Methods for constructing multivalent protein drugs and vaccines mediated by nucleic acid multimerization and uses thereof

Abstract

The application relates to a method for constructing a multivalent protein drug and vaccine mediated by nucleic acid polymerization and application, and particularly provides a multimeric complex based on a mutually matched nucleic acid skeleton, wherein the complex is a multimeric complex composed of 3-6 monomers with the mutually matched nucleic acid skeleton, each monomer being a polypeptide with a nucleic acid single strand; in the multimeric complex, the nucleic acid single strand of each monomer forms a double strand with the nucleic acid single strands of two other monomers through base complementation, thereby forming a mutually matched nucleic acid skeleton structure. The application also provides a pharmaceutical composition containing the multimeric complex, a nucleic acid sequence library for constructing the multimeric complex, and a method for optimizing the mutually matched nucleic acid skeleton. The method can complete multivalency of an existing short-acting protein drug or antigen without the need of reconstructing a fusion protein or chemical modification and cross-linking, and improve the half-life and activity and / or immunogenicity of the protein drug or antigen.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to methods for constructing and applying nucleic acid polymerization-mediated multivalent protein drugs and vaccines. Background Technology

[0002] For many biomolecules, their aggregation or multivalent state directly affects their activity and half-life in vivo. For example, the activation of most immune receptors involves receptor aggregation on the cell membrane, thereby activating downstream signaling pathways within the cell. Therefore, the natural ligands or antibodies of these receptors often significantly enhance their ability to activate receptors when they form multivalent or high-valent states. Additionally, some low-molecular-weight protein drugs (MW 40 kDa), such as cytokines, growth hormones, and synthetic peptides, have high renal clearance and short in vivo half-lives; these protein drugs can also increase their molecular weight and half-life by forming high-valent states.

[0003] Therefore, protein multivalent conversion is a promising technology in the biopharmaceutical field, and many methods exist. However, most chemical cross-linking methods suffer from poor linker specificity and heterogeneous linker polymers. Currently, the most widely used method is to produce proteins through cellular expression as multivalent fusion proteins. This involves fusing drug-functional protein regions with oligomer-forming proteins to create chimeras, such as Fc bivalent fusion proteins and GCN4 trivalent fusion proteins. While these fusion proteins can form uniform oligomers, their cellular expression levels and activity are often lower than the original protein drugs. Furthermore, the presence of the Fc region carries the risk of activating the immune system, inducing cytokine release, and causing cytotoxicity. Therefore, there is an urgent need in this field to develop a simple, flexible, and efficient method to convert validated protein drugs into homogeneous, highly specific multivalent proteins in a non-fusion protein manner.

[0004] Furthermore, protein multivalent presentation is also significant for vaccine development. Firstly, in B-cell-based vaccine design, activating B-cell receptors (BCRs) using viral or bacterial proteins as antigens is a crucial step. Like the immune receptors mentioned above, effective activation of BCRs requires receptor aggregation on the cell membrane; therefore, high-valent antigens have a clear advantage over monomeric antigens in activating B cells. Secondly, high-valent antigens are not necessarily single-antigen oligomers; they can contain different proteins from a particular virus or mutants and subtypes of the same protein from different viral strains. Thus, the presentation of diverse antigens can theoretically induce a polyclonal response in the host's immune system, generating a wider range of neutralizing antibodies. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient and stable assembly framework design for n-order nucleic acid oligomers, which is suitable for the efficient and stable assembly of nucleic acid-coupled protein drugs to form multivalent drugs or vaccines.

[0006] The second objective of this invention is to provide a simple and efficient method for forming protein drugs into multivalent macromolecules to prolong the drug's half-life and increase its activity.

[0007] The third objective of this invention is to provide a simple, flexible, efficient, and modular method for forming multivalent macromolecular complexes of the same or different protein antigens, which can be used to activate immune cells and improve the immunogenicity of vaccines.

[0008] In a first aspect of the present invention, a multimeric complex based on a mutually compatible nucleic acid backbone is provided, wherein the complex is a multimer formed by the combination of n monomers having mutually compatible nucleic acid backbones, wherein each monomer is a polypeptide having the nucleic acid single chain, and n is a positive integer from 3 to 6; in the multimer, the nucleic acid single chain of each monomer and the nucleic acid single chains of the other two monomers form mutually compatible double chains through base complementarity, thereby forming a mutually compatible nucleic acid backbone structure.

[0009] In another preferred embodiment, n is 3, 4, 5, or 6.

[0010] In another preferred embodiment, the complex is a trimer, tetramer, or pentamer, and preferably the structure of the complex is as follows: Figure 1 As shown.

[0011] In another preferred embodiment, the monomer has the structure of Formula I: Z1-W (I) In the formula, Z1 represents the polypeptide moiety; W represents a single-stranded nucleic acid sequence; "-" indicates a connector or key.

[0012] In another preferred example, "-" represents a covalent bond.

[0013] In another preferred embodiment, the nucleic acid sequence is selected from the group consisting of: levorotatory nucleic acids, peptide nucleic acids, locked nucleic acids, thiomodified nucleic acids, 2'-fluoromodified nucleic acids, 5-hydroxymethylcytosine nucleic acids, phosphorodiamidate morpholino nucleic acids, or combinations thereof; In another preferred embodiment, in the polymer, the Z1 of each monomer is the same or different.

[0014] In another preferred embodiment, the W of each monomer in the polymer is different.

[0015] In another preferred embodiment, the monomer has a structure of formula II. D-[LW]m (II) in, D represents the protein drug element portion; W represents the nucleic acid sequence; L indicates no connection or connector; "-" indicates a covalent bond; m can be 1, 2, or 3.

[0016] In another preferred example, m is 1.

[0017] In another preferred embodiment, the monomer has a structure of Formula III. A-[LW]m (III) in, A represents the polypeptide antigen element portion; W represents the nucleic acid sequence; L indicates no connection or connector; "-" indicates a covalent bond; m can be 1, 2, or 3.

[0018] In another preferred example, m is 1.

[0019] In another preferred embodiment, the nucleic acid sequence W has the structure shown in Formula 1: X1-R1-X2-R2-X3 (1) in, R1 is the base complementary pairing region 1; R2 is the base complementary pairing region 2; X1, X2, and X3 are each independently either non-redundant or redundant nucleic acids; "-" is the key.

[0020] In another preferred embodiment, the lengths of R1 and R2 are each independently 10-20 bases, more preferably 14-16 bases.

[0021] In another preferred embodiment, X1 has a length of 0-5 bases.

[0022] In another preferred embodiment, the length of X3 is 0-5 bases.

[0023] In another preferred embodiment, X2 has a length of 0-3 bases.

[0024] In another preferred embodiment, the sequence of X2 is selected from the group consisting of A, AA, AGA, or AAA.

[0025] In another preferred embodiment, R1 of each monomer forms a base-complementary pairing structure with R2 of the left neighbor (or left-side) monomer; while R2 forms a base-complementary pairing structure with R1 of the right neighbor (or right-side) monomer.

[0026] In another preferred embodiment, the monomer sequence is any one of the single-stranded nucleic acid sequences SEQ ID No: 1-60 (see Table 9-1) or a set of sequences thereof that form a trimeric complex based on a mutually compatible nucleic acid backbone.

[0027] In another preferred embodiment, the monomer sequence is any one of the single-stranded nucleic acid sequences SEQ ID No: 61-140 (see Table 9-2) or a set of sequences thereof that form a tetrameric complex based on a mutually compatible nucleic acid backbone.

[0028] In another preferred embodiment, the monomer sequence is any one of the single-stranded nucleic acid sequences SEQ ID No: 141-240 (see Table 9-3) or a set of sequences thereof that form a pentamer complex based on a mutually compatible nucleic acid backbone.

[0029] In another preferred embodiment, the monomer sequence is a phosphorodiamidate morpholino nucleic acid.

[0030] In another preferred embodiment, the monomer sequence is any one of the single-stranded nucleic acid sequences SEQ ID No: 275-278 or a set thereof that form a tetrameric complex based on a mutually compatible nucleic acid backbone.

[0031] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (a) The multimeric complex based on the intercompatible nucleic acid backbone described in the first aspect; and (b) Pharmaceutically acceptable carriers.

[0032] In another preferred embodiment, the pharmaceutical composition includes a vaccine composition.

[0033] In another preferred embodiment, the pharmaceutical composition comprises a therapeutic and / or preventative pharmaceutical composition.

[0034] In another preferred embodiment, the polymeric complex includes a trimeric complex, a tetrameric complex, and a pentameric complex.

[0035] In a third aspect of the invention, a nucleic acid sequence library is provided, the nucleic acid library comprising nucleic acid sequences for forming the multimeric complex based on the intermatched nucleic acid backbone described in the first aspect.

[0036] In another preferred embodiment, the nucleic acid sequence includes: (a) Nucleic acid sequences used to form trimeric complexes based on mutually compatible nucleic acid backbones; (b) Nucleic acid sequences used to form tetrameric complexes based on mutually compatible nucleic acid backbones; and / or (c) Nucleic acid sequences used to form pentamer complexes based on intermatched nucleic acid backbones.

[0037] In another preferred embodiment, the nucleic acid sequence W has the structure shown in Formula 1: X1-R1-X2-R2-X3 (1) in, R1 is the base complementary pairing region 1; R2 is the base complementary pairing region 2; X1, X2, and X3 are each independently either non-redundant or redundant nucleic acids; "-" is the key.

[0038] In a fourth aspect of the invention, the use of the nucleic acid sequence library described in the third aspect is provided for preparing the multimeric complex described in the first aspect or a pharmaceutical composition containing the multimeric complex described therein.

[0039] In a fifth aspect of the invention, a method is provided for determining a single-stranded nucleic acid sequence for forming a multimeric complex based on a mutually compatible nucleic acid backbone, comprising the steps of: (a) Set the annealing algorithm parameters: Set the initial annealing temperature, the final annealing temperature, and the annealing temperature decay coefficient. ; Set optimization constraint parameters: ① The number n of single nucleic acid strands is preferably a positive integer between 3 and 6; ② Pairing sequence length better It consists of 12-16 bases; ③ Dissociation temperature threshold of the pairing region ; ④ Free energy threshold of specific paired region sequences ; ⑤ Non-specific pairing free energy threshold ; ⑥ Connector X2, preferably A, AA and AAA; ⑦ Secondary structure (hairpin) dissociation temperature threshold ; ⑧ The proportion of CG in the paired sequence P CG Better The range is , ⑨ Optionally, for n=4, use a symmetric sequence and initialize the sequence set according to the above parameters. ; (b) Calculate the set mentioned in the previous step. objective function value That is, calculating the non-specific pairing free energy between sequences and between the sequences themselves. The sum of these values ​​yields the non-specific pairing free energy matrix. Search for the minimum value in its upper triangular matrix. and ( ),according to and Nonspecific pairing free energy Random selection or An update operation is performed, resulting in a new nucleic acid sequence, and thus an updated sequence set. ; (c) Determine the set mentioned in the previous step Verify whether the sequences in the sequence satisfy the optimization constraint parameters set in step (a), including the dissociation temperature of the specific pairing region. Specific pairing region sequence free energy Secondary structure dissociation temperature and CG proportions If the above parameters meet the constraints, proceed to step (d); otherwise, repeat step (c). If, at a certain annealing temperature, step (b) is executed 15 times consecutively without achieving the desired result, then... Therefore, to prevent infinite loops, sets Become a set Proceed to the next step; (d) Calculate the set mentioned in the previous step The objective function value E1 is compared. and ,like This indicates that the nonspecific pairing free energy is optimized, and the sequence set... Become a set of sequences ,like This indicates that the nonspecific pairing free energy has not been optimized. In this case, it is necessary to determine whether to accept the result according to the Metropolis criterion. Collect into ;and (e) The annealing temperature is based on the attenuation coefficient set in step (a). Attenuation is performed on the basis of the previous step. Repeat steps (b), (c), and (d), i.e., the Monte Carlo annealing algorithm, until the annealing temperature reaches the annealing termination temperature, as described in the previous step. It becomes a single-stranded nucleic acid sequence used to form multimeric complexes based on intermatched nucleic acid backbones.

[0040] In another preferred embodiment, step (a) of setting the annealing algorithm parameters includes: For example, setting the initial annealing temperature ±2℃, annealing termination temperature ±0.02℃, annealing temperature attenuation coefficient It depends on the situation, but is generally 0.98±0.01; Set optimization constraint parameters: ① The number of single-stranded nucleic acids, n, is a positive integer from 3 to 6. ② Pairing sequence length It depends on the situation (ideally, (12-16 bases) ③ Dissociation temperature threshold of the pairing region It depends on the length of the paired sequence (e.g., When the base is, ; When the base is, ), ④ Free energy threshold of specific paired region sequences It depends on the length of the paired sequence (preferably, When the base is, kcal / mol; When the base is, kcal / mol) ⑤ Non-specific pairing free energy threshold It depends on the sequence length (preferably, kcal / mol) ⑥ Connector x2, depending on the situation (can be A, AA, AAA, etc.). ⑦ Secondary structure (hairpin) dissociation temperature threshold It depends on the situation (ideally, ±2℃ ⑧ Proportion of CG in paired sequences The range of , is , ⑨ Specifically, when n=4, a symmetric sequence can be used, and the sequence set can be initialized according to the above parameters. .

[0041] In another preferred embodiment, each of the single-stranded nucleic acid sequences W has the structure shown in Formula 1: X1-R1-X2-R2-X3 (1) in, R1 is the base complementary pairing region 1; R2 is the base complementary pairing region 2; X1, X2, and X3 are each independently either non-redundant or redundant nucleic acids; "-" is the key.

[0042] In another preferred embodiment, in step (d), the optimized set is a set that satisfies the following conditions: (C1) In the interpaired nucleic acid backbone structure, the free energy of the DNA double-stranded structure formed by the target pairing ( Smaller or smallest; and (C2) In the interpaired nucleic acid backbone structure, non-target pairings Larger or maximized.

[0043] In another preferred embodiment, in step (d), the set of advantages also satisfies the following condition: (C3) Pairing dissociation temperature of regions R1 and R2 ( (when bases are used).

[0044] In another preferred embodiment, in step (c), the free energy of the DNA oligomer (i.e., the mutually compatible nucleic acid backbone structure) is calculated using the nearest neighbor method. ).

[0045] In another preferred embodiment, in step (c), the DNA oligomers (i.e., the mutually paired nucleic acid backbone structures) are decomposed into 10 different nearest-neighbor pairwise interactions, which are: AA / TT; AT / TA; TA / AT; CA / GT; GT / CA; CT / GA; GA / CT; CG / GC; GC / CG; and GG / CC; and based on the enthalpy of these pairwise interactions, ) and entropy ) Calculate the corresponding respective The value is then calculated; the free energies of the paired interactions included in the intermatched nucleic acid backbone structure are then combined (or summed) to obtain the free energy of the intermatched nucleic acid backbone structure.

[0046] In another preferred embodiment, the method includes repeating steps (b), (c), and (d) multiple times (i.e., performing n1 iterations) to obtain the global optimal solution during the iteration process.

[0047] In another preferred embodiment, during the iteration process, poor solutions are accepted to a limited extent according to the Metropolis criterion, and the probability of accepting a poor solution gradually approaches 0, thereby enabling the algorithm to find the global optimal solution as much as possible when it terminates.

[0048] In another preferred embodiment, the simulated annealing algorithm is iterated using the following objective function to optimize the free energy of the non-target pairing regions: For sequence and sequence Non-target pairing free energy This is the sum of the free energies of non-target pairings among all sequences, and its value is negative; the larger this negative value is, the more beneficial it is to reduce non-target pairings.

[0049] In a sixth aspect of the invention, a set of single-stranded nucleic acid sequences for forming a multimeric complex based on a mutually compatible nucleic acid backbone is provided, said set of single-stranded nucleic acid sequences being determined by the method described in the fifth aspect.

[0050] In another preferred embodiment, the set is selected from the following group: (S1) Single-stranded nucleic acid sequence used to form a trimeric complex based on a mutually compatible nucleic acid backbone: Table 9-1 (S2) Single-stranded nucleic acid sequences used to form tetrameric complexes based on mutually compatible nucleic acid backbones: Table 9-2 (S3) Single-stranded nucleic acid sequences used to form pentamer complexes based on mutually compatible nucleic acid backbones: Table 9-3 .

[0051] In a seventh aspect of the invention, an apparatus is provided for determining a single-stranded nucleic acid sequence for forming a multimeric complex based on a mutually compatible nucleic acid backbone, the apparatus comprising: (M1) Input module, which is used to input annealing algorithm parameters, optimization constraint parameters and optional nucleic acid sequence to be optimized; The annealing algorithm parameters include: initial annealing temperature, final annealing temperature, and annealing temperature decay coefficient. ; The optimization constraint parameters include: ① The number n of single nucleic acid strands is preferably a positive integer between 3 and 6; ② Pairing sequence length better It consists of 12-16 bases; ③ Dissociation temperature threshold of the pairing region ; ④ Free energy threshold of specific paired region sequences ; ⑤ Non-specific pairing free energy threshold ; ⑥ Connector X2, preferably A, AA and AAA; ⑦ Secondary structure (hairpin) dissociation temperature threshold ; ⑧ The proportion of CG in the paired sequence P CG Better The range is , (M2) Optimization module, configured to perform the following sub-steps to obtain an optimized single-stranded nucleic acid sequence or a set thereof: (z1) Calculate the initial set objective function value That is, calculating the non-specific pairing free energy between sequences and between the sequences themselves. The sum of these values ​​yields the non-specific pairing free energy matrix. Search for the minimum value in its upper triangular matrix. and ( ),according to and Nonspecific pairing free energy Random selection or An update operation is performed, resulting in a new nucleic acid sequence, and thus an updated sequence set. ; (z2) Determine the set mentioned in the previous step Verify whether the sequences in the dataset meet the set optimization constraints, including the dissociation temperature of the specific pairing regions. Specific pairing region sequence free energy Secondary structure dissociation temperature and CG proportions If the above parameters meet the constraints, proceed to step (z3); otherwise, repeat step (z2). If, at a certain annealing temperature, the conditions are not met after 15 consecutive executions, the process continues. Therefore, to prevent infinite loops, sets Become a set Proceed to the next step; (z3) Calculate the set mentioned in the previous step The objective function value E1 is compared. and ,like This indicates that the nonspecific pairing free energy is optimized, and the sequence set... Become a set of sequences ,like This indicates that the nonspecific pairing free energy has not been optimized. In this case, it is necessary to determine whether to accept the result according to the Metropolis criterion. Collect into ;and (z4) Annealing temperature is based on the set attenuation coefficient. Attenuation is performed on the basis of the previous step. Repeat steps (z1), (z2), and (z3), i.e., the Monte Carlo annealing algorithm, until the annealing temperature reaches the annealing termination temperature, as described in the previous step. To become a single-stranded nucleic acid sequence used to form multimeric complexes based on intermatched nucleic acid backbones; and (M3) Output module, which is used to output optimized single-stranded nucleic acid sequences or sets thereof.

[0052] In another preferred embodiment, the optimization constraint parameters further include: for tetramers (n=4), using symmetric sequences, and initializing the sequence set according to the above parameters. ; It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0053] Figure 1 A schematic diagram of the polymer is shown.

[0054] Figure 2 The flowchart of the annealing algorithm involved in this patent is shown.

[0055] Figure 3 A schematic diagram of trimeric sequence-specific pairing is shown.

[0056] Figure 4 The image shows a gel electrophoresis diagram of the trimeric optimized sequence nucleic acid backbone assembly.

[0057] Figure 5 The diagram shows a sequence-specific pairing pattern after tetramer optimization.

[0058] Figure 6 The graph shows the sum of free energies of non-specific pairing regions between tetramer optimized sequences.

[0059] Figure 7 The image shows a gel electrophoresis diagram of the tetrameric optimized sequence nucleic acid backbone assembly.

[0060] Figure 8 A schematic diagram of the transformation of tetramer into pentamer is shown.

[0061] Figure 9 The graph shows the sum of free energies of the unpaired regions of the sequence after optimization of pentamer transformation scheme 1.

[0062] Figure 10 The diagram shows a schematic of the gel electrophoresis assembly of the nucleic acid backbone after optimization of pentamer transformation scheme 1.

[0063] Figure 11 The diagram shows the sequence-specific pairing after optimization of pentamer scheme 2.

[0064] Figure 12 The graph shows the sum of free energies of the unpaired regions of the sequence after optimization of pentamer transformation scheme 2.

[0065] Figure 13 The diagram shows a schematic of the gel electrophoresis assembly of the nucleic acid backbone after optimization of pentamer transformation scheme 2.

[0066] Figure 14 The coupling of G-CSF with L-DNA was shown.

[0067] Figure 15 The purification effect of the (L-DNA)-G-CSF conjugate is shown in the figure.

[0068] Figure 16 The assembly diagrams of monovalent, divalent, and trivalent G-CSF complexes are shown.

[0069] Figure 17 The effect of the L-DNA tetramer framework on the in vitro activity of G-CSF was demonstrated.

[0070] Figure 18 The in vitro activity evaluation of divalent and trivalent G-CSF assembled from L-DNA tetramers is shown.

[0071] Figure 19 The images show: a. purification of SM(PEG)2-PMO1 using HiTrap Capto MMC; b. electrophoretic gel image of the coupling efficiency of SM(PEG)2-PMO1 with anti-HSA nanobody.

[0072] Figure 20 The positive ion mode liquid chromatography-mass spectrometry shows the identification of PMO1(a), SM(PEG)2-PMO1(b), anti-HSANb(c), and anti-HSA Nb-PMO1(d).

[0073] Figure 21 Showing the use of Superdex TM75 Increase 10 / 300 GL Separation of nanobodies and PMO-nanobody conjugates.

[0074] Figure 22 Electrophoretic gel images and schematic diagrams of NAPPA-PMO assembled samples are shown. Left: pmo-NAPPA4- HSA(1,2,3); Right: pmo-NAPPA4- HSA(1).

[0075] Figure 23 The binding activity of anti-HSA Nb, anti-HSA Nb-PMO1, and pmo-NAPPA4-HSA(1) to human serum albumin protein was demonstrated by ELISA.

[0076] Figure 24 The experiment on the resistance of pmo-NAPPA4-HSA(1) to nuclease degradation is shown. Left: SDS-PAGE electrophoresis image of pmo-NAPPA4-HSA(1) after treatment with three nucleases; Right: Agarose gel electrophoresis image of DDNA-NAPPA4 after treatment with three nucleases. Detailed Implementation

[0077] Through extensive and in-depth research, the inventors have developed for the first time a multivalent protein drug, its library, preparation method, and applications. Using the drug library and preparation method of this invention, short-acting protein drugs can be rapidly, efficiently, cost-effectively, and with high yields to form multivalent complexes, thereby increasing drug half-life, or monomeric antigens can be converted into high-valent antigens to enhance their immunogenicity. This invention is based on these findings.

[0078] Specifically, this invention provides a multivalent protein drug comprising n protein drug units, wherein each drug unit includes a drug element portion of the same type and different nucleic acid element portions linked to the drug element portion; n is a positive integer ≥2; the n different nucleic acid element portions form n polymers through nucleic acid base complementarity, thereby constituting the multivalent protein drug; the multivalent protein drug of this invention forms a stable pairing structure of complementary nucleic acid bases only through rapid assembly (e.g., 1 minute) (rather than complex peptide bonds or other chemical modifications). Experiments show that the drug of this invention can increase its molecular weight through high valence, thereby prolonging its half-life in animals.

[0079] Alternatively, based on the same implementation method, the drug element can also be an antigen used for vaccine development; the difference is that each antigen unit includes the same or different antigen element parts and different nucleic acid element parts connected to the antigen element parts; n different nucleic acid element parts form n polymers through nucleic acid base complementarity, thereby constituting the multivalent antigen.

[0080] Finally, this invention provides a highly optimized nucleic acid sequence library, including a set of nucleic acid sequences that are efficiently and accurately assembled into 2-5 polymers, for rapid and accurate self-assembly of the aforementioned drug or antigen units into multivalent macromolecular complexes.

[0081] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those used in this invention. The same meaning as commonly understood by one of ordinary skill in the art. As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0082] Drug D In this invention, the drug element is a protein drug or a polypeptide drug.

[0083] Typically, the protein drugs include, but are not limited to, cytokines, hormones (such as insulin, growth hormone, etc.), antibody drugs, and peptides.

[0084] In a preferred embodiment of the present invention, the protein drug is G-CSF for the treatment of leukopenia.

[0085] Antigen library A This invention provides an antigen library, the antigen library comprising N antigen units; The antigen unit includes an antigen element portion and a nucleic acid element portion connected to the antigen element portion; different nucleic acid element portions form n-mers through nucleic acid base complementarity, thereby constituting a multivalent antigen; The antigen element is a protein antigen or a polypeptide antigen. Typically, the protein and polypeptide antigens mentioned include, but are not limited to, viral and bacterial proteins, their structural regions, and fragments; In this invention, the antigen element is selected from M different antigen proteins in the library, M N; M different antigen proteins contain different proteins from a certain virus or mutants of the same protein from different viral strains; In a preferred embodiment of the present invention, the protein antigen is derived from the novel coronavirus SARS-CoV-2; specifically, it is a high-valent antigen formed from the receptor-binding domain (RBD) of the viral spike protein.

[0086] L-nucleotides Left-handed nucleic acids (L-RNAs) are mirror images of naturally occurring right-handed nucleic acids (D-RNAs). They can be divided into left-handed DNA (L-DNA) and left-handed RNA (L-RNA). The left-handed (chiral center) is mainly located in the deoxyribose or ribose portion of the nucleic acid, exhibiting a mirror-image inversion. Therefore, left-handed nucleic acids cannot be degraded by the ubiquitous nucleases in blood plasma (such as exonucleases and endonucleases).

[0087] Preparation method 1. Design and preparation of L-nucleic acid chain frameworks According to the present invention, the L-nucleic acid framework is formed by two or more L-nucleic acid single strands through base pairing. The 5' or 3' end of each L-nucleic acid single strand is activated to a group that can be subsequently modified (e.g., NH2), and then one end of a linker (e.g., SMCC, SBAP, etc.) is coupled to the activated group on the L-nucleic acid single strand. The L-nucleic acid with the linker can be assembled into the desired L-nucleic acid framework. In another preferred embodiment, the activated functional group (e.g., aldehyde, maleimide, etc.) at the 5' or 3' end of the L-nucleic acid single strand is already included in the nucleic acid synthesis. After confirming that the L-nucleic acid with the linker can successfully self-assemble into a framework, the L-nucleic acid single strand with the linker can be coupled with an antibody for subsequent assembly. The L-nucleic acid framework of the present invention can be prepared essentially by the following steps.

[0088] 1.1 Design of rapidly self-assembled L-nucleic acid single strands Determine the required number of multivalents, n (e.g., trimers, tetramers); determine the required number of L-nucleotide single strands, n, based on the number of multivalents, n; design the corresponding number of L-nucleotide single strand sequences, and adjust the stability of the target nucleic acid framework by optimizing base pairing to reduce the possibility of non-specific pairing between nucleic acid strands. Details of nucleic acid sequence design are described in the invention description and embodiments.

[0089] 1.2 Activation of L-DNA or L-RNA Activation of L-nucleotides involves modification of their 5' (X1) or 3' (X3) end active groups and subsequent conjugation with linkers. Modification of the active groups can be custom-made by nucleic acid synthesis companies; the linkers generally possess bifunctional groups, meaning one end can couple to the active group of the nucleic acid, and the other end can be linked to specific sites on the protein (such as NH3, SH).

[0090] According to a preferred embodiment of the present invention, all L-nucleotides constituting the framework are modified with an aldehyde at their 5' ends, thereby completing the activation of the L-nucleotides and enabling subsequent coupling to the N-terminus of the protein. -amine.

[0091] 2. Preparation method of protein-L-nucleic acid complex First, the 5' or 3' end of the L-nucleic acid is modified with an aldehyde. Then, under low pH (5-6) conditions, the aldehyde group of the L-nucleic acid is specifically linked to the N-terminus NH3 of the protein through a reductive amination reaction.

[0092] Algorithms and Algorithm-Optimized Nucleic Acid Sequences The present invention also provides a method and apparatus for determining single-stranded nucleic acid sequences for forming multimeric complexes based on mutually compatible nucleic acid backbones. Preferably, the method includes the preferred algorithm of the present invention.

[0093] Typically, the computer-optimized nucleic acid sequence library of the present invention may include: (a) nucleic acid sequences that can self-assemble into trimers through complementary base pairing; (b) nucleic acid sequences that can self-assemble into tetramers through complementary base pairing; and (c) nucleic acid sequences that can self-assemble into pentamers through complementary base pairing.

[0094] The complex formed by representative nucleic acid sequences is, for example... Figure 1 The shown molecules are trimers, tetramers, and pentamers.

[0095] Preferably, the nucleic acid sequence W of the present invention has the structure shown in Formula 1: W = X1 - R1 - X2 - R2 - X3 (1) in, R1 is the base complementary pairing region 1; R2 is the base complementary pairing region 2; X1, X2, and X3 are each independently either non-redundant or have no nucleic acid. R1 and R2 are 14-16 bases in length; X1 and X3 have a length of 0-5 bases; X2 has a length of 0-3 bases and a sequence of A, AA, AGA or AAA; In this process, R1 of a nucleic acid sequence forms a target pair with R2 of a different nucleic acid sequence, while R2 of a nucleic acid sequence forms a target pair with R1 of another nucleic acid sequence.

[0096] In this invention, self-pairing of any region of the nucleic acid sequence is considered non-target pairing and should be avoided in the design.

[0097] Preferably, the nucleic acid sequence of the present invention can be designed or optimized using a computer algorithm of Simulated Annealing (SA).

[0098] The computer algorithm described above calculates the free energy of the DNA double-stranded structure formed by the target pairing. Minimize, while removing non-target pairs maximize; Specifically, the stability of a DNA double-stranded structure depends on the nearest neighbor base pairs in the sequence; any Watson-Crick DNA double-stranded structure may contain 10 different nearest neighbor interactions, and these paired interactions are: AA / TT; AT / TA; TA / AT; CA / GT; GT / CA; CT / GA; GA / CT; CG / GC; GC / CG; GG / CC; More specifically, the 10 base pairs mentioned above The value, at any temperature, can be expressed through enthalpy. ) and entropy The enthalpy, entropy, and free energy data for the 10 sequences were calculated. Table 1 shows the enthalpy, entropy, and free energy data for the 10 sequences.

[0099] Table 1. Thermodynamic data for 10 sequences Note: , and All measurements were taken under conditions of 1 M NaCl, 25°C, and pH 7. From the IDT website ( https: / / sg.idtdna.com / calc / analyzer Measured.

[0100] The enthalpy values ​​of DNA oligomers are given by the thermodynamic values ​​in Table 1. and free energy value Both can be effectively predicted using the nearest neighbor method. Taking the complementary pairing of GGAATTCC / CCTTAAGG as an example, the nearest neighbor method is used to calculate... = -14.63 kcal / mol.

[0101] The annealing algorithm described above optimizes... The value was also determined, and the nucleic acid sequence pairing and dissociation temperature was also measured. The constraints ensure that regions R1 and R2 are within the specified range. ( (when bases are used).

[0102] The nearest neighbor model, based on thermodynamic calculations, makes accurate predictions about the stability of DNA double helixes. For a given base sequence, this model predicts based on the nearest neighbor base pairs. The calculation of its melting temperature involves enthalpy. ) and entropy The calculation method is as follows: (1) Where R is a constant (1.987 cal K). -1 mol -1 ), CT is a chain concentration given as 0.1 µM, It is the entropy value of the DNA double strand at a given sodium ion concentration. It is the enthalpy value under given conditions.

[0103] Simulated annealing is a general probabilistic algorithm, a method for approximating optimization problems based on the Monte Carlo approach. It aims to find an approximate optimal solution within a large search space within a given time. The idea behind simulated annealing originates from the annealing process of solid materials in physics: the solid is first heated sufficiently, then slowly cooled. During heating, the internal energy of particles within the solid increases due to free movement. As the temperature gradually decreases, the particles tend to become more ordered, reaching an equilibrium state at each temperature. If the rate of temperature decrease is slow enough near the freezing point, the ground state is reached, and the internal energy is minimized. According to the Metropolis criterion, particles at temperature... T The probability of reaching equilibrium is ,in E For temperature T Internal energy at that time To change the amount, K This is the Boltzmann constant. A similar process exists for combinatorial optimization problems, where the microscopic state of the solid is considered. i The simulation is a solution X, and the objective function is equivalent to the state. i Internal energy of time and with control parameters T Simulate solid temperature. For T For each value, the iterative process of "generating a new solution → calculating the difference in the objective function → determining whether to accept → accepting / discarding" is repeated, and the values ​​are gradually reduced. T The algorithm accepts poor solutions to a limited extent during the iteration process according to the Metropolis criterion, and the probability of accepting poor solutions gradually approaches 0, so that the algorithm can find the global optimal solution as much as possible when it terminates.

[0104] In this invention, the free energy of the non-target pairing regions between multimeric nucleic acid single strands is simulated as internal energy. While ensuring the free energy and dissociation temperature of the target pairing regions between nucleic acid single strands, the free energy of the non-target pairing regions is optimized through iterative simulated annealing algorithms. Finally, the optimized single strands are more conducive to multimeric assembly. The optimization objective function used is as follows: For sequence and sequence Non-target pairing free energy The sum of the free energies of non-target pairings among all sequences is negative; a larger value of this sum is more beneficial for reducing non-target pairings. Therefore, the objective function is constructed based on the idea of ​​minimizing energy using a degenerate algorithm. Previously, a negative sign was added, converting it to a positive number. A smaller value is more conducive to reducing non-target pairings.

[0105] A flowchart of a representative algorithm of the present invention is shown below. Figure 2 As shown.

[0106] In this invention, the simulated annealing algorithm introduces a random factor. In each iteration, it will accept a solution that is worse than the current one with a certain probability. Therefore, it may escape the local optimum and reach the global optimum.

[0107] Multivalent macromolecular complexes The present invention also provides a nucleic acid multimer-mediated protein drug formation multivalent macromolecular complex designed using the above algorithm, wherein the complex has improved drug half-life and activity.

[0108] Preferably, the nucleic acid sequence is a set of nucleic acid sequences specifically assembled into n-mers in a nucleic acid sequence library; Preferably, in this invention, the protein drug is a protein drug that needs to form multiple valences to increase its half-life or activity.

[0109] Typically, each nucleic acid strand of the nucleic acid sequence set is linked to the protein drug to form a protein drug-nucleic acid chain unit, having the structure shown in Formula 2: D-[LW i ], i=1 to n (2) in, D represents the protein drug element portion; Each W i Independently, it is a nucleic acid sequence; the nucleic acid sequence is selected from the group consisting of: levorotatory nucleic acids, peptide nucleic acids, locked nucleic acids, thiomodified nucleic acids, 2'-fluoromodified nucleic acids, 5-hydroxymethylcytosine nucleic acids, or combinations thereof; the nucleic acid sequence has the structure shown in Formula 1 and is selected from the above-mentioned group of nucleic acid sequences capable of forming n-mers; L is the connector; the connector portion is in W. i The synthesis or preparation of [the substance] is already included, linked in W i On X1 or X3 (see Equation 1); "-" indicates a covalent bond; In another preferred embodiment, the pharmaceutical element portion is selected from the group consisting of protein drugs and peptide drugs that require increased molecular weight to improve half-life, and protein drugs and peptide drugs that require multivalent formation to improve activity. In another preferred embodiment, L has an aldehyde, NHS ester, or similar functional group near the D end for attaching to the N end of D. -amine or lysine -amine; In another preferred embodiment, L near the D end has a maleimide functional group or a haloacetyl (such as bromoacetyl, iodoacetyl, etc.) functional group for connecting the free thiol (-SH) functional group on D; In another preferred embodiment, D is selected from the group consisting of: natural proteins, recombinant proteins, chemically modified proteins, and synthetic polypeptides; In another preferred embodiment, D may be site-specifically modified or have non-natural amino acids added to link the LW of formula 1. i ; The protein drugs are respectively linked to different LWs that can assemble into n-mers. i This forms protein drug self-assembly units, D-[L-W1], D-[L-W2], ..., D-[LW n ]; Protein drug self-assembly units, D-[L-W1], D-[L-W2], ..., D-[LW n By mixing in equimolar amounts in solution, they are assembled into multivalent protein drug molecular complexes.

[0110] This invention also provides a method for using nucleic acid multimers designed with the above algorithm to mediate the formation of multivalent macromolecular complexes of one or more antigens, thereby improving the effect of vaccines in in vivo in inducing the production of neutralizing antibodies; The nucleic acid sequence mentioned above is a set of nucleic acid sequences specifically assembled into n-mers in a nucleic acid sequence library; Wherein, the antigen is an antigen or an antigen library; the antigen library includes M different antigen proteins, 1 M n; Each nucleic acid strand of the nucleic acid sequence set is linked to an antigen in the antigen library to form an antigen-nucleic acid chain unit, having the structure shown in Formula 3: A k -[LW i ], i=1-n, k=1-M (3) in, A k For antigen k in the antigen library; an A k Each corresponds to one or more LW i (For example: A1-[L-W1], A1-[L-W2], A2-[L-W3], A3-[L-W4]); The other aspects of Equation 3 are the same as those of Equation 3 above; The antigen proteins are respectively linked to different LW proteins that can assemble into n-mers. i This forms antigen self-assembly units, such as A1-[L-W1], A2-[L-W2], ..., A3-[L-W1] N ]; Antigen self-assembly units are mixed in solution in equal molar amounts to assemble into multivalent antigen complexes.

[0111] The main advantages of this invention are: (1) The present invention can make existing short-acting protein drugs multivalent without the need to reconstruct fusion proteins or complex chemical modifications and cross-linking, thereby improving their half-life and activity; the aldehyde modification of L-nucleic acid can specifically link the N-terminal amine of the protein to form protein drug units that can self-assemble into oligomers. (2) The protein drug unit (protein-nucleic acid linker) of the present invention can complete the multivalent conversion of protein drugs within one minute using the levorotatory nucleic acid chain; (3) In vaccine development, this invention can form monomeric protein antigens at high titers, thereby improving their immunogenicity; (4) In terms of vaccine development, the present invention can also assemble antigen mutants and subtypes of different viral or bacterial strains into multivariate high-valent antigens to induce a wider range of neutralizing antibodies.

[0112] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0113] Example 1: Design, synthesis and verification of trimeric nucleic acid backbone assembly Design three lines that can be followed Figure 1 (A) Shape-paired nucleic acids. Specifically, Nucleic acid single strand and Nucleic acid single strands perform specific complementary pairings, but do not pair with other nucleic acid single strands; similarly, , respectively with , It performs specific complementary pairing without pairing with other single-stranded nucleic acids, and the free energy of specific complementary pairing ( It is much smaller than the nonspecific pairing free energy. ), the free energy of specific complementary pairing ( Less than -29 kcal / mol, and non-specific pairing free energy ( All values ​​are greater than -7 kcal / mol, thus the trimer form is the most stable in the reaction system. The specific steps for trimer optimization are illustrated in the diagram. Figure 2 The annealing parameters described are: initial annealing temperature. Annealing termination temperature Annealing temperature attenuation coefficient =0.98 (the initial annealing temperature decays to 0.98 of the current value with each annealing cycle); the optimization parameter constraint is: pairing sequence length. L =16 bases, dissociation temperature threshold Paired sequence free energy threshold: kcal / mol, nonspecific pairing free energy threshold: kcal / mol. The specific implementation steps for optimization are as follows: Sequence initialization: Initialize the paired sequence according to the parameters. Based on complementary base pairing, , and Six sequences were processed as follows Figure 1 The concatenation shown in (A) ultimately yields the initialization sequence set. .

[0114] A new solution emerges: A new solution Both are sets of sequences The updated result. First, calculate... This is to find the set The two sequences that have the greatest impact on target pairing in non-specific pairing and Then according to Random selection of non-target pairing regions or An update was performed, resulting in a new nucleic acid sequence. The dissociation temperature of the paired regions of this nucleic acid sequence was then examined. Is the temperature greater than 54℃, and is the free energy of the pairing region ( Is it less than -29 kcal / mol? If the dissociation temperature and pairing region free energy have not been reached... The constraints are then repeatedly updated if the dissociation temperature and the pairing region free energy are reached. The constraints of ) are applied according to the base pairing principle. The sequence is updated, resulting in a new set of sequences. If, after fifteen updates, the resulting new nucleic acid sequence still fails to reach the dissociation temperature and pairing region free energy... To prevent infinite loops, the set... Becoming a new interpretation .

[0115] Optimize the judgment: Calculate the sets according to Formula 2 respectively. and objective function value and ,like This indicates that the update of the non-target pairing free energy ( ) was optimized New interpretation become .like This indicates that the update resulted in a worsening solution. Based on the Metropolis criterion, the probability is calculated. At the same time, randomly generated , ,if If the solution is good, accept the worsened solution; otherwise, reject it. Finally, the initial annealing temperature decreases to 0.98, generating a new solution, until the annealing termination temperature is reached, resulting in the optimized sequence set. .

[0116] The optimized sequences in Table 2_1 were obtained through the above algorithm. The purpose of the 5' end A in each sequence is for the modification of active groups and subsequent coupling with linkers. The non-target pairing free energy ( The matrix table and the target pairing region parameter index table contain statistics on some key parameter values. See the diagram illustrating sequence-specific pairing after trimer optimization for details. Figure 3 From the corresponding nucleic acid backbone, run gel electrophoresis ( Figure 4 As can be seen, Lane 9 is an artificially designed trimer, with unclear bands and trailing patterns, while Lane 10 is an optimized sequence. , and The main bands formed indicate the formation of trimers and exhibit extremely high stability.

[0117] Table 2_1 Initialized and optimized sequences of trimer Table 2_3 Parameter indices of target pairing region in the trimer-optimized sequence Example 2: Design, synthesis and verification of tetrameric nucleic acid backbone assembly Design four lines that can be followed Figure 1 (B) Shape-paired nucleic acids. Among them, single-stranded nucleic acids... , , and Each with a single strand of nucleic acid , , and It performs specific complementary pairing without pairing with other single-stranded nucleic acids, and the free energy of specific complementary pairing ( It is much smaller than the nonspecific pairing free energy. ), the free energy of specific complementary pairing ( The non-specific pairing free energy is less than -27.4 kcal / mol. All values ​​are greater than -7 kcal / mol, thus the tetramer form is the most stable in the reaction system. The specific steps for tetramer optimization are illustrated in the diagram. Figure 2 The annealing parameters described are: initial annealing temperature. Annealing termination temperature Annealing temperature attenuation coefficient (The initial annealing temperature decays to 0.98 of the current temperature with each annealing cycle); the optimization parameter constraint is: pairing sequence length. L =14 bases, dissociation temperature threshold Paired sequence free energy threshold: kcal / mol, nonspecific pairing free energy threshold kcal / mol.

[0118] The specific implementation steps for optimization are as follows: Sequence initialization: Initialize the paired sequence according to the parameters. Based on complementary base pairing, , , and Eight sequences are processed as follows Figure 1 The concatenation shown in (B) ultimately yields the initialization sequence set. During the tetramer experiment, a fixed nuclear structure was found to effectively improve the assembly efficiency of nucleic acid sequences. Based on nucleic acid sequence formula 1, nucleic acid sequence W2 was developed, which has the structure of formula 4: W2=X1-Q1-C1-X2-C2-Q2-X3 (4) C1 and C2 are the fixed core structure parts, and Q1 and Q2 are sequences other than the fixed core structure.

[0119] Table 3 Nucleic acid sequences containing fixed nuclear structures , , and New solution generation: Same as in Example 1. The difference is that if a fixed core structure is used, the update does not include the fixed core structure portion; parameter constraints must be strictly adhered to, and the dissociation temperature of the new nucleic acid sequence pairing region (…) The temperature should be greater than 52℃, and the free energy of the pairing region should be ( It is less than -27.4 kcal / mol.

[0120] Optimization judgment: Same as the optimization judgment in Example 1.

[0121] Table 4_1 shows the optimized sequences obtained through the above algorithm, and their specific pairing diagram is as follows: Figure 5 As shown in Figure 6, the broken-line graph of the unpaired target free energy during the optimization process of the tetramer optimized sequence is presented. This invention seeks to avoid affecting the free energy of the optimized sequence without adding connectives when connecting elements are included. This has a significant impact, regardless of whether the free energy of the sequence is optimized by adding connectives. The sum of the matrices was maximized. Therefore, during the optimization process, the objective function value of the sequence without the addition of linker optimization was statistically analyzed, and only the sequence with the addition of linker optimization was subjected to final detection. The corresponding nucleic acid backbone was then used to run gel electrophoresis. Figure 7 As can be seen, Lane 15 is a band of artificially designed tetramers, which exhibits a tailing phenomenon, while Lane 16 is the sequence after algorithm optimization. , , and The main band formed is around 100 bp, indicating the formation of a tetramer and exhibiting extremely high stability.

[0122] The above tetramer assembly steps primarily optimize the non-specific pairing free energy between sequences, while the secondary structures formed by the sequence folding themselves also significantly influence tetramer assembly. If the dissociation temperature of the secondary structures formed by the sequences is too high, once such stable secondary structures are formed, it is difficult to break this state, making tetramer assembly difficult. Therefore, it is necessary to control the dissociation temperature of the secondary structures corresponding to the four sequences assembling the tetramer to prevent it from becoming too high. For tetramers, it is necessary to control the dissociation temperature of the secondary structures of the four nucleic acid sequences. If a symmetrical structure is used ( Figure 1 In respectively with (Maintaining symmetry) Similar secondary structures will appear between pairs of sequences, and the dissociation temperatures of the two secondary structures are not much different. In this case, it is only necessary to control the secondary structure of the two nucleic acid sequences to achieve the previous effect. Therefore, symmetry is beneficial for controlling the secondary structure in tetramer optimization.

[0123] Table 4_1 Initialized and optimized tetramer sequences Table 4_3 Parameters of Tetramer Optimized Sequence Pairing Region Example 3: Design, synthesis and verification of pentamer nucleic acid backbone assembly The optimized tetramer sequence exhibits excellent assembly performance, largely because no pairing occurs at the center of the tetramer. In other words, the fixed core structure provides the tetramer with ample freedom, preventing the formation of complex complexes in the central region. Therefore, to integrate the tetramer sequence and fixed core structure in the optimization of the pentamer sequence, two schemes utilizing the tetramer sequence and fixed core structure from Example 2 were explored and designed.

[0124] The first transformation plan: Design five possible... Figure 8 (B) Shape-paired nucleic acids: This scheme preserves a portion of the tetramer sequence and the complete, fixed nuclear structure from Example 2, and the nuclear structure is not opened. In the original tetramer... and Except for the nuclear structure, the tetramer is opened and two 14-nucleotide sequences are added. and Among them, single-stranded nucleic acids , , , and Each with a single strand of nucleic acid , , , and It performs specific complementary pairing without pairing with other single-stranded nucleic acids. The free energy of specific complementary pairing ( The non-specific pairing free energy is less than -27.4 kcal / mol. The concentrations are all greater than -7 kcal / mol, thus the pentamer form is the most stable in the reaction system. The specific implementation steps for optimizing the first pentamer conversion scheme are illustrated below. Figure 2 The annealing parameters described are: initial annealing temperature. Annealing termination temperature Annealing temperature attenuation coefficient (The initial annealing temperature decays to 0.9 of the current value with each iteration; due to the use of tetramer partial sequences, the update region is smaller, resulting in faster annealing temperature decay); the optimization parameter constraint is: paired sequence length. L =14 bases, dissociation temperature threshold Paired sequence free energy threshold: kcal / mol, nonspecific pairing free energy threshold kcal / mol.

[0125] This scheme uses a complete tetrameric fixed core structure, developing nucleic acid sequence W5 based on nucleic acid sequence formula 4, and nucleic acid sequence W6 based on nucleic acid sequence formula 4. W5 has the structure of formula 7, and W6 has the structure of formula 8. W3=X1-R1-X2-C1-X2-C2-Q1-X3 (5) W4=X1-Q1-C1-X2-C2-X2-R1-X3 (6) This scheme includes four structural sequences: Equation 1, Equation 4, Equation 5, and Equation 6.

[0126] Table 5 Nucleic acid sequences containing partial nuclear structures , , , and The specific implementation steps for optimization are as follows: Initialize the pairing sequence according to the parameters. Based on complementary base pairing, and , , , , , and The same sequence derived from the tetramer optimized sequence of Example 2. and spliced ​​together , and The assembly is obtained according to formula 5. , and spliced ​​together , and spliced ​​together , and The assembly is obtained according to formula 6. Finally, the initial sequence set is obtained. .

[0127] A new solution is generated: random in , , and We select one sequence to update, thus obtaining a new nucleic acid sequence. We then test whether the dissociation temperature of this nucleic acid sequence is greater than 52℃, and simultaneously measure the free energy of the pairing region (…). If the dissociation temperature and pairing region free energy are not reached, then... The constraints are then repeatedly updated if the dissociation temperature and the pairing region free energy are reached. The constraints of ) are applied according to the base pairing principle. The sequence is updated, resulting in a new set of sequences. If, after fifteen updates, the resulting new nucleic acid sequence still fails to reach the dissociation temperature and pairing region free energy... To prevent infinite loops, the set... Becoming a new interpretation Because this scheme uses a complete fixed core structure and a portion of the tetramer sequence, the fixed core structure and the retained tetramer sequence portion must remain unchanged during the generation of new solutions.

[0128] Optimization judgment: Same as the optimization judgment in Example 1. The difference is that the initial annealing temperature decays to 0.9 of the current value.

[0129] The optimized sequences of the first pentamer transformation scheme in Table 6_1 were obtained through this algorithm. Figure 9 A broken line graph showing the sum of free energy values ​​of non-target pairing regions between sequences during this optimization process. and , and , and The free energy of the non-target pairing region between them was not included in the statistics. Figure 10 Lane 9 is the main band formed by the optimized sequence assembly, indicating the formation of a pentamer and exhibiting extremely high stability.

[0130] Table 6_1 Initialized and optimized sequences of the first transformation scheme for pentamer The second transformation scheme: Design five steps that can be followed Figure 8(C) Shape-paired nucleic acids: This scheme only retains the fixed nuclear structure sequence of the tetramer. To accommodate the pentamer, the nuclear structure... and The first part is opened, and the other parts need to be randomly generated and then optimized as pentamers. Among them, the nucleic acid single strands... , , , and Each with a single strand of nucleic acid , , , and It performs specific complementary pairing without pairing with other single-stranded nucleic acids. The free energy of specific complementary pairing ( The non-specific pairing free energy is less than -27.4 kcal / mol. The concentrations are all greater than -7.2 kcal / mol, thus the pentamer form is the most stable in the reaction system. The specific implementation steps for optimizing the second pentamer conversion scheme are illustrated below. Figure 2 The annealing parameters described are: initial annealing temperature. Annealing termination temperature Annealing temperature attenuation coefficient (The initial annealing temperature decays to 0.98 of the current temperature with each annealing cycle); the optimization parameter constraint is: pairing sequence length. L =14 bases, dissociation temperature threshold Paired sequence free energy threshold: kcal / mol, nonspecific pairing free energy threshold kcal / mol. The specific implementation steps for optimization are as follows: Sequence initialization: Initialize the paired sequences according to the optimization parameter constraints. and a set of sequences of length 9 excluding the core structure. Based on the principle of complementary base pairing, we obtain , , , and By concatenating the sequences according to Table 7, we finally obtain the initialization sequence set. .

[0131] New solution generation: Same as in Example 1. The difference is that, because this scheme uses a tetramer-based fixed core structure, the fixed core structure must remain unchanged during the new solution generation process. Simultaneously, strict adherence to parameter constraints is maintained, updating the dissociation temperature of the nucleic acid sequence pairing region to be greater than 52°C, and simultaneously limiting the free energy of the pairing region (…). It is less than -27.4 kcal / mol.

[0132] Optimization judgment: Same as the optimization judgment in Example 1.

[0133] The optimized sequences of the second transformation scheme for pentamers in Table 8_1 were obtained through this algorithm. A schematic diagram of their specific pairing is shown below. Figure 11 As shown, Figure 12 This is a line graph showing the sum of free energy values ​​in the non-target pairing regions between sequences during this optimization process. Figure 13 Lane35 is a sequence , , , and The main band formed, although the pentamer has some tailing, shows good assembly effect.

[0134] Table 8.1 Initial and optimized sequences of the second transformation scheme for pentamer In addition, Examples 1, 2 and 3 were repeated to obtain the single-stranded nucleic acid sequences and sets thereof for forming trimeric, tetrameric and pentamer complexes based on intermatched nucleic acid backbones, as shown in Tables 9-1, 9-2 and 9-3 (see above).

[0135] Example 4: Conjugation of G-CSF to L-DNA The coupling of G-CSF and L-DNA employs a reductive amination reaction to selectively couple L-DNA with an aldehyde-modified 5' end to the N-terminus of G-CSF. The G-CSF buffer was replaced with acetate buffer (20 mM acetate, 150 mM NaCl, pH 5.0) using dilution or gel filtration chromatography, and the sample was concentrated to 30 mg / mL. 100 OD (1 OD = 33 µg) of aldehyde-modified L-DNA powder was dissolved in 60 µL of acetate buffer. 30 mg of sodium cyanoborohydride was dissolved in acetate buffer, adjusting the concentration to 800 mM. 50 µL, 60 µL, and 20 µL of the above concentrations of G-CSF, L-DNA, and sodium cyanoborohydride were mixed thoroughly and incubated at room temperature in the dark for 48 hours by rotation. The coupling effect was verified by polyacrylamide gel electrophoresis of samples before and after the reaction. The (L-DNA)-(G-CSF) conjugate showed a significant shift in the electrophoretic gel image compared to the uncoupled G-CSF, and the coupling efficiency reached 70%–80%. Figure 14 ).

[0136] Example 5: Purification of (L-DNA)-(G-CSF) conjugates The purification of (L-DNA)-(G-CSF) conjugates involves two steps. The first step uses Hitrap Q HP to remove unreacted G-CSF and the (L-DNA)2-(G-CSF) conjugate with two L-DNA strands linked to it. Figure 15 a). The reaction mixture obtained in Example 5 was diluted 10-fold with the loading buffer of the Q column and then loaded onto the column. Ten column volumes of loading buffer were used to elute the mixture to remove unreacted G-CSF. Then, 50 column volumes of elution were performed using a 0-100% linear gradient to separate the (L-DNA)-(G-CSF) conjugate and the (L-DNA)2-(G-CSF) conjugate. Polyacrylamide gel electrophoresis was used to identify the component type of each A280 absorption peak. Figure 15 (b) Collect the (L-DNA)-(G-CSF) conjugate (containing unreacted nucleic acids). In the second step, use Hiscrease CaptoMMC to remove the unreacted nucleic acids, finally obtaining a highly pure (L-DNA)-(G-CSF) conjugate. Figure 15 c). Load the samples collected in step one directly onto a Hiscrew Capto MMC column, elute with loading buffer for 10 column volumes to remove unreacted nucleic acids, and then elute the (L-DNA)-(G-CSF) conjugate with 100% elution buffer.

[0137] The purification conditions are shown in the table below: The purity of the (L-DNA)-(G-CSF) conjugate sample obtained by the two-step purification method was determined by 2% agarose gel electrophoresis. Figure 15 d) The gel image shows that there is only one nucleic acid band in the sample and the (L-DNA)-(G-CSF) conjugate is significantly shifted on the electrophoretic gel image compared to the unconjugated L-DNA, indicating that the unreacted nucleic acid and (L-DNA)2-(G-CSF) conjugate have been completely removed.

[0138] Example 6: Assembly of monovalent, divalent and trivalent G-CSF complexes The nucleic acid concentrations of S1-G-CSF, S3-G-CSF, S4-G-CSF, S2, S3, and S4 were determined using Nanodrop. Appropriate amounts of the above components were taken according to the structural design of monovalent, divalent, and trivalent protein complexes and mixed in a 1:1:1:1 molar ratio. After mixing, each assembly unit automatically assembled according to the base pairing principle. The assembly effect and sample purity were assessed by polyacrylamide gel electrophoresis before and after assembly. Figure 16 ).

[0139] Example 7: In vitro activity evaluation of G-CSF M-NFS-60 cells (mouse leukemia lymphocytes / G-CSF-dependent cells) were seeded in resuscitation medium (RPMI 1640 + 10% FBS + 15 ng / mL G-CSF + 1X penicillin-streptomycin) and resuscitated at 37°C with 5% CO2. Cells were passaged two to three times after reaching a confluence of 80%-90%, and then seeded into 96-well plates for plating. Corning 3599# 96-well plates were used for plating experiments, with a cell density of 6000 cells / well. Different samples (GCSF, NAPPA4-GCSF, NAPPA4-GCSF2, NAPPA4-GCSF3) were serially diluted to working concentrations of (0.001, 0.01, 0.1, 1, 10, 100 ng / mL) with a final volume of 100 μL. PBS was used as a control. After culturing in a constant temperature incubator for 48 hours, 10 μL of CCK8 solution was added to each well, and the culture plate was incubated in the incubator for 1-4 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell proliferation rate of different samples was calculated.

[0140] Cell proliferation rate (%) = [A(drug-treated) - A(0-drug-treated)] / [A(0-drug-treated) - A(blank)] × 100 A (Drug Addition): Absorbance of the pores containing cells, CCK solution, and drug solution. A (Blank): Absorbance of wells containing culture medium and CCK8 solution but without cells. A (0 drug added): Absorbance of the well containing cells and CCK8 solution but no drug solution. The activity of a G-CSF-linked L-DNA tetramer framework was evaluated using the above activity assay methods. It was found that the L-DNA tetramer framework had no effect on the activity of G-CSF. Figure 17 Using the same activity assay, it was found that divalent and trivalent G-CSF assembled with L-DNA tetramers had no negative impact on the activity of G-CSF. Figure 18 ).

[0141] Example 8: Conjugation and purification of SM(PEG)2-PMO conjugates In this embodiment, morpholine phosphate (PMO) nucleic acids were used for the experiment. Specifically, the following four PMO single-stranded sequences (from 5' to 3') were selected. Chain 1 (PMO1): SEQ ID NO: 275 5'- AGCAGCCTCGTTGAATCGCCAAGACACC -3' Chain 2 (PMO2): SEQ ID NO: 276 5'- AGGTGTCTTGGCGAAAGTTGCTCCGACG -3' Chain 3 (PMO3): SEQ ID NO: 277 5'- ACGTCGGAGCAACTAAGCGGTTCTGTGG -3' Chain 4 (PMO4): SEQ ID NO: 278 5'-ACCACAGAACCGCTATCAACGAGGCTGC-3' The 5' end is modified with an NH2 group, which is used to couple the NHS active group of SM(PEG)2.

[0142] The PMO single chain containing 5'-terminal NH2 modification was dissolved in phosphate buffer (50 mM NaH2PO4, 150 mM NaCl, pH 7.4) to prepare a stock solution with a final concentration of 1 mM. SM(PEG)2 (linker molecule) powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a fresh 250 mM SM(PEG)2 stock solution. 10–50 molar volumes of SM(PEG)2 stock solution were added to the PMO single chain stock solution, and the mixture was rapidly mixed and reacted at room temperature for 30 min–2 h. After the reaction was complete, 10% (v / v) of 1 M Tris-HCl (pH 7.0) was added to the reaction solution, and the mixture was incubated at room temperature for 20 min to terminate the reaction with excess SM(PEG)2. After incubation, SM(PEG)2-PMO was purified using HitrapCapto MMC. Unreacted SM(PEG)2 flowed directly through the column without binding. SM(PEG)2-PMO bound to the column was eluted with buffer (25 mM BICINE, 200 mM NH4Cl, 1 M Arginine monohydrochloride, pH 8.5). The elution results are as follows. Figure 19 As shown in a.

[0143] PMO samples before and after coupling were analyzed using positive ion mode liquid chromatography-mass spectrometry. For example... Figure 20 a and Figure 20 As shown in b, the results indicate that the molecular weight of the finally obtained SM(PEG)2-PMO is consistent with the theoretical value, and the coupling reaction efficiency is high.

[0144] Example 9: Preparation of Nanobody Mutants A cysteine ​​mutation was introduced at the carboxyl terminus of the nanobody for nucleic acid conjugation. The gene sequence of the anti-HSA nanobody was optimized to use yeast-preferred codons and then subcloned into the pPICZ alpha A plasmid. The amino acid sequence of the anti-HSA nanobody is SEQ ID NO: 279. A His tag was added to the N-terminus of the nanobody to facilitate purification.

[0145] SEQ ID NO: 279, Amino acid sequence of anti-HSA nanobody mutant: HHHHHHAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGSC After linearization, the plasmid was electroporated into Pichia pastoris strain X33, and strains with high copies of the target gene were selected using Zeocin concentration gradient YPD agar plates. Single clones were cultured in GMGY at 30℃ and 250 rpm to obtain sufficient cells. Then, the target nanobody was induced to be secreted and expressed by GMMY at 20℃ and 250 rpm, with 1% methanol added every 24 hours.

[0146] High-copy-selected strains can achieve nanobody expression yields of 40-80 mg / L in laboratory-grade glass flasks.

[0147] SDS-PAGE analysis revealed that the culture supernatant contained a large amount of target nanobody monomers and nanobody dimers after 72 hours of induction. The nanobodies in the culture supernatant were purified using a His-tagged affinity column.

[0148] Example 10: Conjugation and purification of nanobody-PMO conjugates The His-tagged affinity chromatography-eluted nanobody sample (Example 9) was dialyzed using a dialysis buffer containing a reducing agent (20 mM Tris, 15 mM NaCl, pH 7.4). During dialysis, the C-terminal thiol groups were reduced, and small impurity molecules such as free -SH groups were removed. The reduced nanobody was mixed with SM(PEG)2-PMO single chain (prepared in Example 8) at a molar ratio of 1:1 to 2, and reacted at room temperature for 2 hours after thorough mixing.

[0149] Identification via SDS-PAGE, such as Figure 19 The results from b show that the coupling efficiency can reach over 90%.

[0150] Unreacted SM(PEG)2-PMO single chains were removed using a His-tagged affinity column, and nanobodies and nanobodies-PMO mixtures were collected.

[0151] Using Superdex TM 75 Increase 10 / 300 GL to separate nanobodies and nanobodies-PMOs, such as Figure 21 As shown, nanobodies and nanobodies-PMOs were effectively separated.

[0152] Analysis of nanobodies before and after nucleic acid conjugation using positive ion mode liquid chromatography-mass spectrometry, such as Figure 20 Results c and d show that the molecular weight of the finally obtained nanobody-PMO is consistent with the theoretical value.

[0153] Example 11: NAPPA-PMO drug self-assembly The self-assembly process of NAPPA-PMO drugs is introduced below using pmo-NAPPA4-HSA(1) as an example.

[0154] The concentrations of anti-HSA Nb-PMO1, PMO2, PMO3, and PMO4 were determined respectively. Appropriate amounts of the above components were preheated at 37°C for 5 min, then mixed at a 1:1 molar ratio at 37°C and incubated for 1 min. This completed the assembly of pmo-NAPPA4-HSA(1).

[0155] The assembly of pmo-NAPPA4-HSA(1,2,3) is similar, and the required assembly modules are anti-HSA Nb-PMO1, anti-HSA Nb-PMO2, anti-HSA Nb-PMO3 and PMO4.

[0156] Under low-temperature conditions, SDS-PAGE was used to identify the assembly of the samples, such as... Figure 22 The results showed that the assembled sample bands were uniform.

[0157] Example 12: Verification of the binding activity of nanobody-PMO monomer and assembled NAPPA-PMO drug The following uses pmo-NAPPA4-HSA(1) as an example to detect the binding ability of Anti-HSA nanobody, Anti-HSA Nb-PMO1 monomer and assembled pmo-NAPPA4-HSA(1) to HSA protein (Bipsys, HSA-H5220) using ELISA.

[0158] 100 ng of HSA protein was coated into each well of a 96-well microplate and incubated overnight at 4°C. After washing with washing buffer (PBS containing 0.05% Tween-20) and blocking with blocking buffer (PBS containing 3% BSA and 0.05% Tween-20), serially diluted Anti-HSA nanobody, PMO-conjugated Anti-HSA Nb-PMO1 nanobody, and assembled pmo-NAPPA4-HSA(1) were added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-conjugated rabbit anti-camel VHH antibody (GenScript, A02016) diluted 1:5000 was added and incubated at room temperature for 1 hour. After washing three times, tetramethylbenzidine substrate solution (Beyotime, P0209) was added for color development. The color development was stopped using stop solution (Beyotime, P0215). The absorbance at 450 nm of each well was read using a microplate reader (Molecular Devices, SpectraMax i3x) and the corresponding EC50 was calculated.

[0159] like Figure 23 The calculation results show that the EC50 values ​​of Anti-HSA nanobody, Anti-HSA Nb-PMO1 and assembled pmo-NAPPA4-HSA(1) bound to HSA protein are 0.577 nM, 0.391 nM and 0.529 nM, respectively. This indicates that the PMO conjugation method does not affect the binding activity of nanobody to corresponding antigen, and the PMO assembly method also does not affect the binding activity of nanobody to corresponding antigen.

[0160] Example 13: Nuclease Degradation Resistance Experiment of NAPPA-PMO Drug To verify whether PMO, as a nucleic acid derivative, can withstand degradation by various nucleases, and to validate whether the assembled NAPPA-PMO drug is also resistant to nucleases or depolymerization, the following experiment was designed. Three common nucleases, DNase I (Thermo Scientific, EN0523), T7 Endonuclease I (NEB, M0302S), and S1 Nuclease (Thermo Scientific, EN0321), were used to incubate the PMO assembled sample pmo-NAPPA4-HSA(1) and the D-DNA assembled sample DDNA-NAPPA4 (control) at 37 degrees Celsius for 1 hour. The incubated pmo-NAPPA4-HSA(1) and DDNA-NAPPA4 were analyzed by SDS-PAGE and 2% agarose gel electrophoresis, respectively.

[0161] like Figure 24As shown, pmo-NAPPA4-HSA(1) is not degraded by the three nucleases (left figure), while DDNA-NAPPA4 is completely degraded by DNase I and S1 Nuclease and cleaved into shorter fragments by T7 Endonuclease I. Therefore, the experiment shows that the NAPPA-PMO drug can withstand common nuclease degradation.

[0162] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Ansheng (Shanghai) Pharmaceutical Technology Co., Ltd. <120> Construction methods and applications of nucleic acid polymerization-mediated multivalent protein drugs and vaccines <130> P2023-1022 <150> CN2020113403773 <151> 2020-11-25 <160> 279 <170> PatentIn version 3.5 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-1 Single-stranded nucleic acid sequence number S1 <400> 1 acacctggtt gttggataaa tcgttgaagg ctagga 36 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-1 Single-stranded nucleic acid sequence number S2 <400> 2 atcctagccttcaacgaaaa aactagagtc cgccga 36 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-1 Single-stranded nucleic acid sequence number S3 <400> 3 atcggcggac tctagttaaa atccaacaac caggtg 36 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-2 Single-stranded nucleic acid sequence number S1 <400> 4 atgcgttgag ttccagtaaa ggcaacatca ccacat 36 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-2 Single-stranded nucleic acid sequence number S2 <400> 5 aatgtggtga tgttgccaaa tctgaatcct cgtgct 36 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-2 Single-stranded nucleic acid sequence number S3 <400> 6 aagcacgagg attcagaaaa actggaactc aacgca 36 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-3 Single-stranded nucleic acid sequence number S1 <400> 7 attccaatcg tcctgtgaaa agttccgctc tgagtt 36 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-3 Single-stranded nucleic acid sequence number S2 <400> 8 aaactcagag cggaactaaa ctggcagatg gatgaa 36 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-3 Single-stranded nucleic acid sequence number S3 <400> 9 attcatccat ctgccagaaa cacaggacga ttggaa 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-4 Single-stranded nucleic acid sequences numbered S1 <400> 10 acgaggcaag ttctgtgaaa atgactacca ggtccg 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-4 Single-stranded nucleic acid sequences numbered S2 <400> 11 acggacctgg tagtcataaa atccactgac gctgaa 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-4 Single-stranded nucleic acid sequences numbered S3 <400> 12 attcagcgtc agtggataaa cacagaactt gcctcg 36 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-5 contains single-stranded nucleic acid sequences numbered S1. <400> 13 atagttcgtt gctcggaaaa ggcattgaga ggacct 36 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-5 contains single-stranded nucleic acid sequences numbered S2. <400> 14 aaggtcctct caatgccaaa atggtgatgt cgcttg 36 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-5 contains single-stranded nucleic acid sequences numbered S3. <400> 15 acaagcgaca tcaccataaa tccgagcaac gaacta 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-6 Single-stranded nucleic acid sequences numbered S1 <400> 16 agtcgtgtgc ttccaagaaa tagccaggtg aggact 36 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-6 Single-stranded nucleic acid sequences numbered S2 <400> 17 aagtcctcac ctggctaaaa aacagcggag tgtcat 36 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-6 Single-stranded nucleic acid sequences numbered S3 <400> 18 aatgacactc cgctgttaaa cttggaagca cacgac 36 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-7 Single-stranded nucleic acid sequences numbered S1 <400> 19 aacgcatcgc ttgatagaaa agaggagcac ggttat 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-7 Single-stranded nucleic acid sequences numbered S2 <400> 20 aataaccgtg ctcctctaaa gtaggcaatc caccat 36 <210> twenty one <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-7 Single-stranded nucleic acid sequences numbered S3 <400> twenty one aatggtggat tgcctacaaa ctatcaagcg atgcgt 36 <210> twenty two <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-8 Single-stranded nucleic acid sequences numbered S1 <400> twenty two agtcgttcca ccgaacaaaa tggctctggt cattga 36 <210> twenty three <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-8 Single-stranded nucleic acid sequences numbered S2 <400> twenty three atcaatgacc agagccaaaa aatcgcacatctcagg 36 <210> twenty four <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-8 Single-stranded nucleic acid sequences numbered S3 <400> twenty four acctgagatg tgcgattaaa tgttcggtgg aacgac 36 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-9 Single-stranded nucleic acid sequences numbered S1 <400> 25 agcggagtga ccatagtaaa aggcaggaca ttgttc 36 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-9 Single-stranded nucleic acid sequences numbered S2 <400> 26 agaacaatgt cctgcctaaa gtgctcgtcg tgaaga 36 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-9 Single-stranded nucleic acid sequences numbered S3 <400> 27 atcttcacga cgagcacaaa actatggtca ctccgc 36 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-10 Single-stranded nucleic acid sequence number S1 <400> 28 aattggaccg ctctactaaa atggcaccac agtcaa 36 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-10 Single-stranded nucleic acid sequence number S2 <400> 29 attgactgtg gtgccataaa caggctatca gcatcc 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-10 Single-stranded nucleic acid sequence number S3 <400> 30 aggatgctga tagcctgaaa agtagagcgg tccaat 36 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-11 Single-stranded nucleic acid sequence number S1 <400> 31 accattgagc cagtgataaa aaccgttgtg agttgc 36 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-11 Single-stranded nucleic acid sequence number S2 <400> 32 agcaactcac aacggttaaa tcgcacacct gtcgta 36 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-11 Single-stranded nucleic acid sequence number S3 <400> 33 atacgacagg tgtgcgaaaa atcactggct caatgg 36 <210> 34 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-12 Single-stranded nucleic acid sequence number S1 <400> 34 aagtgaagaa gcagcctaaa gttgtcatcg cacacc 36 <210> 35 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-12 Single-stranded nucleic acid sequence number S2 <400> 35 aggtgtgcga tgacaacaaa atgtcgtaac cgtgga 36 <210> 36 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-12 Single-stranded nucleic acid sequence number S3 <400> 36 atccacggtt acgacataaa aggctgcttc ttcact 36 <210> 37 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-13 Single-stranded nucleic acid sequence number S1 <400> 37 aatagcgtct tgagcctaaa tggaggacat accgac 36 <210> 38 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-13 Single-stranded nucleic acid sequence number S2 <400> 38 agtcggtatg tcctccaaaa ggtcacagtt gctgct 36 <210> 39 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-13 Single-stranded nucleic acid sequence number S3 <400> 39 aagcagcaac tgtgaccaaa aggctcaaga cgctat 36 <210> 40 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-14 Single-stranded nucleic acid sequence number S1 <400> 40 atgccgtgtt cagattcaaa tgtgcgtctg gattga 36 <210> 41 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-14 Single-stranded nucleic acid sequence number S2 <400> 41 atcaatccag acgcacaaaa agacaggtgg tccgat 36 <210> 42 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-14 Single-stranded nucleic acid sequence number S3 <400> 42 aatcggacca cctgtctaaa gaatctgaac acggca 36 <210> 43 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-15 Single-stranded nucleic acid sequence number S1 <400> 43 attcaggaca gcgtcataaa accgactgga gcaact 36 <210> 44 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-15 Single-stranded nucleic acid sequence number S2 <400> 44 aagttgctcc agtcggtaaa gatgccttcg tgtgag 36 <210> 45 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-15 Single-stranded nucleic acid sequence number S3 <400> 45 actcacacga aggcatcaaa atgacgctgt cctgaa 36 <210> 46 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-16 Single-stranded nucleic acid sequences numbered S1 <400> 46 agcagccaag gttatctaaa caatgacacg gaggat 36 <210> 47 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-16 Single-stranded nucleic acid sequence number S2 <400> 47 aatcctccgt gtcattgaaa gtgattcgca ccagac 36 <210> 48 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-16 Single-stranded nucleic acid sequence number S3 <400> 48 agtctggtgc gaatcacaaa agataacctt ggctgc 36 <210> 49 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-17 Single-stranded nucleic acid sequence number S1 <400> 49 accaccgtgt atgacctaaa agtgacagca catcgc 36 <210> 50 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-17 Single-stranded nucleic acid sequence number S2 <400> 50 agcgatgtgc tgtcactaaa acaggctcta cgagga 36 <210> 51 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-17 Single-stranded nucleic acid sequence number S3 <400> 51 atcctcgtag agcctgtaaa aggtcataca cggtgg 36 <210> 52 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-18 Single-stranded nucleic acid sequence number S1 <400> 52 aactacggag cgaagataaa tcctgaccaa cttgct 36 <210> 53 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-18 Single-stranded nucleic acid sequence number S2 <400> 53 aagcaagttg gtcaggaaaa gactggctga acacga 36 <210> 54 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-18 Single-stranded nucleic acid sequence number S3 <400> 54 atcgtgttca gccagtcaaa atcttcgctc cgtagt 36 <210> 55 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-19 Single-stranded nucleic acid sequence number S1 <400> 55 agttcctgat ccagcctaaa catccttgtc ttgcca 36 <210> 56 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-19 Single-stranded nucleic acid sequence number S2 <400> 56 atggcaagac aaggatgaaa cacgaccgct tagaag 36 <210> 57 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-19 Single-stranded nucleic acid sequence number S3 <400> 57 acttctaagc ggtcgtgaaa aggctggatc aggaac 36 <210> 58 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-20 Single-stranded nucleic acid sequence number S1 <400> 58 atatcgcact ccagcataaa ccgtgtgaac atcagg 36 <210> 59 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-20 Single-stranded nucleic acid sequence number S2 <400> 59 acctgatgtt cacacggaaa agcctacgag acttgg 36 <210> 60 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 3-20 Single-stranded nucleic acid sequence number S3 <400> 60 accaagtctc gtaggctaaa atgctggagt gcgata 36 <210> 61 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-1 Single-stranded nucleic acid sequence number S1 <400> 61 aagcgtcgtg aatccaaatg agcctgccaa tg 32 <210> 62 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-1 Single-stranded nucleic acid sequence number S2 <400> 62 acattggcag gctcaaaacc gaagtcaacg ct 32 <210> 63 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-1 Single-stranded nucleic acid sequence number S3 <400> 63 aagcgttgac ttcggaaaac tatggacggcga 32 <210> 64 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-1 Single-stranded nucleic acid sequence number S4 <400> 64 atcgccgtcc atagtaaagg attcacgacg ct 32 <210> 65 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-2 Single-stranded nucleic acid sequence number S1 <400> 65 aatggcgagc aatccaaatg agcctggacc aa 32 <210> 66 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-2 Single-stranded nucleic acid sequence number S2 <400> 66 attggtccag gctcaaaacc gaacgctgtg at 32 <210> 67 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-2 Single-stranded nucleic acid sequence number S3 <400> 67 aatcacagcg ttcggaaaac tatcgtgcgg ca 32 <210> 68 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-2 Single-stranded nucleic acid sequence number S4 <400> 68 atgccgcacg atagtaaagg attgctcgcc at 32 <210> 69 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-3 Single-stranded nucleic acid sequence number S1 <400> 69 atgaccacgc aatccaaatg agccaacctc ca 32 <210> 70 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-3 Single-stranded nucleic acid sequence number S2 <400> 70 atggaggttg gctcaaaacc gaacagcagc tt 32 <210> 71 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-3 Single-stranded nucleic acid sequence number S3 <400> 71 aaagctgctg ttcggaaaac tatctgccgc ct 32 <210> 72 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-3 Single-stranded nucleic acid sequence number S4 <400> 72 aaggcggcag atagtaaagg attgcgtggt ca 32 <210> 73 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-4 Single-stranded nucleic acid sequence number S1 <400> 73 atgtcgcacc aatccaaatg agcaagcctc gt 32 <210> 74 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-4 Single-stranded nucleic acid sequence number S2 <400> 74 aacgaggctt gctcaaaacc gaacgctgtc at 32 <210> 75 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-4 Single-stranded nucleic acid sequence number S3 <400> 75 aatgacagcg ttcggaaaac tatgtggcgg ca 32 <210> 76 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-4 Single-stranded nucleic acid sequence number S4 <400> 76 atgccgccac atagtaaagg attggtgcga ca 32 <210> 77 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-5 contains single-stranded nucleic acid sequences numbered S1. <400> 77 atgctggcac aatccaaatg agcgacgagg tt 32 <210> 78 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-5 contains single-stranded nucleic acid sequences numbered S2. <400> 78 aaacctcgtc gctcaaaacc gaagtgccag tt 32 <210> 79 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-5 contains single-stranded nucleic acid sequences numbered S3. <400> 79 aaactggcac ttcggaaaac tatgaggcgg ct 32 <210> 80 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-5 contains single-stranded nucleic acid sequences numbered S4. <400> 80 aagccgcctc atagtaaagg attgtgccag ca 32 <210> 81 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-6 Single-stranded nucleic acid sequences numbered S1 <400> 81 atgtcgcacc aatccaaatg agcaggttgg ca 32 <210> 82 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-6 Single-stranded nucleic acid sequences numbered S2 <400> 82 atgccaacct gctcaaaacc gaacgctgtc aa 32 <210> 83 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-6 Single-stranded nucleic acid sequences numbered S3 <400> 83 attgacagcg ttcggaaaac tatcagccgc ct 32 <210> 84 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-6 Single-stranded nucleic acid sequences numbered S4 <400> 84 aaggcggctg atagtaaagg attggtgcga ca 32 <210> 85 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-7 Single-stranded nucleic acid sequences numbered S1 <400> 85 atgtggtcgc aatccaaatg agcacctgcc aa 32 <210> 86 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-7 Single-stranded nucleic acid sequences numbered S2 <400> 86 attggcaggt gctcaaaacc gaacgtgacg at 32 <210> 87 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-7 Single-stranded nucleic acid sequences numbered S3 <400> 87 aatcgtcacg ttcggaaaac tatcaacgcc gc 32 <210> 88 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-7 Single-stranded nucleic acid sequences numbered S4 <400> 88 agcggcgttg atagtaaagg attgcgacca ca 32 <210> 89 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-8 Single-stranded nucleic acid sequences numbered S1 <400> 89 aagcgtcgtc aatccaaatg agcacggcaa tg 32 <210> 90 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-8 Single-stranded nucleic acid sequences numbered S2 <400> 90 acattgccgt gctcaaaacc gaagtgaacg ct 32 <210> 91 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-8 Single-stranded nucleic acid sequences numbered S3 <400> 91 aagcgttcac ttcggaaaac tatggctcgc ct 32 <210> 92 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-8 Single-stranded nucleic acid sequences numbered S4 <400> 92 aaggcgagcc atagtaaagg attgacgacg ct 32 <210> 93 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-9 Single-stranded nucleic acid sequences numbered S1 <400> 93 atgtggcgac aatccaaatg agcaagcctc ca 32 <210> 94 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-9 Single-stranded nucleic acid sequences numbered S2 <400> 94 atggaggctt gctcaaaacc gaagacgctg tt 32 <210> 95 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-9 Single-stranded nucleic acid sequences numbered S3 <400> 95 aaacagcgtc ttcggaaaac tatcgtgcgg ca 32 <210> 96 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-9 Single-stranded nucleic acid sequences numbered S4 <400> 96 atgccgcacg atagtaaagg attgtcgcca ca 32 <210> 97 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-10 Single-stranded nucleic acid sequence number S1 <400> 97 atgctgccac aatccaaatg agcctggaac ca 32 <210> 98 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-10 Single-stranded nucleic acid sequence number S2 <400> 98 atggttccag gctcaaaacc gaacgcagtc at 32 <210> 99 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-10 Single-stranded nucleic acid sequence number S3 <400> 99 aatgactgcg ttcggaaaac tatcgccgct ct 32 <210> 100 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-10 Single-stranded nucleic acid sequence number S4 <400> 100 aagagcggcg atagtaaagg attgtggcag ca 32 <210> 101 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-11 Single-stranded nucleic acid sequence number S1 <400> 101 atgcgtcgtc aatccaaatg agcttggcaa gg 32 <210> 102 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-11 Single-stranded nucleic acid sequence number S2 <400> 102 accttgccaa gctcaaaacc gaacgtgctg tt 32 <210> 103 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-11 Single-stranded nucleic acid sequence number S3 <400> 103 aaacagcacg ttcggaaaac tatggagcgg ct 32 <210> 104 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-11 Single-stranded nucleic acid sequence number S4 <400> 104 aagccgctcc atagtaaagg attgacgacg ca 32 <210> 105 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-12 Single-stranded nucleic acid sequence number S1 <400> 105 aactgccagc aatccaaatg agcctcgttc ca 32 <210> 106 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-12 Single-stranded nucleic acid sequence number S2 <400> 106 atggaacgag gctcaaaacc gaagttggca gt 32 <210> 107 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-12 Single-stranded nucleic acid sequence number S3 <400> 107 aactgccaac ttcggaaaac tatcgccgct tg 32 <210> 108 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-12 Single-stranded nucleic acid sequence number S4 <400> 108 acaagcggcg atagtaaagg attgctggca gt 32 <210> 109 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-13 Single-stranded nucleic acid sequence number S1 <400> 109 atgcgtcgtc aatccaaatg agcctccagg tt 32 <210> 110 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-13 Single-stranded nucleic acid sequence number S2 <400> 110 aaacctggag gctcaaaacc gaatgacacg ct 32 <210> 111 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-13 Single-stranded nucleic acid sequence number S3 <400> 111 aagcgtgtca ttcggaaaac tatggcggca gt 32 <210> 112 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-13 Single-stranded nucleic acid sequence number S4 <400> 112 aactgccgcc atagtaaagg attgacgacg ca 32 <210> 113 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-14 Single-stranded nucleic acid sequence number S1 <400> 113 aagcgtcgtg aatccaaatg agccatcgtc ca 32 <210> 114 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-14 Single-stranded nucleic acid sequence number S2 <400> 114 atggacgatg gctcaaaacc gaatgtgctg gt 32 <210> 115 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-14 Single-stranded nucleic acid sequence number S3 <400> 115 aaccagcaca ttcggaaaac tatgcggcaa cc 32 <210> 116 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-14 Single-stranded nucleic acid sequence number S4 <400> 116 aggttgccgc atagtaaagg attcacgacg ct 32 <210> 117 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-15 Single-stranded nucleic acid sequence number S1 <400> 117 attgccagga tgctgaatca cggtcggaca 30 <210> 118 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-15 Single-stranded nucleic acid sequence number S2 <400> 118 atgtccgacc gtgatagtcg cagaaggcat 30 <210> 119 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-15 Single-stranded nucleic acid sequence number S3 <400> 119 aatgccttct gcgacatagt acaacgccgc 30 <210> 120 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-15 Single-stranded nucleic acid sequence number S4 <400> 120 agcggcgttg tactaacagc atcctggcaa 30 <210> 121 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-16 Single-stranded nucleic acid sequence number S1 <400> 121 aggcgatcac aatccaaatg agcgtgttac gg 32 <210> 122 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-16 Single-stranded nucleic acid sequence number S2 <400> 122 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 123 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-16 Single-stranded nucleic acid sequence number S3 <400> 123 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 124 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-16 Single-stranded nucleic acid sequence number S4 <400> 124 aagcagccgc atagtaaagg attgtgatcg cc 32 <210> 125 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-17 Single-stranded nucleic acid sequence number S1 <400> 125 atggtccaac acgctaagcc tcaccgtctt 30 <210> 126 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-17 Single-stranded nucleic acid sequence number S2 <400> 126 aaagacggtg aggctatcgc acaacctggt 30 <210> 127 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-18 Single-stranded nucleic acid sequence number S3 <400> 127 aaccaggttg tgcgaatcgg agtggcagaa 30 <210> 128 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-17 Single-stranded nucleic acid sequence number S4 <400> 128 attctgccac tccgaaagcg tgttggacca 30 <210> 129 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-18 Single-stranded nucleic acid sequence number S1 <400> 129 aaccttggtg tgcgaaactc ctggcagcaa 30 <210> 130 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-18 Single-stranded nucleic acid sequence number S2 <400> 130 attgctgcca ggagtaagcg tgtggttcca 30 <210> 131 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-18 Single-stranded nucleic acid sequence number S3 <400> 131 atggaaccacacgctatgaggaccgtcgtt 30 <210> 132 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-18 Single-stranded nucleic acid sequence number S4 <400> 132 aaacgacggt cctcaatcgc acaccaaggt 30 <210> 133 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-19 Single-stranded nucleic acid sequence number S1 <400> 133 atgccaagtc cgagaatgct gcgaactggt 30 <210> 134 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-19 Single-stranded nucleic acid sequence number S2 <400> 134 aaccagttcg cagcaaagag cctgaaccgt 30 <210> 135 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-19 Single-stranded nucleic acid sequence number S3 <400> 135 aacggttcag gctctaacga cgcttgacca 30 <210> 136 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-19 Single-stranded nucleic acid sequence number S4 <400> 136 atggtcaagc gtcgtatctc ggacttggca 30 <210> 137 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-20 Single-stranded nucleic acid sequence number S1 <400> 137 aagcagcctc gttgaatcgc caagacacct 30 <210> 138 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-20 Single-stranded nucleic acid sequence number S2 <400> 138 aaggtgtctt ggcgaaagtt gctccgacga 30 <210> 139 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-20 Single-stranded nucleic acid sequence number S3 <400> 139 atcgtcggag caactaagcg gttctgtgga 30 <210> 140 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 4-20 Single-stranded nucleic acid sequence number S4 <400> 140 atccacagaa ccgctatcaa cgaggctgct 30 <210> 141 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-1 Single-stranded nucleic acid sequence number S1 <400> 141 atcaggcgac ctcttaaaac caccatcgtt gc 32 <210> 142 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-1 Single-stranded nucleic acid sequence number S2 <400> 142 agcaacgatg gtggtaaaaa tccaaatgag cgtgttacgg 40 <210> 143 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-1 Single-stranded nucleic acid sequence number S3 <400> 143 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 144 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-1 Single-stranded nucleic acid sequence number S4 <400> 144 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 145 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-1 Single-stranded nucleic acid sequence number S5 <400> 145 aagcagccgc atagtaaagg attaaaaaga ggtcgcctga 40 <210> 146 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-2 Single-stranded nucleic acid sequence number S1 <400> 146 aggcgacgat gtcttaaaac ctggttgctg ga 32 <210> 147 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-2 Single-stranded nucleic acid sequence number S2 <400> 147 atccagcaac caggtaaaaa tccaaatgag cgtgttacgg 40 <210> 148 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-2 Single-stranded nucleic acid sequence number S3 <400> 148 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 149 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-2 Single-stranded nucleic acid sequence number S4 <400> 149 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 150 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-2 Single-stranded nucleic acid sequence number S5 <400> 150 aagcagccgc atagtaaagg attaaaaaga catcgtcgcc 40 <210> 151 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-3 Single-stranded nucleic acid sequence number S1 <400> 151 atggaacctg gtgctaaatg ctcgcctgtc aa 32 <210> 152 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-3 Single-stranded nucleic acid sequence number S2 <400> 152 attgacaggc gagcaaaaaa tccaaatgag cgtgttacgg 40 <210> 153 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-3 Single-stranded nucleic acid sequence number S3 <400> 153 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 154 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-3 Single-stranded nucleic acid sequence number S4 <400> 154 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 155 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-3 Single-stranded nucleic acid sequence number S5 <400> 155 aagcagccgc atagtaaagg attaaaagca ccaggttcca 40 <210> 156 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-4 Single-stranded nucleic acid sequence number S1 <400> 156 atggtcaggc gacttaaaag gacgaggttg ct 32 <210> 157 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-4 Single-stranded nucleic acid sequence number S2 <400> 157 aagcaacctc gtcctaaaaa tccaaatgag cgtgttacgg 40 <210> 158 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-4 Single-stranded nucleic acid sequence number S3 <400> 158 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 159 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-4 Single-stranded nucleic acid sequence number S4 <400> 159 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 160 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-4 Single-stranded nucleic acid sequence number S5 <400> 160 aagcagccgc atagtaaagg attaaaaagt cgcctgacca 40 <210> 161 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-5 Single-stranded nucleic acid sequence number S1 <400> 161 atgctggacc accttaaatc agatggaggc ga 32 <210> 162 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-5 Single-stranded nucleic acid sequence number S2 <400> 162 atcgcctcca tctgaaaaaa tccaaatgag cgtgttacgg 40 <210> 163 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-5 Single-stranded nucleic acid sequence number S3 <400> 163 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 164 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-5 Single-stranded nucleic acid sequence number S4 <400> 164 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 165 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-5 Single-stranded nucleic acid sequence number S5 <400> 165 aagcagccgc atagtaaagg attaaaaagg tggtccagca 40 <210> 166 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-6 contains single-stranded nucleic acid sequences numbered S1. <400> 166 aaacgtccag gagctaaatc tcgtcgcctg aa 32 <210> 167 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-6 contains single-stranded nucleic acid sequences numbered S2. <400> 167 attcaggcga cgagaaaaaa tccaaatgag cgtgttacgg 40 <210> 168 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-6 contains single-stranded nucleic acid sequences numbered S3. <400> 168 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 169 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-6 contains single-stranded nucleic acid sequences numbered S4. <400> 169 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 170 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-6 Single-stranded nucleic acid sequences numbered S5 <400> 170 aagcagccgc atagtaaagg attaaaagct cctggacgtt 40 <210> 171 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-7 contains single-stranded nucleic acid sequences numbered S1. <400> 171 accacgacca ttgctaaaaa cttcaggcga cg 32 <210> 172 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-7 Single-stranded nucleic acid sequences numbered S2 <400> 172 acgtcgcctg aagttaaaaa tccaaatgag cgtgttacgg 40 <210> 173 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-7 Single-stranded nucleic acid sequences numbered S3 <400> 173 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 174 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-7 Single-stranded nucleic acid sequences numbered S4 <400> 174 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 175 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-7 Single-stranded nucleic acid sequences numbered S5 <400> 175 aagcagccgc atagtaaagg attaaaagca atggtcgtgg 40 <210> 176 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-8 Single-stranded nucleic acid sequences numbered S1 <400> 176 aaggcgaggt cttcaaaatg gttgctggac ga 32 <210> 177 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-8 Single-stranded nucleic acid sequences numbered S2 <400> 177 atcgtccagc aaccaaaaaa tccaaatgag cgtgttacgg 40 <210> 178 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-8 Single-stranded nucleic acid sequences numbered S3 <400> 178 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 179 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-8 Single-stranded nucleic acid sequences numbered S4 <400> 179 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 180 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-8 Single-stranded nucleic acid sequences numbered S5 <400> 180 aagcagccgc atagtaaagg attaaatgaa gacctcgcct 40 <210> 181 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-9 Single-stranded nucleic acid sequences numbered S1 <400> 181 atcaaggcga ccagtaaaaa gctcctcgac ga 32 <210> 182 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-9 Single-stranded nucleic acid sequences numbered S2 <400> 182 atcgtcgagg agcttaaaaa tccaaatgag cgtgttacgg 40 <210> 183 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-9 Single-stranded nucleic acid sequences numbered S3 <400> 183 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 184 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-9 Single-stranded nucleic acid sequences numbered S4 <400> 184 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 185 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-9 Single-stranded nucleic acid sequences numbered S5 <400> 185 aagcagccgc atagtaaagg attaaaactg gtcgccttga 40 <210> 186 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-10 Single-stranded nucleic acid sequences numbered S1 <400> 186 attcaggcga ctcctaaaag cacgacgatg gt 32 <210> 187 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-10 Single-stranded nucleic acid sequences numbered S2 <400> 187 aaccatcgtc gtgctaaaaa tccaaatgag cgtgttacgg 40 <210> 188 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-10 Single-stranded nucleic acid sequences numbered S3 <400> 188 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 189 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-10 Single-stranded nucleic acid sequence number S4 <400> 189 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 190 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-10 Single-stranded nucleic acid sequence number S5 <400> 190 aagcagccgc atagtaaagg attaaaagga gtcgcctgaa 40 <210> 191 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-11 Single-stranded nucleic acid sequence number S1 <400> 191 aagcacctgc aatccaaatc gccaggacaa gt 32 <210> 192 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-11 Single-stranded nucleic acid sequence number S2 <400> 192 aacttgtcct ggcgaaaatg agcaaccatg cc 32 <210> 193 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-11 Single-stranded nucleic acid sequence number S3 <400> 193 aggcatggtt gctcaaaacc gaacgtcgtg at 32 <210> 194 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-11 Single-stranded nucleic acid sequence number S4 <400> 194 aatcacgacg ttcggaaaac tatggagcgg ct 32 <210> 195 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-11 Single-stranded nucleic acid sequence number S5 <400> 195 aagccgctcc atagtaaagg attgcaggtg ct 32 <210> 196 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-12 Single-stranded nucleic acid sequence number S1 <400> 196 aacctgctgc aatccaaatc gccacctcaa ga 32 <210> 197 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-12 Single-stranded nucleic acid sequence number S2 <400> 197 atcttgaggt ggcgaaaatg agcctggacg tt 32 <210> 198 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-12 Single-stranded nucleic acid sequence number S3 <400> 198 aaacgtccag gctcaaaacc gaactggtgc tt 32 <210> 199 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-12 Single-stranded nucleic acid sequence number S4 <400> 199 aaagcaccag ttcggaaaac tatgccgctc ct 32 <210> 200 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-12 Single-stranded nucleic acid sequence number S5 <400> 200 aaggagcggc atagtaaagg attgcagcag gt 32 <210> 201 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-13 Single-stranded nucleic acid sequence number S1 <400> 201 aagctggtgc aatccaaatc gcctcctgac aa 32 <210> 202 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-13 Single-stranded nucleic acid sequence number S2 <400> 202 attgtcagga ggcgaaaatg agcaaggttg gc 32 <210> 203 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-13 Single-stranded nucleic acid sequence number S3 <400> 203 agccaacctt gctcaaaacc gaacgcagat gt 32 <210> 204 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-13 Single-stranded nucleic acid sequence number S4 <400> 204 aacatctgcg ttcggaaaac tatggagcgg ca 32 <210> 205 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-13 Single-stranded nucleic acid sequence number S5 <400> 205 atgccgctcc atagtaaagg attgcaccag ct 32 <210> 206 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-14 Single-stranded nucleic acid sequence number S1 <400> 206 atgcacgcac aatccaaatc gccatcagag gt 32 <210> 207 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-14 Single-stranded nucleic acid sequence number S2 <400> 207 aacctctgat ggcgaaaatg agctgcctcc at 32 <210> 208 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-14 Single-stranded nucleic acid sequence number S3 <400> 208 aatggaggca gctcaaaacc gaacgtcgtc at 32 <210> 209 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-14 Single-stranded nucleic acid sequence number S4 <400> 209 aatgacgacg ttcggaaaac tatcgagcgg ct 32 <210> 210 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-14 Single-stranded nucleic acid sequence number S5 <400> 210 aagccgctcg atagtaaagg attgtgcgtg ca 32 <210> 211 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-15 Single-stranded nucleic acid sequence number S1 <400> 211 aagcgtcgtg aatccaaatc gccatcagac ca 32 <210> 212 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-15 Single-stranded nucleic acid sequence number S2 <400> 212 atggtctgat ggcgaaaatg agcaaggctc gt 32 <210> 213 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-15 Single-stranded nucleic acid sequence number S3 <400> 213 aacgagcctt gctcaaaacc gaaccagctt gt 32 <210> 214 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-15 Single-stranded nucleic acid sequence number S4 <400> 214 aacaagctgg ttcggaaaac tatgcggcag gt 32 <210> 215 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-15 Single-stranded nucleic acid sequence number S5 <400> 215 aacctgccgc atagtaaagg attcacgacg ct 32 <210> 216 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-16 Single-stranded nucleic acid sequences numbered S1 <400> 216 atcagcacgc aatccaaatc gccagttcaa cc 32 <210> 217 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-16 Single-stranded nucleic acid sequences numbered S2 <400> 217 aggttgaact ggcgaaaatg agcaagcagg ct 32 <210> 218 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-16 Single-stranded nucleic acid sequence number S3 <400> 218 aagcctgctt gctcaaaacc gaacgtggtg tt 32 <210> 219 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-16 Single-stranded nucleic acid sequence number S4 <400> 219 aaacacccacg ttcggaaaac tatggagcgg ca 32 <210> 220 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-16 Single-stranded nucleic acid sequence number S5 <400> 220 atgccgctcc atagtaaagg attgcgtgct ga 32 <210> 221 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-17 Single-stranded nucleic acid sequence number S1 <400> 221 aagctgcacc aatccaaatc gccagaaggt ca 32 <210> 222 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-17 Single-stranded nucleic acid sequence number S2 <400> 222 atgaccttct ggcgaaaatg agcacgacgc at 32 <210> 223 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-17 Single-stranded nucleic acid sequence number S3 <400> 223 aatgcgtcgt gctcaaaacc gaacaacctg ct 32 <210> 224 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-17 Single-stranded nucleic acid sequence number S4 <400> 224 aagcaggttg ttcggaaaac tatggagcgg ca 32 <210> 225 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-17 Single-stranded nucleic acid sequence number S5 <400> 225 atgccgctcc atagtaaagg attggtgcag ct 32 <210> 226 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-18 Single-stranded nucleic acid sequence number S1 <400> 226 aacgctcgtc aatccaaatc gcctcaggac aa 32 <210> 227 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-18 Single-stranded nucleic acid sequence number S2 <400> 227 attgtcctga ggcgaaaatg agccaacgac ct 32 <210> 228 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-18 Single-stranded nucleic acid sequence number S3 <400> 228 aaggtcgttg gctcaaaacc gaagctggtg tt 32 <210> 229 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-18 Single-stranded nucleic acid sequence number S4 <400> 229 aaacaccagc ttcggaaaac tatgccgcac ct 32 <210> 230 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-18 Single-stranded nucleic acid sequence number S5 <400> 230 aaggtgcggc atagtaaagg attgacgagc gt 32 <210> 231 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-19 Single-stranded nucleic acid sequence number S1 <400> 231 aagtgcgtcg aatccaaatc gccaagacct ca 32 <210> 232 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-19 Single-stranded nucleic acid sequence number S2 <400> 232 atgaggtctt ggcgaaaatg agcaggctgg aa 32 <210> 233 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-19 Single-stranded nucleic acid sequence number S3 <400> 233 attccagcct gctcaaaacc gaagcaacgt gt 32 <210> 234 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-19 Single-stranded nucleic acid sequence number S4 <400> 234 aacacgttgc ttcggaaaac tatgccgctc ct 32 <210> 235 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-19 Single-stranded nucleic acid sequence number S5 <400> 235 aaggagcggc atagtaaagg attcgacgca ct 32 <210> 236 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-20 Single-stranded nucleic acid sequence number S1 <400> 236 atcacgcagc aatccaaatc gccatcacaa cg 32 <210> 237 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-20 Single-stranded nucleic acid sequence number S2 <400> 237 acgttgtgat ggcgaaaatg agcacgagcc tt 32 <210> 238 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-20 Single-stranded nucleic acid sequence number S3 <400> 238 aaaggctcgt gctcaaaacc gaaggttgca ct 32 <210> 239 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-20 Single-stranded nucleic acid sequence number S4 <400> 239 aagtgcaacc ttcggaaaac tatgccgctc ca 32 <210> 240 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sequence set 5-20 Single-stranded nucleic acid sequence number S5 <400> 240 atggagcggc atagtaaagg attgctgcgt ga 32 <210> 241 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Trimer initialization S1 sequence <400> 241 agtgatccga agtcgacaaa cgtattagcg ctcgat 36 <210> 242 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Trimer-initialized S2 sequence <400> 242 aatcgagcgc taatacgaaa gtgcaatgcg tcgatg 36 <210> 243 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Trimer initialization S3 sequence <400> 243 acatcgacgc attgcacaaa gtcgacttcg gatcac 36 <210> 244 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Trimer-optimized S1 sequence <400> 244 accaccgtgt atgacctaaa agtgacagca catcgc 36 <210> 245 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Trimer-optimized S2 sequence <400> 245 agcgatgtgc tgtcactaaa acaggctcta cgagga 36 <210> 246 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Trimer-optimized S3 sequence <400> 246 atcctcgtag agcctgtaaa aggtcataca cggtgg 36 <210> 247 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer-initialized S1 sequence <400> 247 aacctggtac aatccaaatg agctacacta gc 32 <210> 248 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer-initialized S2 sequence <400> 248 agctagtgta gctcaaaacc gaagtatcga tt 32 <210> 249 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer initialization S3 sequence <400> 249 aaatcgatac ttcggaaaac tatagtgagt tg 32 <210> 250 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer-initialized S4 sequence <400> 250 acaactcact atagtaaagg attgtaccag gt 32 <210> 251 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer-optimized S1 sequence <400> 251 aggcgatcac aatccaaatg agcgtgttac gg 32 <210> 252 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer-optimized S2 sequence <400> 252 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 253 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer-optimized S3 sequence <400> 253 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 254 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Tetramer-optimized S4 sequence <400> 254 aagcagccgc atagtaaagg attgtgatcg cc 32 <210> 255 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The first transformation scheme for pentamer initializes the S1 sequence. <400> 255 atcacagagc gcgtaaaaac gccactcatg ga 32 <210> 256 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> The first transformation scheme for pentamer initializes the S2 sequence. <400> 256 atccatgagt ggcgtaaaaa tccaaatgag cgtgttacgg 40 <210> 257 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The first transformation scheme for pentamer initializes the S3 sequence. <400> 257 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 258 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The first transformation scheme for pentamer initializes the S4 sequence. <400> 258 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 259 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> The first transformation scheme for pentamer initializes the S5 sequence. <400> 259 aagcagccgc atagtaaagg attaaatacg cgctctgtga 40 <210> 260 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> S1 sequence after optimization of the first transformation scheme of pentamer <400> 260 attcaggcga ctcctaaaag cacgacgatg gt 32 <210> 261 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> The S2 sequence after optimization of the first transformation scheme of the pentamer <400> 261 aaccatcgtc gtgctaaaaa tccaaatgag cgtgttacgg 40 <210> 262 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> S3 sequence after optimization of the first transformation scheme of pentamer <400> 262 accgtaacac gctcaaaacc gaagtgccaa tt 32 <210> 263 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The S4 sequence after optimization of the first transformation scheme of the pentamer <400> 263 aaattggcac ttcggaaaac tatgcggctg ct 32 <210> 264 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> S5 sequence after optimization of the first transformation scheme of pentamer <400> 264 aagcagccgc atagtaaagg attaaaagga gtcgcctgaa 40 <210> 265 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The second transformation scheme for pentamer initializes the S1 sequence. <400> 265 atgagtgcgc aatccaaatc gccagtcatg ca 32 <210> 266 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The second transformation scheme for pentamer initializes the S2 sequence. <400> 266 atgcatgact ggcgaaaatg agcgctcgtt ga 32 <210> 267 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The second transformation scheme for pentamer initializes the S3 sequence. <400> 267 atcaacgagc gctcaaaacc gaagtgccaa ct 32 <210> 268 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The second transformation scheme for pentamer initializes the S4 sequence. <400> 268 aagttggcac ttcggaaaac tatcgcgcga ct 32 <210> 269 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The second transformation scheme for pentamer initializes the S5 sequence. <400> 269 aagtcgcgcg atagtaaagg attgcgcact ca 32 <210> 270 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The S1 sequence after optimization of the second transformation scheme of the pentamer <400> 270 atcacgcagc aatccaaatc gccatcacaa cg 32 <210> 271 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The S2 sequence after optimization of the second transformation scheme of the pentamer <400> 271 acgttgtgat ggcgaaaatg agcacgagcc tt 32 <210> 272 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The S3 sequence after optimization of the second transformation scheme of the pentamer <400> 272 aaaggctcgt gctcaaaacc gaaggttgca ct 32 <210> 273 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The S4 sequence after optimization of the second transformation scheme of the pentamer <400> 273 aagtgcaacc ttcggaaaac tatgccgctc ca 32 <210> 274 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> The S5 sequence after optimization of the second transformation scheme for the pentamer <400> 274 atggagcggc atagtaaagg attgctgcgt ga 32 <210> 275 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PMO single-stranded nucleic acid sequence <400> 275 agcagcctcg ttgaatcgcc aagacacc 28 <210> 276 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PMO single-stranded nucleic acid sequence <400> 276 aggtgtcttg gcgaaagttg ctccgacg 28 <210> 277 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PMO single-stranded 3-nucleotide sequence <400> 277 acgtcggagc aactaagcgg ttctgtgg 28 <210> 278 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PMO single-stranded 4-nucleotide sequence <400> 278 accacagaac cgctatcaac gaggctgc 28 <210> 279 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of anti-HSA nanobody mutant <400> 279 His His His His His Ala Val Gln Leu Val Glu Ser Gly Gly Gly 1 5 10 15 Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 20 25 30 Phe Thr Phe Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 35 40 45 Lys Glu Pro Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr 50 55 60 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70 75 80 Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 85 90 95 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Ser Cys 115 120

Claims

1. A method for determining a single-stranded nucleic acid sequence for forming a multimeric complex based on a mutually compatible nucleic acid backbone, characterized in that, Including the following steps: (a) Set the annealing algorithm parameters: Set the initial annealing temperature, the final annealing temperature, and the annealing temperature decay coefficient. ; Set optimization constraint parameters: ① The number of single-stranded nucleic acids, n, where n is a positive integer from 3 to 6; ② Pairing sequence length ,in It consists of 12-16 bases; ③ Dissociation temperature threshold of the pairing region The It depends on the length of the paired sequence; where, When the base is, ; When the base is, ; ④ Free energy threshold of specific paired region sequences The It depends on the length of the paired sequence; where, When the base is, kcal / mol; When the base is, kcal / mol; ⑤ Non-specific pairing free energy threshold ,in kcal / mol; ⑥ Connecting element X2, wherein X2 is selected from the following group: A, AA, AAA; ⑦ Secondary structure dissociation temperature threshold The ; ⑧ Proportion of CG in paired sequences Its range is , Initialize the sequence set based on the above parameters. ; (b) Calculate the set mentioned in the previous step. objective function value That is, calculating the non-specific pairing free energy between sequences and between the sequences themselves. The sum of these values ​​yields the non-specific pairing free energy matrix. Search for the minimum value in its upper triangular matrix. and ,in ,according to and Nonspecific pairing free energy Random selection or An update operation is performed, resulting in a new nucleic acid sequence, and thus an updated sequence set. ; (c) Determine the set mentioned in the previous step Verify whether the sequences in the sequence satisfy the optimization constraint parameters set in step (a), including the dissociation temperature of the specific pairing region. Specific pairing region sequence free energy Secondary structure dissociation temperature and CG proportions If the above parameters meet the constraints, proceed to step (d); otherwise, repeat step (c). If, at a certain annealing temperature, step (b) is executed 15 times consecutively without achieving the desired result, then... To prevent infinite loops, sets Become a set Proceed to the next step; (d) Calculate the set mentioned in the previous step The objective function value E1 is compared. and ,like This indicates that the nonspecific pairing free energy is optimized, and the sequence set... Become a set of sequences ,like This indicates that the nonspecific pairing free energy has not been optimized. In this case, it is necessary to determine whether to accept the result according to the Metropolis criterion. Collect into ;and (e) The annealing temperature is based on the attenuation coefficient set in step (a). Attenuation is performed on the basis of the previous step. Repeat steps (b), (c), and (d), i.e., the Monte Carlo annealing algorithm, until the annealing temperature reaches the annealing termination temperature, as described in the previous step. To become a single-stranded nucleic acid sequence used to form multimeric complexes based on intermatched nucleic acid backbones; Each single-stranded nucleic acid sequence has the structure shown in Equation 1: X1-R1-X2-R2-X3 (1) in, R1 is the base complementary pairing region 1; R2 is the base complementary pairing region 2; X1, X2, and X3 are each independently either non-redundant or redundant nucleic acids; "-" is the key; Furthermore, in step (d), the optimized set is the set that satisfies the following conditions: (C1) The free energy of the DNA double-stranded structure formed by target pairing between sequences in the nucleic acid backbone structure. Less than Or the smallest; and (C2) In the nucleic acid backbone structure, non-target pairings between sequences kcal / mol or maximize it.

2. The method as described in claim 1, characterized in that, In step (a), setting the annealing algorithm parameters includes: ⑨ For n=4, use a symmetric sequence.

3. The method as described in claim 1, characterized in that, In step (d), the optimized set also satisfies the following condition: (C3) When the bases are in the R1 and R2 regions, the pairing dissociation temperature is... .

4. The method as described in claim 1, characterized in that, In step (c), the free energy of the matched nucleic acid backbone structure is calculated using the nearest neighbor method. ).

5. The method as described in claim 1, characterized in that, In step (c), the interpaired nucleic acid backbone structure is decomposed into 10 different nearest-neighbor pairwise interactions: AA / TT; AT / TA; TA / AT; CA / GT; GT / CA; CT / GA; GA / CT; CG / GC; GC / CG; and GG / CC; and the enthalpy of these pairwise interactions is used to determine the interaction. Entropy The corresponding calculations yielded their respective results. The value is then calculated; the free energies of the paired interactions included in the intermatched nucleic acid backbone structure are combined or summed to obtain the free energy of the intermatched nucleic acid backbone structure.

6. The method as described in claim 1, characterized in that, The method involves repeating steps (b), (c), and (d) multiple times to obtain the global optimal solution during the iteration process.

7. The method as described in claim 6, characterized in that, During the iteration process, according to the Metropolis criterion, poor solutions are accepted to a limited extent and the probability of accepting a poor solution gradually approaches 0, so that the algorithm can find the global optimal solution as much as possible when it terminates.

8. The method as described in claim 1, characterized in that, The simulated annealing algorithm is iterated using the following objective function to optimize the free energy of the non-target pairing region: For sequence and sequence Non-target pairing free energy This is the sum of the free energies of non-target pairings among all sequences, and its value is negative; the larger this negative value is, the more beneficial it is to reduce non-target pairings.

9. A set of single-stranded nucleic acid sequences for forming multimeric complexes based on mutually compatible nucleic acid backbones, characterized in that, The set of single-stranded nucleic acid sequences is determined using the method of claim 1, and the set is selected from the set of single-stranded nucleic acid sequences shown in the group consisting of those used to form tetrameric complexes based on intermatched nucleic acid backbones: 。 10. A multimeric complex based on a mutually compatible nucleic acid backbone, characterized in that, The complex is a polymer formed by n monomers with mutually compatible nucleic acid backbones, wherein each monomer is a polypeptide with a single nucleic acid chain, and n is 4; in the polymer, the nucleic acid single chain of each monomer forms a mutually compatible double chain with the nucleic acid single chains of the other two monomers through base complementarity, thereby forming a mutually compatible nucleic acid backbone structure. The single-stranded nucleic acid sequence in the multimer complex is composed of the set of single-stranded nucleic acid sequences as described in claim 9.

11. The polymeric complex of claim 10, characterized in that, The monomer has the structure of Formula I: Z1-W (I) In the formula, Z1 is the polypeptide portion; W represents a single-stranded nucleic acid sequence; "-" indicates a connector or key.

12. The polymeric complex according to claim 11, characterized in that, "-" indicates a covalent bond.

13. The polymeric complex of claim 11, characterized in that, The nucleic acid sequence is selected from the following group: levorotatory nucleic acid, peptide nucleic acid, locked nucleic acid, thiomodified nucleic acid, 2'-fluoromodified nucleic acid, 5-hydroxymethylcytosine nucleic acid, phosphorodiamidate morpholino nucleic acid, or combinations thereof.

14. The polymeric complex of claim 11, characterized in that, In the polymer, the Z1 of each monomer is the same or different.

15. The polymeric complex of claim 11, characterized in that, In the polymer, the W of each monomer is different.

16. The polymeric complex of claim 10, characterized in that, The monomer described has a structure of formula II: D-[LW]m (II) in, D represents the protein drug element portion; W represents the nucleic acid sequence; L indicates no connection or connector; "-" indicates a covalent bond; m can be 1, 2, or 3.

17. The polymeric complex of claim 16, characterized in that, m is 1.

18. The polymeric complex of claim 10, characterized in that, The monomer described has a structure of Formula III: A-[LW]m (III) in, A represents the polypeptide antigen element portion; W represents the nucleic acid sequence; L indicates no connection or connector; "-" indicates a covalent bond; m can be 1, 2, or 3.

19. The polymeric complex of claim 18, characterized in that, m is 1.

20. The polymeric complex according to any one of claims 11-19, characterized in that, The nucleic acid sequence W has the structure shown in Formula 1: X1-R1-X2-R2-X3 (1) in, R1 is the base complementary pairing region 1; R2 is the base complementary pairing region 2; X1, X2, and X3 are each independently either non-redundant or redundant nucleic acids; "-" is the key.

21. The polymeric complex of claim 20, characterized in that, R1 and R2 are each 10-20 bases long independently.

22. The polymeric complex of claim 20, characterized in that, X1 has a length of 0-5 bases.

23. The polymeric complex of claim 20, characterized in that, The length of X3 is 0-5 bases.

24. The polymeric complex of claim 20, characterized in that, The length of X2 is 0-3 bases.

25. The polymeric complex of claim 20, characterized in that, The sequence of X2 is selected from the following group: A, AA, AGA or AAA.

26. The polymeric complex of claim 20, characterized in that, Each monomer's R1 forms a base-complementary pairing structure with the R2 of its left neighboring monomer; while R2 forms a base-complementary pairing structure with the R1 of its right neighboring monomer.

27. The polymeric complex according to any one of claims 10-19 and 21-26, characterized in that, The monomer sequence is phosphorodiamidate morpholino nucleic acid.

28. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) The multimeric complex based on the intermatched nucleic acid backbone according to any one of claims 10-27; and (b) Pharmaceutically acceptable carriers.

29. The pharmaceutical composition of claim 28, characterized in that, The pharmaceutical compositions include vaccine compositions.

30. The pharmaceutical composition of claim 28, characterized in that, The pharmaceutical compositions described herein include therapeutic and / or preventative pharmaceutical compositions.

31. A nucleic acid sequence library, characterized in that, The nucleic acid library includes nucleic acid sequences for forming multimeric complexes based on intermatched nucleic acid backbones as described in any one of claims 10-27.

32. The nucleic acid sequence library as described in claim 31, characterized in that, The nucleic acid sequence W has the structure shown in Formula 1: X1-R1-X2-R2-X3 (1) in, R1 is the base complementary pairing region 1; R2 is the base complementary pairing region 2; X1, X2, and X3 are each independently either non-redundant or redundant nucleic acids; "-" is the key.

33. The use of a nucleic acid sequence library as described in claim 31 or 32, characterized in that, Used for preparing the polymeric complex of any one of claims 10-27 or a pharmaceutical composition containing the polymeric complex of any one of claims 10-27.

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