange protection was determined.
[0058] Example 4
[0059] Preferably, a method for analyzing the retardation of nuclease cross-linking sites was implemented. Figure 10The conceptual diagram illustrates this process. Specifically, when chromatin is digested by micrococcal nuclease (MNase), the gene structure exhibits a multiple of nucleosomes. Genomic DNA, after histone removal via proteinase K, appears as arithmetic bands of 150 bp, 300 bp, 450 bp, 600 bp… at 150 bp intervals on an agarose gel. This native DNA is then digested into mononucleotides by exonucleases, and the bands disappear. If naked linker DNA in the chromatin has cross-linked with psoralen, enzyme digestion stops at the cross-linking site, thus maintaining its original arithmetic banding pattern. Naked chromatin, after being labeled with Biotin-TAT-Amotosalen (BTA), Amotosalen-PEG3-Biotin (APB), and Amotosalen·HCl, is sequentially digested with micrococcal nuclease and proteinase K. Traditional methods for cutting non-crosslinked DNA use lambda exonucleases. These enzymes recognize the 5' phosphate group of the DNA sequence and cleave until the crosslinking site stops. Therefore, a typical digestion combination is PNK (for 5' phosphorylation) - lambda exonuclease (cuts linear double-stranded DNA, stopping at the crosslinking site) - S1 nuclease (digests single-stranded DNA), a process that takes two hours. In contrast, this method uses Bal 31 nuclease from marine bacteria. This enzyme has exonuclease function and can efficiently digest DNA from the 5' and 3' ends until the crosslinking site stops, a process that takes only 3 minutes and yields results consistent with traditional digestion combinations. See details... Figure 11 The agarose gel images of both are shown.
[0060] Preferably, a biotinylated dot blot method was implemented, in which the chromatin labeled with Biotin-TAT-Amotosalen was used to perform a DNA dot blot experiment to evaluate DNA samples with different quality gradients. Figure 12 Based on the immunoblotting results, in the HRP-labeled secondary antibody detection, the Biotin-TAT-Amotosalen labeled group showed a dose-related biotin signal compared to the Amotosalen·HCl labeled group, while there was no significant difference between the Amotosalen·HCl labeled group and the blank group. This specific labeling ability stems from the fact that Biotin-TAT-Amotosalen can recognize and label DNA sequences in open chromatin regions, enabling them to be accurately recognized by biotin antibodies.
[0061] Example 5
[0062] Preferably, using A549 cells as a model, the results of the co-localization experiment under a confocal microscope are as follows: Figure 13Compared with the APB control group, the Pearson colocalization coefficient of green fluorescence and blue fluorescence of nuclear dye Hoechst 33342 in the Biotin-TAT-Amotosalen group reached 0.84±0.03, which was significantly higher than 0.17±0.05 in the APB control group. Therefore, Biotin-TAT-Amotosalen may be able to break through the nuclear membrane barrier, providing a spatial basis for its use as a marker molecule for open chromatin regions.
[0063] Preferably, using A549 cells as a model, flow cytometry was used to detect the cell uptake efficiency, and the results were as follows: Figure 14 Quantitative analysis showed that the intracellular fluorescence intensity of Biotin-TAT-Amotosalen was 1.7 times higher than that of the APB control group, indicating its significant transmembrane transport efficiency. This property, by increasing the intracellular concentration of psoralen, thereby enhancing the spatiotemporal efficiency of the photocrosslinking reaction, provides a pharmacokinetic basis for BTA in nuclear entry and labeling.
[0064] application
[0065] Disruptions in chromatin accessibility are a significant manifestation in the development of various diseases, including cardiovascular diseases, cancer, digestive system diseases, nervous system diseases, endocrine disorders, respiratory diseases, and infectious diseases. By revealing the dynamic regulatory mechanisms of gene expression, chromatin accessibility provides crucial theoretical support for understanding the laws governing life activities and analyzing the occurrence and development of diseases. Generally, open regions of chromatin contain numerous transcription start sites, promoters, and other elements. This method uses psoralen chemical labeling and enrichment to obtain open region fragments, which are then amplified, sequenced using next-generation sequencing, and subjected to conventional bioinformatics analysis. Figure 15 The heatmap shown indicates that the psoralen enrichment peaks aggregate within a 10kb range upstream and downstream of the TSS.
[0066] The above embodiments are merely illustrative examples to clearly illustrate the present invention and are not intended to limit the implementation of the invention. Those skilled in the art will recognize that other variations or modifications can be made 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 biotinylated polypeptide conjugate drug, characterized in that... The amino terminus of the polypeptide is modified with biotin, the coupling method is a click reaction, and the drug is a poorly soluble drug.
2. The polypeptide according to claim 1, characterized in that... It is a TAT transmembrane peptide with the amino acid sequence YGRKKRRQRRR.
3. The polypeptide according to claim 1, characterized in that... It was synthesized by solid-phase synthesis, with the addition of a non-natural amino acid Fmoc-Lys(N3)-OH with an azide side chain to the synthon, and biotin modified at the amino terminus.
4. The poorly soluble drug according to claim 1, characterized in that... It is psoralen, synthesized from trimethylsalin and modified with cyclooctyne (DBCO).
5. The poorly soluble drug according to claim 1, characterized in that... It undergoes a 2+2 cycloaddition reaction with thymine of nucleic acids under 365nm light and undergoes decrosslinking under 254nm light.
6. The coupling method according to claim 1, characterized in that... Cycloalkyne-azide click reaction (SPAAC).
7. The biotinylated polypeptide conjugate according to claim 1, characterized in that... It can be used for the photoreversible labeling of nucleic acids.
8. The biotinylated polypeptide conjugate according to claim 1, characterized in that... It has good water solubility, strong affinity for DNA, high cellular uptake rate, nuclear colocalization, and can be enriched by streptavidin magnetic beads.
9. The biotinylated polypeptide conjugate according to claim 1, characterized in that... It can be used for DNA-specific labeling of open regions in living chromatin.
10. The marked light reversibility according to claim 7, characterized in that... Crosslinking at 254 nm occurs under the protection of acridine orange.
11. The chromatin open region labeling according to claim 10, characterized in that... It can be analyzed by the Bal 31 nuclease crosslinking site blocking method.