Signal peptide, polypeptide-nucleic acid conjugate and synthetic method and application thereof

Abstract

The invention discloses a signal peptide, a polypeptide-nucleic acid conjugate and a synthesis method and application thereof, and belongs to the technical field of molecular biology. The signal peptide is one of NPXY, YXXphi, [DE] XXX [LI] and DXXLL; wherein X is any amino acid, phi is any large-volume hydrophobic side chain amino acid, [DE] represents one of D and E, and [LI] represents one of L and I. In addition, the invention also provides application of the signal peptide in preparation of a compound capable of penetrating through a cell membrane or a body tissue barrier by being connected with a nucleic acid fragment. The invention also provides a polypeptide-nucleic acid conjugate which is a conjugate of any one of the signal peptides and siRNA (small interfering Ribonucleic Acid). After several signal peptides provided by the invention are coupled with the siRNA, the siRNA can be promoted to escape into cytoplasm from an endosome, so that the siRNA drug transfection efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a signal peptide, a polypeptide-nucleic acid conjugate, its synthesis method, and its application. Background Technology

[0002] With the emergence of RNA interference (RNAi), a groundbreaking technology in molecular biology, small interfering RNA (siRNA), an important initiator in the RNAi pathway, has attracted widespread attention. siRNA regulates gene expression by silencing its complementary target mRNA, which is of great significance for the treatment of various diseases. Because it acts directly on the gene expression regulation level, siRNA drugs have many advantages over protein-targeting therapies, including shorter development cycles, higher success rates, lower in vivo toxicity, and longer duration of action. However, as a drug, siRNA needs to overcome the vascular barrier, evade renal metabolic clearance and the phagocytic system, cross the endothelial barrier, achieve endocytosis and endosome escape, and avoid degradation by nucleases. Research data shows that the intracellular escape rate of siRNA drugs after endocytosis is <2%, which is a key issue leading to a significant reduction in the therapeutic efficacy of siRNA drugs.

[0003] In recent years, numerous drug delivery vectors and strategies have been proposed to address the delivery challenges of siRNA drugs, including both viral and non-viral methods. Viral vectors, due to their strong biotoxicity and immunogenicity, have faced significant research limitations, leading to the wider research and application of non-viral delivery systems. Of the six currently marketed siRNA drugs, all utilize lipid nanoparticles (LNPs) and N-acetylgalactosamine (GalNAc) as delivery systems. GalNAc, through conjugation, forms structurally more stable small-molecule conjugates with siRNA, giving it a dominant position in the siRNA drug field. However, both LNPs and GalNAc currently on the market have limitations in liver targeting, and while they have shown good efficacy at the extracellular delivery level, significant challenges remain regarding intracellular escape. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a signal peptide, a polypeptide-nucleic acid conjugate, a method for synthesizing the same, and its application, thereby solving the technical problem of how to achieve efficient intracellular escape of siRNA and improve the transfection efficiency of siRNA drugs in the prior art.

[0005] To achieve the above-mentioned technical objectives, the present invention provides a signal peptide, wherein the signal peptide is one of NPXY, YXXФ, [DE]XXX[LI], DXXLL or one of the sequences containing E, D, T and S; wherein X is any amino acid, Ф is any bulky hydrophobic side chain amino acid, N is asparagine, P is proline, Y is tyrosine, E is glutamic acid, L is leucine, I is isoleucine, D is aspartic acid, S is serine, T is threonine, [DE] represents either D or E, and [LI] represents either L or I.

[0006] In any embodiment, the amino acid sequence of the signal peptide is one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0007] Furthermore, the present invention also proposes the application of the above-mentioned signal peptide in the preparation of a complex that can penetrate cell membranes or body tissue barriers by linking it with nucleic acid fragments.

[0008] In any embodiment, the nucleic acid fragment is siRNA.

[0009] The present invention also proposes a polypeptide-nucleic acid conjugate, which is a conjugate of any of the above-mentioned signal peptides and siRNA.

[0010] Furthermore, the present invention also proposes a method for synthesizing the above-mentioned signal peptide, comprising the following steps:

[0011] Using Wang resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-amino acid-Wang resin. After deprotection, the process was repeated to obtain Fmoc-peptide-Wang resin. The linker was condensed, the protecting group was removed, and the thiol donor was condensed. The reaction was then carried out with trifluoroacetic acid. The cleavage solution was dried under nitrogen, precipitated with anhydrous diethyl ether, and centrifuged to obtain the precipitate. The precipitate was then dissolved in water and acetonitrile, purified by HPLC, and lyophilized to obtain the signal peptide.

[0012] In any embodiment, the condensation linker comprises: condensed [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid as a linker.

[0013] In any embodiment, the condensed mercapto donor comprises condensed 3-(triphenylmethylmercapto)propionic acid.

[0014] In addition, the present invention also proposes a method for preparing the above-mentioned polypeptide-nucleic acid conjugate, comprising the following steps: mixing the signal peptide and the sense strand in a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, separating the reaction product by electrophoresis after the first oscillation, cutting off the target band, eluting through a column, and lyophilizing to obtain the polypeptide-nucleic acid conjugate single-chain product; mixing the polypeptide-nucleic acid conjugate single-chain product with the corresponding antisense strand at the same concentration, and oscillating for the second time to obtain the polypeptide-nucleic acid conjugate.

[0015] In any embodiment, the temperature of the first oscillation is 35-40°C, and the duration is 16-18 hours.

[0016] In any embodiment, the temperature of the second oscillation is 90-95°C, and the duration is 1-2 minutes.

[0017] Compared with the prior art, the beneficial effects of the present invention include: after the signal peptides proposed in the present invention are coupled with siRNA, the signal peptides recognize intracellular transport protein receptors, which can promote the escape of siRNA from the endosome into the cytoplasm, thereby improving the transfection efficiency of siRNA drugs. Attached Figure Description

[0018] Figure 1 This is the analytical liquid chromatography spectrum of Mpa-AEEA-KDPDEVETES prepared in Example 1 of this invention.

[0019] Figure 2 This is the mass spectrum of Mpa-AEEA-KDPDEVETES prepared in Example 1 of this invention.

[0020] Figure 3 This is the mass spectrum of Mpa-AEEA-FDNPVY obtained in Example 2 of this invention.

[0021] Figure 4 This is the mass spectrum of Mpa-AEEA-SYKGLPPE prepared in Example 3 of this invention.

[0022] Figure 5 This is the mass spectrum of the Mpa-AEEA-SDKQTLL prepared in Example 4 of this invention.

[0023] Figure 6 This is the mass spectrum of Mpa-AEEA-SFHDDSDEDLLHI obtained in Example 5 of this invention.

[0024] Figure 7 This is the mass spectrum of Mpa-AEEA-GYHDDSDELLE obtained in Example 6 of this invention.

[0025] Figure 8This is the mass spectrum of Mpa-AEEA-RDRDYDEDDEDDI obtained in Example 7 of this invention.

[0026] Figure 9 This is the mass spectrum of the KDPDEVETES-sense obtained in Example 8 of this invention.

[0027] Figure 10 This is the mass spectrum of FDNPVY-siRNA prepared in Example 8 of this invention.

[0028] Figure 11 This is the mass spectrum of SYKGLPPE-siRNA prepared in Example 8 of this invention.

[0029] Figure 12 This is the mass spectrum of the SDKQTLL-siRNA prepared in Example 8 of this invention.

[0030] Figure 13 This is the mass spectrum of GYHDDSDELLE-sense obtained in Example 8 of this invention.

[0031] Figure 14 This is the mass spectrum of the RDRDYDEDDEDDI-sense obtained in Example 8 of this invention.

[0032] Figure 15 This is a gel electrophoresis image of the purity of the polypeptide-nucleic acid conjugate prepared in Example 8 of this invention.

[0033] Figure 16 This is a bar chart showing the percentage of GFP fluorescence expression in Hela cells at different concentrations in each group of Example 9 of the present invention.

[0034] Figure 17 These are the EC50 curves fitted to the GFP fluorescence expression of Hela cells at different concentrations in each group of Example 9 of this invention.

[0035] Figure 18 This is a bar chart showing the contrast between stronger Cy5 fluorescence in HeLa cells under different incubation times in Example 10 of the present invention.

[0036] Figure 19 This is a gel electrophoresis image of the purity of the polypeptide-nucleic acid conjugate in Example 10 of the present invention.

[0037] Figure 20 This is the mass spectrum of KDPDEVETES-siRNA-Cy5 from Example 10 of the present invention.

[0038] Figure 21 This describes the knockdown effect of nucleic acid drugs on GFP protein in cells under different inhibitor treatments in Example 11 of the present invention.

[0039] Figure 22 The graphs show the statistical levels of GFP mRNA expression in HeLa cells in Example 12 of this invention: A. 1 nM; B. 100 nM.

[0040] Figure 23 The EC50 curves are obtained by fitting the GFP fluorescence expression of Hela cells at different concentrations in Example 13 of this invention.

[0041] Figure 24 This describes the knockdown effect of nucleic acid drugs on GFP protein in cells under different inhibitor treatments in Example 14 of the present invention.

[0042] Figure 25 This is an immunofluorescence image of the nucleic acid drug and early endosomes in cells, and a statistical graph of fluorescence overlap rate, from Example 15 of this invention.

[0043] Figure 26 This is an immunofluorescence image of the nucleic acid drug and late-stage endosomes in cells, and a statistical graph of fluorescence overlap rate, as shown in Example 15 of this invention.

[0044] Figure 27 This is an immunofluorescence image of the nucleic acid drug and late-stage endosomes in cells, and a statistical graph of fluorescence overlap rate, from Example 15 of this invention.

[0045] Figure 28 This is an immunofluorescence image of the nucleic acid drug and late-stage endosomes in cells, and a statistical graph of fluorescence overlap rate, from Example 15 of this invention.

[0046] Figure 29 This is an immunofluorescence image and fluorescence overlap rate statistical graph of the nucleic acid drug and lysosomes in cells in Example 15 of the present invention.

[0047] Figure 30 This is an immunofluorescence image and fluorescence overlap rate statistical graph of the nucleic acid drug and lysosomes in cells in Example 15 of the present invention.

[0048] Figure 31 This is an immunofluorescence image and fluorescence overlap rate statistical graph of the nucleic acid drug and circulating endosomal in cells in Example 15 of the present invention.

[0049] Figure 32 This is an immunofluorescence image and fluorescence overlap rate statistical diagram of the nucleic acid drug and the trans-Golgi network structure (TGN) in cells in Example 15 of the present invention. Detailed Implementation

[0050] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0051] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0052] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0053] Current research indicates that the endocytosis and subsequent transport of cell surface receptors into the cell interior are mediated by specific short-sequence peptide signals within the protein domains of these receptors in the cytoplasm. These signal peptides are mainly mediated by four types: tyrosine-based sorting signal peptides, dileucine-based sorting signal peptides, acidic group-based sorting signal peptides, and ubiquitination of lysine residues in the cytoplasm. Based on existing sorting signal peptide types, short-sequence sorting signal peptides related to transport proteins between early endosomes (EE), late endosomes (LE), and the trans Golgi network (TGN) were designed. The signal peptide-siRNA conjugate proposed in this invention, after entering the cell via currently mature endocytosis delivery vectors (such as Lipo3000 and LNP), promotes the escape of siRNA from the endosomes into the cytoplasm by mediating the transport of EE, LE, and TGN, thereby improving the siRNA drug transfection efficiency.

[0054] This specific embodiment provides a signal peptide, which is one of NPXY, YXXФ, [DE]XXX[LI], DXXLL or one of the sequences containing E, D, T and S; wherein X is any amino acid, Ф is any bulky hydrophobic side chain amino acid, N is asparagine, P is proline, Y is tyrosine, E is glutamic acid, L is leucine, I is isoleucine, D is aspartic acid, S is serine, T is threonine, [DE] represents either D or E, and [LI] represents either L or I.

[0055] In some embodiments, sequences containing E, D, T, and S are called acidic clusters. The main characteristic sequence of this type of acidic cluster contains E, D, T, and S amino acids. T and S in the secondary sequence play an important role, but there is no fixed order.

[0056] In some embodiments, the amino acid sequence of the signal peptide is one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0057] The amino acid sequence and related pathways of the signal peptide proposed in this invention are shown in Table 1.

[0058] Table 1. Amino acid sequences and related pathways of signal peptides

[0059]

[0060] The structural formula of the signal peptide is shown in Table 2 below.

[0061] Table 2

[0062]

[0063] This specific embodiment also proposes an application of the above-mentioned signal peptide in the preparation of a complex that can penetrate cell membranes or body tissue barriers by linking it with a nucleic acid fragment, wherein the nucleic acid fragment is preferably siRNA.

[0064] This specific embodiment also proposes a polypeptide-nucleic acid conjugate, which is a conjugate of any of the above-mentioned signal peptides and siRNA.

[0065] This specific embodiment also proposes a method for synthesizing the above-mentioned signal peptide, including the following steps:

[0066] Using Wang Resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-amino acid-Wang Resin (Wang Resin is also known as Wang Resin). After deprotection and repeated cycles, Fmoc-polypeptide-Wang Resin was obtained. The linker was condensed, the protecting group was removed, and a thiol donor was condensed. The mixture was then cleaved with trifluoroacetic acid. The cleavage solution was dried under nitrogen, precipitated with anhydrous diethyl ether, and centrifuged to obtain the precipitate. The precipitate was then dissolved in water and acetonitrile, purified by HPLC, and lyophilized to obtain the signal peptide. The condensation linker included condensed [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid as a linker; the condensation thiol donor included condensed 3-(triphenylmethylmercapto)propionic acid.

[0067] This specific embodiment also proposes a method for preparing a peptide-nucleic acid conjugate, comprising the following steps: mixing the signal peptide and the sense strand in a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, shaking for the first time at 35-40°C for 16-18 hours, separating the reaction product by electrophoresis, cutting off the target band, eluting through a column, and lyophilizing to obtain the peptide-nucleic acid conjugate single-chain product; mixing the peptide-nucleic acid conjugate single-chain product with the corresponding antisense strand at an equal concentration, shaking for the second time at 90-95°C for 1-2 minutes to obtain the peptide-nucleic acid conjugate.

[0068] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0069] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0070] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0071] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0072] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0073] Example 1: Preparation of sorting signal peptide Mpa-AEEA-KDPDEVETES:

[0074] 1. The sorting signal peptide sequence designed based on intracellular transporter protein is Mpa-AEEA-KDPDEVETES.

[0075] 2. Using Wang Resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-S(tbu)-Wang Resin. Deprotection was then performed, and the process was repeated to sequentially condense Fmoc-K(Boc)D(otbu)PD(otbu)E(otbu)VE(otbu)T(tbu)E(otbu)S(tbu)-Wang. Resin was used to condense [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (AEEA) as a linker, the protecting group was removed, and 3-(triphenylmethylmercapto)propionic acid (MPa) was condensed. The cleavage reaction was carried out with trifluoroacetic acid (TFA), the cleavage solution was dried under nitrogen, and anhydrous diethyl ether was used to precipitate the peptide. After centrifugation, the precipitate was dissolved in water and acetonitrile in a certain ratio, purified by HPLC, and lyophilized to obtain the target peptide chain NH2-Mpa-AEEA-Lys-Asp-Pro-Asp-Glu-Val-Glu-Thr-Glu-Ser-COOH.

[0076] 3. Characterization of the sorting signal peptide Mpa-AEEA-KDPDEVETES is shown in [reference needed]. Figure 1 and Figure 2 This demonstrates the preparation of the signal peptide.

[0077] Example 2: Preparation of the sorting signal peptide Mpa-AEEA-FDNPVY:

[0078] 1. The sorting signal peptide sequence designed based on intracellular transporter protein is Mpa-AEEA-FDNPVY.

[0079] 2. Using Wang resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-Y-Wang Resin. After deprotection, the reaction was repeated to obtain Fmoc-FD(OtBu)N(Trt)PVY-Wang Resin. [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (Fmoc-NH-AEEA-CH2COOH) was condensed as a linker, the protecting group was removed, and 3-(triphenylmethylmercapto)propionic acid (MPa) was condensed. The reaction was cleaved with trifluoroacetic acid (TFA), the cleavage solution was dried under nitrogen, and anhydrous diethyl ether was used to precipitate the precipitate. After centrifugation, the precipitate was dissolved in water and acetonitrile in a certain ratio, purified by HPLC, and lyophilized to obtain the target peptide chain NH2-Mpa-AEEA-Phe-Asp-Asn-Pro-Val-Tyr-COOH.

[0080] 3. Characterization of the sorting signal peptide Mpa-AEEA-FDNPVY is shown in [reference needed]. Figure 3 This demonstrates the preparation of the signal peptide.

[0081] Example 3: Preparation of the sorting signal peptide Mpa-AEEA-SYKGLPPE:

[0082] 1. The sorting signal peptide sequence designed based on intracellular transporter protein is Mpa-AEEA-SYKGLPPE.

[0083] 2. Using Wang Resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-E(otbu)-Wang Resin. After deprotection, the reaction was repeated to obtain Fmoc-SYK(Boc)GLPPE(otbu)-Wang Resin. [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (Fmoc-NH-AEEA-CH2COOH) was condensed as a linker, the protecting group was removed, and 3-(triphenylmethylmercapto)propionic acid (MPa) was condensed. The reaction was cleaved with trifluoroacetic acid (TFA), the cleavage solution was dried under nitrogen, and anhydrous diethyl ether was used to precipitate the precipitate. After centrifugation, the precipitate was dissolved in water and acetonitrile in a certain ratio, purified by HPLC, and lyophilized to obtain the target peptide chain NH2-Mpa-AEEA-Ser-Tyr-Lys-Gly-Leu-Pro-Pro-Glu-COOH.

[0084] 3. Characterization of the sorting signal peptide Mpa-AEEA-SYKGLPPE is shown in [reference needed]. Figure 4 .

[0085] Example 4: Preparation of the sorting signal peptide Mpa-AEEA-SDKQTLL:

[0086] 1. The sorting signal peptide sequence designed based on intracellular transporter protein is Mpa-AEEA-SDKQTLL.

[0087] 2. Using Wang resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-L-Wang Resin. After deprotection, the reaction was repeated to obtain Fmoc-SD(OtBu)K(Boc)Q(Trt)T(tBu)LL-Wang Resin. [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (Fmoc-NH-AEEA-CH2COOH) was condensed as a linker, the protecting group was removed, and 3-(triphenylmethylmercapto)propionic acid (MPa) was condensed. The mixture was then cleaved with trifluoroacetic acid (TFA), the cleavage solution was dried under nitrogen, and the precipitate was obtained by anhydrous diethyl ether. After centrifugation, the precipitate was dissolved in water and acetonitrile in a specific ratio, purified by HPLC, and lyophilized to obtain the target peptide chain NH2-Mpa-AEEA-Ser-Asp-Lys-Gln-Thr-Leu-Leu-COOH.

[0088] 3. Characterization of the sorting signal peptide Mpa-AEEA-SDKQTLL is shown in [reference needed]. Figure 5 .

[0089] Example 5: Preparation of the sorting signal peptide Mpa-AEEA-SFHDDSDEDLLHI:

[0090] 1. The sorting signal peptide sequence designed based on intracellular transporter protein is Mpa-AEEA-SFHDDSDEDLLHI.

[0091] 2. Using Wang resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-I-Wang Resin. After deprotection, the process was repeated, and the condensation was sequentially repeated to obtain Fmoc-S(tbu)FH(Trt)D(otbu)D(otbu)S(tbu)D(otbu)E(otbu)D(otbu)LLH(Trt)I-Wang Resin was used to condense [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (Fmoc-NH-AEEA-CH2COOH) as a linker, the protecting group was removed, and 3-(triphenylmethylmercapto)propionic acid (MPa) was condensed. The reaction was cleaved with trifluoroacetic acid (TFA), the cleavage solution was dried under nitrogen, and anhydrous diethyl ether was used to precipitate the peptide. After centrifugation, the precipitate was dissolved in water and acetonitrile in a certain ratio, purified by HPLC, and lyophilized to obtain the target peptide chain NH2-Mpa-AEEA-Ser-Phe-His-Asp-Asp-Ser-Asp-Glu-Asp-Leu-Leu-His-Ile-COOH.

[0092] 3. Characterization of the sorting signal peptide Mpa-AEEA-SFHDDSDEDLLHI is shown in [the original text]. Figure 6 .

[0093] Example 6: Preparation of the sorting signal peptide Mpa-AEEA-GYHDDSDELLE:

[0094] 1. The sorting signal peptide sequence designed based on intracellular transporter protein is Mpa-AEEA-GYHDDSDELLE.

[0095] 2. Using Wang Resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-E(otbu)-Wang Resin. After deprotection, the process was repeated, and the condensation was repeated to obtain Fmoc-GYH(Trt)D(otbu)D(otbu)S(tbu)D(otbu)E(otbu)LLE(otbu)-Wang Resin was used to condense [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (Fmoc-NH-AEEA-CH2COOH) as a linker, the protecting group was removed, and 3-(triphenylmethylmercapto)propionic acid (MPa) was condensed. The cleavage reaction was carried out with trifluoroacetic acid (TFA), the cleavage solution was dried under nitrogen, and anhydrous diethyl ether was used to precipitate the peptide. After centrifugation, the precipitate was dissolved in water and acetonitrile in a certain ratio, purified by HPLC, and lyophilized to obtain the target peptide chain NH2-Mpa-AEEA-Gly-Tyr-His-Asp-Asp-Ser-Asp-Glu-Asp-Leu-Leu-Glu-COOH.

[0096] 3. Characterization of the sorting signal peptide Mpa-AEEA-GYHDDSDELLE is shown in [reference needed]. Figure 7 .

[0097] Example 7: Preparation of the sorting signal peptide Mpa-AEEA-RDRDYDEDDEDDI:

[0098] 1. The sorting signal peptide sequence designed based on intracellular transporter protein is Mpa-AEEA-RDRDYDEDDEDDI.

[0099] 2. Using Wang resin as the starting resin carrier, Fmoc-I-Wang was obtained by sequentially condensing the corresponding Fmoc-amino acids in the polypeptide sequence via solid-phase synthesis. Resin was used to deprotect the peptide, and the process was repeated to condense Fmoc-R(Pbf)D(otbu)R(Pbf)D(otbu)YD(otbu)E(otbu)D(otbu)D(otbu)E(otbu)D(otbu)I-WangResin. This peptide was then condensed with [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (Fmoc-NH-AEEA-CH2COOH) as a linker, deprotected, and condensed with 3-(triphenylmethylmercapto)propionic acid (MPa). The cleavage reaction was carried out with trifluoroacetic acid (TFA), the cleavage solution was dried under nitrogen, and the precipitate was obtained by anhydrous diethyl ether. After centrifugation, the precipitate was dissolved in water and acetonitrile in a specific ratio, purified by HPLC, and lyophilized to obtain the target peptide chain NH2-Mpa-AEEA-Arg-Asp-Arg-Asp-Tyr-Asp-Glu-Asp-Asp-Glu-Asp-As p-Ile-COOH.

[0100] 3. Characterization of the sorting signal peptide Mpa-AEEA-RDRDYDEDDEDDI is shown in [reference needed]. Figure 8 .

[0101] Example 8: Preparation of polypeptide-nucleic acid conjugates:

[0102] 1. Mix Mpa-AEEA-KDPDEVETES, Mpa-AEEA-FDNPVY, Mpa-AEEA-SYKGLPPE, Mpa-AEEA-SDKQTLL, Mpa-AEEA-SFHDDSDEDLLHI, Mpa-AEEA-GYHDDSDELLE, and Mpa-AEEA-RDRDYDEDDEDDI with maleimide-linked EGFP siRNA positive strand (Mal-sense) at a 1:10 ratio, add 20 Mm of Tris-HCl buffer, and the total volume of the system is 120 μL. Shake at 40 °C for 16 hours. The reaction products were separated by non-denaturing acrylamide gel electrophoresis, developed under 254 nm UV light and the target band was cut off. The target product was eluted with 0.3 M sodium acetate for 3 hours, passed through a nucleic acid desalting column (NAP-10), and the target nucleic acid was eluted with two column volumes of nuclease-free water to remove sodium acetate. The product was then lyophilized to obtain peptide-nucleic acid conjugated single-chain products KDPDEVETES-sense, FDNPVY-sense, SYKGLPPE-sens, SDKQTLL-sense, SFHDDSDEDLLHI-sense, GYHDDSDELLE-sense, and RRDYDEDDEDDI-sense.

[0103] 2. Dilute KDPDEVETES-sense, FDNPVY-sense, SYKGLPPE-sens, SDKQTLL-sense, SFHDDSDEDLLHI-sense, GYHDDSDELLE-sense, and RDRDYDEDDEDDI-sense with their corresponding EGFP siRNA antisense strands to equal concentrations using 50mM Tris-HCl. Mix thoroughly at a volume ratio of 1:1, shake at 95°C for 1 minute, and allow to return to room temperature naturally within 30 minutes to obtain the peptide-nucleic acid conjugates KDPDEVETES-siRNA, FDNPVY-siRNA, SYKGLPPE-siRNA, SDKQTLL-siRNA, SFHDDSDEDLLHI-siRNA, GYHDDSDELLE-siRNA, and RDRDYDEDDEDDI-siRNA.

[0104] 3. Characterization of polypeptide-nucleic acid conjugates is shown in [reference needed]. Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 and Figure 15 This demonstrates the preparation of seven polypeptide-nucleic acid conjugates and also indicates the high purity of these seven polypeptide-nucleic acid conjugates.

[0105] Example 9 Transfection Experiment I:

[0106] 1. Prepare solutions of siRNA, KDPDEVETES-siRNA, FDNPVY-siRNA, SYKGLPPE-siRNA, SDKQTLL-siRNA, SFHDDSDEDLLHI-siRNA, GYHDDSDELLE-siRNA, and RDRDYDEDDEDDI-siRNA at different concentration gradients: 0.015, 0.045, 0.137, 0.412, 1.235, 3.704, 11.11, 33.33, 100.00, and 300 nM.

[0107] 2. Stable EGFP-expressing human cervical cancer HeLa cells (EGFP HeLa) were seeded in 24-well plates, with 50,000 cells seeded in each well. After overnight culture, each group of nucleic acid drugs was prepared using Opti-MEM medium according to the above concentration gradient. Then, each group was added to an equal volume of Lipo3000 dilution medium, and the mixture was stirred up and down 30-50 times. After standing for 10 minutes, the mixture was added to the corresponding wells. After culturing for 16 hours, the medium was changed, and the cells were cultured for another 48 hours. After cell digestion, the GFP fluorescence intensity of each group of cells was detected by flow cytometry.

[0108] 3. The weaker the GFP fluorescence in HeLa cells, the better the transfection effect. The statistical results of transfection rates for siRNA and KDPDEVETES-siRNA, FDNPVY-siRNA, SYKGLPPE-siRNA, SDKQTLL-siRNA, SFHDDSDEDLLHI-siRNA, GYHDDSDELLE-siRNA, and RDRDYDEDDEDDI-siRNA are shown in [the table below]. Figure 16 In each group, GFP fluorescence decreased with increasing concentration, indicating that the transfection effect increased with increasing drug concentration. The EC50 results fitted by Graphpad software are shown below. Figure 17, siRNA and KDPDEVETES-siRNA, FDNPVY-siRNA, SYKGLPPE-siRNA, SDKQTLL-siRNA, SFHDDSDEDLLHI-siRNA, GYHDDS The EC50s of DELLE-siRNA and RDRDYDEDDEDDI-siRNA were 3.930, 0.3411, 3.167, 1.912, 0.7151, 0.7432, 0.5241, and 0.9023 respectively.

[0109] Example 10 Cell Uptake Experiment:

[0110] 1. Mpa-AEEA-KDPDEVETES was mixed with the positive strand of RNA (Mal-sense-Cy5) linked to maleimide (Mal) at the 5' end and anthocyanin 5 (Cy5) at the 3' end at a ratio of 1:10. 20 Mm of Tris-HCl buffer was added, resulting in a total volume of 120 μL. The mixture was shaken at 40 °C for 16 hours. The reaction products were separated by non-denaturing acrylamide gel electrophoresis. The target band was developed under 254 nm UV light and excised. The target product was eluted with 0.3 M sodium acetate for 3 hours, passed through a nucleic acid desalting column (NAP-10), and eluted with two column volumes of nuclease-free water to remove the sodium acetate. The product was then lyophilized to obtain the peptide-nucleic acid conjugated single-chain product KDPDEVETES-sense-Cy5.

[0111] 2. Dilute the EGFP siRNA sense strand (siRNA-sense-Cy5) and KDPDEVETES-sense-Cy5 with the EGFP siRNA anti-sense strand (anti-sense) to equal concentrations using 50mM Tris-HCl, mix thoroughly at a volume ratio of 1:1, shake at 95℃ for 1 minute, and allow to return to room temperature naturally within 30 minutes to obtain siRNA-Cy5 and KDPDEVETES-siRNA-Cy5.

[0112] 3. Prepare siRNA-Cy5 and KDPDEVETES-siRNA-Cy5 at a concentration of 250 nM. Seed human cervical cancer HeLa cells (HeLa) in 24-well plates, seeding 50,000 cells per well. After culturing overnight, prepare each group of nucleic acid drugs in Opti-MEM medium at the above concentration, and then add them to an equal volume of Lipo3000 dilution medium. Mix by pipetting 30-50 times, let stand for 10 min, and then add them to the corresponding wells. After culturing for 0.5, 1, 2, 4, 8, and 16 hours, change the medium to digest the cells, and use flow cytometry to detect the Cy5 fluorescence intensity of each group of cells.

[0113] 4. The stronger the Cy5 fluorescence in HeLa cells, the better the cellular uptake. Statistical results of cellular uptake of siRNA-Cy5 and KDPDEVETES-siRNA-Cy5 are shown in [the table below]. Figure 18 As the uptake time increased, the Cy5 fluorescence in the cells became stronger, indicating that the amount of drug entering the cells increased. Both experimental groups reached a high level at 16 hours. Comparing the uptake levels of siRNA-Cy5 and KDPDEVETES-siRNA-Cy5 groups, it can be seen that the uptake effect was basically the same, which ruled out the difference in transfection effect due to the difference in uptake effect.

[0114] 5. Characterization of polypeptide-nucleic acid conjugates is shown in [reference needed]. Figures 19-20 Gel electrophoresis images showed the purity of siRNA-Cy5 and KDPDEVETES-siRNA-Cy5, and mass spectra were used to qualitatively identify KDPDEVETES-siRNA-Cy5.

[0115] Example 11 Inhibition of the endocytic pathway experiment I:

[0116] 1. Incubate EGFP HeLa cells cultured in 24-well plates with the pathway inhibitors configured according to Table 3 below.

[0117] Table 3

[0118]

[0119] 2. Add 100 nM siRNA and KDPDEVETES-siRNA to equal volumes of lipo3000 dilution buffer, mix by pipetting 30-50 times, let stand for 10 min, and then add to the corresponding wells of the plate. After culturing for 16 hours, change the medium and continue culturing for 48 hours. After digesting the cells, use flow cytometry to detect the GFP fluorescence intensity of each group of cells.

[0120] 3. The weaker the GFP fluorescence in HeLa cells, the better the transfection effect. See the statistical results of transfection rate below. Figure 21 The results from each group show that even after the addition of a single endocytosis pathway inhibitor, the fluorescence of the KDPDEVETES-siRNA group was still significantly reduced compared to the control group, indicating that KDPDEVETES-siRNA is not mediated by a single endocytosis pathway.

[0121] Example 12 Transfection Experiment II:

[0122] 1. siRNA, KDPDEVETES-siRNA, FDNPVY-siRNA, SYKGLPPE-siRNA, SDKQTLL-siRNA, SFHDDSDEDLLHI-siRNA, GYHDDSDELLE-siRNA, and RDRDYDEDDEDDI-siRNA were prepared using liposomes (LNPs). The liposomes were prepared from ionizable lipids (DLin-MC3-DMA), disterol phosphatidylcholine (DSPC), cholesterol, and PEG-DMG (molar ratio of 50:10:38.5:1.5). After mixing by pipetting, vesicles were spontaneously formed. The concentrations of each nucleic acid drug were prepared to be 1 nM and 100 nM, respectively.

[0123] 2. Stable human cervical cancer HeLa cells (EGFP HeLa) expressing EGFP were seeded in 96-well plates, with 10,000 cells seeded in each well. After culturing overnight, the cells were added to the corresponding wells according to the concentration gradient described above. After culturing for 16 hours, the medium was changed, and the cells were cultured for another 48 hours.

[0124] 3. RNA was extracted from each well, and cDNA was obtained by reverse transcription. The expression level of GFP mRNA in HeLa cells of each group was measured by quantitative real-time PCR using SYBR Green dye. The statistical results are shown below. Figure 22 The expression levels of GFP mRNA in the KDPDEVETES-siRNA, FDNPVY-siRNA, SYKGLPPE-siRNA, SFHDDSDEDLLHI-siRNA, and GYHDDSDELLE-siRNA groups were significantly lower than those in the control group, with the KDPDEVETES-siRNA group showing the most significant decrease. Furthermore, the mRNA expression level remained significantly lower even when the drug concentration was reduced to 1 nM, which is consistent with the results of Example 9. This further demonstrates the good transfection effect of KDPDEVETES-siRNA at the mRNA level.

[0125] Example 13 Transfection Experiment III:

[0126] 1. siRNA and KDPDEVETES-siRNA were prepared using liposomes (LNPs). The liposomes used were prepared from ionizable lipids (DLin-MC3-DMA), disterol phosphatidylcholine (DSPC), cholesterol, and PEG-DMG (molar ratio of 50:10:38.5:1.5). After mixing by pipetting, vesicles were spontaneously formed. The concentrations of each nucleic acid drug were prepared as follows: 0.005, 0.015, 0.045, 0.137, 0.412, 1.235, 3.704, 11.11, 33.33, and 100.00 nM.

[0127] 2. Stable human cervical cancer HeLa cells (EGFP HeLa) expressing EGFP were seeded in 96-well plates, with 10,000 cells seeded in each well. After culturing overnight, the cells were added to the corresponding wells according to the concentration gradient described above. After culturing for 16 hours, the medium was changed, and the cells were cultured for another 48 hours.

[0128] 3. RNA was extracted from each well, and cDNA was obtained by reverse transcription. The expression level of GFP mRNA in HeLa cells of each group was measured by quantitative real-time PCR using SYBR Green dye. The results were statistically analyzed and EC50 values ​​were fitted. The results are shown in the table below. Figure 23 The EC50 values ​​of the fitted siRNA and KDPDEVETES-siRNA were 1.740 and 0.07833, respectively, which is more than 20 times different. This indicates that at the mRNA level, the transfection efficiency of KDPDEVETES-siRNA is significantly higher than that of the positive control group siRNA.

[0129] Example 14 Inhibition of Transport Pathways Experiment II:

[0130] 1. Incubate EGFP HeLa cells cultured in 24-well plates with pathway inhibitors.

[0131] 2. siRNA and KDPDEVETES-siRNA were prepared using liposomes (LNPs). The liposomes used were prepared from ionizable lipids (DLin-MC3-DMA), disterol phosphatidylcholine (DSPC), cholesterol, and PEG-DMG (molar ratio of 50:10:38.5:1.5). After mixing by pipetting, vesicles spontaneously formed. The concentration of each nucleic acid drug was prepared at 100 nM. After pipetting 30-50 times, the mixture was allowed to stand for 10 min and then added to the corresponding well plates. After culturing for 16 hours, the medium was changed, and the cells were cultured for another 48 hours. After cell digestion, the GFP fluorescence intensity of each group of cells was detected by flow cytometry.

[0132] 3. The weaker the GFP fluorescence in HeLa cells, the better the transfection effect. See the statistical results of transfection rate below. Figure 24 The results from each group show that even after adding a single endocytosis pathway inhibitor, the fluorescence of the KDPDEVETES-siRNA group was still significantly reduced compared to the control group, indicating that KDPDEVETES-siRNA is not mediated by a single endocytosis pathway. In addition, the transfection effect of KDPDEVETES-siRNA was significantly inhibited by the addition of Nocodazole inhibitor, which inhibits late endosome maturation, in the last group. This result suggests that KDPDEVETES-siRNA transfection may be mediated by endosome escape.

[0133] Example 15 Intracellular localization experiment of peptide-nucleic acid drugs:

[0134] 1. siRNA-Cy5 and KDPDEVETES-siRNA-Cy5 were prepared using liposomes (LNPs). The liposomes used were prepared from ionizable lipids (DLin-MC3-DMA), disterol phosphatidylcholine (DSPC), cholesterol, and PEG-DMG (molar ratio of 50:10:38.5:1.5). After mixing by pipetting, vesicles spontaneously formed. The concentration of each nucleic acid drug was prepared at 100 nM. After pipetting 30-50 times, the mixture was allowed to stand for 10 min and then added to the corresponding well plates. After culturing for 16 hours, the culture medium was removed and the plates were washed twice with PBS.

[0135] 2. Cover cells thoroughly with 4% paraformaldehyde and fix at room temperature for 15 min, then wash with PBS for 5 min × 3 times. Cover cells with 0.1-0.2% Triton X-100 and incubate at room temperature for 20 min, then wash with PBS for 5 min × 3 times; incubate with 3% BSA for 60 min, then wash with PBS for 5 min × 3 times; incubate with primary antibody diluted with 3% BSA overnight at 4℃, then warm to room temperature for 20 min the next day, then wash with PBS for 5 min × 3 times. Incubate with secondary antibody diluted with 3% BSA at room temperature in the dark for 1 h, then wash with PBS for 5 min × 3 times. Finally, add the nuclear dye bisbenzimidazole H 33342 trihydrochloride (Hoechst 33342) and incubate for 5 min, then wash with PBS 3 times. Add culture medium to cover the cell surface, observe the cells under a laser confocal microscope and take images. Use FIJI to count the fluorescence overlap rate in each channel and plot a bar chart. See the experimental fluorescence images and overlap rate statistics below. Figure 25 , 26 Fluorescence colocalization analysis was performed, and the Manders' coefficient (M2: fraction of Cy5 overlapping RAB5 / EEA1) showed that the fluorescence ratio of the KDPDEVETES-siRNA-Cy5 group overlapping with the early endosome was lower than that of the siRNA-Cy5 group, indicating that the content of KDPDEVETES-siRNA-Cy5 in the early endosome was lower than that of the siRNA-Cy5 group. Figure 27 , Figure 28 The fluorescence ratio of KDPDEVETES-siRNA-Cy5 group that overlapped with late endosomes was higher than that of siRNA-Cy5 group, indicating that the content of KDPDEVETES-siRNA-Cy5 in early endosomes was higher than that of siRNA-Cy5 group. Figure 29 , Figure 30The fluorescence ratio of KDPDEVETES-siRNA-Cy5 group overlapping with lysosomes was lower than that of siRNA-Cy5 group, indicating that the content of KDPDEVETES-siRNA-Cy5 in lysosomes was lower than that of siRNA-Cy5 group. Figure 31 The proportion of fluorescence overlapping with circulating endosomal structures in the KDPDEVETES-siRNA-Cy5 group was higher than that in the siRNA-Cy5 group, but no significant difference was observed. Figure 32 The proportion of fluorescence overlapping with TGN in the KDPDEVETES-siRNA-Cy5 group was higher than that in the siRNA-Cy5 group, indicating that the content of KDPDEVETES-siRNA-Cy5 in TGN was significantly higher than that in the siRNA-Cy5 group. In conclusion, it can be inferred that KDPDEVETES-siRNA-Cy5 may improve the efficiency of drug escape from endosomes by mediating the endosome-to-TGN transport pathway, thereby improving transfection efficiency.

[0136] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A signal peptide, characterized in that, The signal peptide is one of NPXY, YXXФ, [DE]XXX[LI], DXXLL or one of the sequences containing E, D, T and S; wherein X is any amino acid, Ф is any bulky hydrophobic side chain amino acid, N is asparagine, P is proline, Y is tyrosine, E is glutamic acid, L is leucine, I is isoleucine, D is aspartic acid, S is serine, T is threonine, [DE] represents either D or E, and [LI] represents either L or I.

2. The signal peptide according to claim 1, characterized in that, The amino acid sequence of the signal peptide is one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:

7.

3. The use of the signal peptide of claim 1 in the preparation of a complex capable of penetrating cell membranes or body tissue barriers by linking it with a nucleic acid fragment.

4. The application according to claim 3, characterized in that, The nucleic acid fragment is siRNA.

5. A polypeptide-nucleic acid conjugate, characterized in that, It is a conjugate of any signal peptide and siRNA as described in any one of claims 1-2.

6. A method for synthesizing the signal peptide according to any one of claims 1-2, characterized in that, Includes the following steps: Using Wang resin as the starting resin carrier, the corresponding Fmoc-amino acids in the polypeptide sequence were sequentially condensed via solid-phase synthesis to obtain Fmoc-amino acid-Wang resin. After deprotection, the process was repeated to obtain Fmoc-peptide-Wang resin. The linker was condensed, the protecting group was removed, and the thiol donor was condensed. The reaction was then carried out with trifluoroacetic acid. The cleavage solution was dried under nitrogen, precipitated with anhydrous diethyl ether, and centrifuged to obtain the precipitate. The precipitate was then dissolved in water and acetonitrile, purified by HPLC, and lyophilized to obtain the signal peptide.

7. The method for synthesizing the signal peptide according to claim 6, characterized in that, The condensation linker includes: condensed [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid as a linker.

8. The method for synthesizing the signal peptide according to claim 6, characterized in that, The condensed mercapto donor includes condensed 3-(triphenylmethylmercapto)propionic acid.

9. A method for preparing the polypeptide-nucleic acid conjugate according to claim 5, characterized in that, Includes the following steps: The signal peptide and the sense strand were mixed in a tris(hydroxymethyl)aminomethane hydrochloride buffer solution. After the first oscillation, the reaction products were separated by electrophoresis, the target band was cut off, eluted through a column, and lyophilized to obtain the polypeptide-nucleic acid conjugate single-chain product. The polypeptide-nucleic acid conjugate single-chain product was mixed with the corresponding antisense strand at the same concentration and oscillated a second time to obtain the polypeptide-nucleic acid conjugate.

10. The method for preparing the polypeptide-nucleic acid conjugate according to claim 8 is characterized in that, The temperature of the first oscillation is 35-40℃, and the duration is 16-18 hours; and / or, the temperature of the second oscillation is 90-95℃, and the duration is 1-2 minutes.

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