WO2025105357A1 - Method for producing modified nucleic acid molecule-peptide complex, nucleic acid display method or ribosome display method - Google Patents

Abstract

The method of the present disclosure is a method for producing a modified nucleic acid molecule-peptide complex in which a functional molecule has been introduced into the peptide moiety of a nucleic acid molecule-peptide complex in which a nucleic acid molecule and a peptide encoded by the nucleic acid molecule are linked.

Description

Method for producing modified nucleic acid molecule-peptide complex, nucleic acid display method or ribosome display method

The present invention relates to a method for producing a modified nucleic acid molecule-peptide complex. The present invention also relates to a nucleic acid display method or a ribosome display method that includes the method for producing the modified complex.

Until now, small molecule drugs have been the main therapeutic drug, but antibodies have attracted attention as molecular targeted drugs, and the development of antibody drugs has increased rapidly in recent years. Among them, peptide drugs, which are medium molecule drugs with molecular weights of several thousand to tens of thousands, have attracted attention. Since peptide drugs can be produced as molecular targeted drugs using evolutionary molecular engineering technology, research and development of peptide drugs using evolutionary molecular engineering technology has been actively carried out in recent years. On the other hand, new functional peptides can be created by introducing functional low molecular weight compounds into peptides. Therefore, by introducing such low molecular weight compounds in the construction of a random sequence library at the beginning of the evolutionary molecular engineering process and selecting from it, including the peptide sequence, it is possible to impart molecular recognition functions derived from the peptide sequence and functionality derived from low molecules.

For example, Non-Patent Documents 1 and 2 disclose the selection of candidate molecules for peptide drugs by synthesizing functional- or functional compound-carrying molecular compound-carrying (misacylated) tRNA using organic synthesis methods and displaying it in a cell-free translation system. Non-Patent Document 3 discloses the selection of candidate molecules for peptides that recognize the target polysaccharide paramylon and emit fluorescence by synthesizing environmentally responsive fluorescent group-carrying (misacylated) tRNA using organic synthesis methods and displaying it in a cell-free translation system.

Med. Chem. Commun., 5 (9), 1400-1403 (2014) Bull. Chem. Soc. Jpn., 89, 444-446 (2016) Scientific Reports, 8, 8271 (2018)

In conventional evolutionary molecular engineering technology, new functional molecules, even though they are small molecular weight compounds, have a relatively large molecular weight and a bulky three-dimensional structure, and therefore do not pass through the ribosome tunnel. Therefore, when constructing a library using conventional evolutionary molecular engineering technology, it was not possible to directly introduce them into the library using a cell-free translation system.

One aspect of the present invention aims to realize a method for introducing compounds with relatively large molecular weights or bulky three-dimensional structures into libraries used for nucleic acid display or ribosome display.

After extensive research, the inventors discovered a method for introducing a compound with a relatively large molecular weight or a compound with a bulky three-dimensional structure to a specific reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex by a chemical synthesis reaction in a water-soluble polar organic solvent. They also discovered that this method can be used to introduce a compound with a relatively large molecular weight or a compound with a bulky three-dimensional structure into a library used in nucleic acid display or ribosome display, leading to the completion of the present invention.

In order to solve the above problems, a method for producing a modified nucleic acid molecule-peptide complex according to one embodiment of the present invention is a method for producing a modified nucleic acid molecule-peptide complex by introducing a functional molecule into the peptide portion of a nucleic acid molecule-peptide complex in which a nucleic acid molecule and a peptide encoded by the nucleic acid molecule are linked, and the method includes a step of introducing a functional molecule into a reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent.

According to one aspect of the present invention, even compounds with relatively large molecular weights or bulky three-dimensional structures can be introduced into libraries used for nucleic acid display or ribosome display.

FIG. 1 is a schematic diagram of ribosome display according to one embodiment of the present invention. FIG. 1 is a schematic diagram of ribosome display according to one embodiment of the present invention. FIG. 1 is a schematic diagram showing a click reaction in a ribosome display according to one embodiment of the present invention. FIG. 1 is a schematic diagram of cDNA display according to one embodiment of the present invention. FIG. 1 shows the fluorescence intensities of peptides 1 to 3. FIG. 1 shows the fluorescence intensities of peptides 1 to 3. FIG. 1 shows the results of SPR analysis of peptide 2. FIG. 1 shows the results of observation of HT-1080 cells and MCF-704 cells using a confocal laser microscope. FIG. 1 shows the intracellular fluorescence pattern of peptide 2 in T98G cells. FIG. 2 shows the fluorescence profiles of heat shocked and untreated T98G cells stained with peptide 2.

One aspect of the present invention is described below, but the present invention is not limited to this. The present invention is not limited to each of the configurations described below, and various modifications are possible within the scope of the claims. In addition, embodiments and examples obtained by appropriately combining the technical means disclosed in the embodiments and examples are also included in the technical scope of the present invention. In addition, in this specification, "A to B" indicates greater than or equal to A and less than or equal to B, unless otherwise specified.

[Method for Producing Modified Nucleic Acid Molecule-Peptide Conjugate]
A method for producing a modified nucleic acid molecule-peptide complex according to one aspect of the present invention is a method for producing a modified nucleic acid molecule-peptide complex by introducing a functional molecule into the peptide portion of the nucleic acid molecule-peptide complex. Hereinafter, this method may be referred to as the "method for producing the modified complex of this embodiment."

The method for producing a modified substance of this embodiment performs a chemical synthesis reaction in a water-soluble polar organic solvent, so that even functional molecules with relatively large molecular weights or bulky three-dimensional structures can be efficiently introduced into the peptide portion of a nucleic acid molecule-peptide complex. A library containing the modified substance produced by the method for producing a modified substance of this embodiment can be used in the nucleic acid display method or ribosome display method described below.

(Nucleic acid molecule-peptide complex)
Examples of the nucleic acid molecule of the nucleic acid molecule-peptide complex include RNA molecules such as mRNA, DNA molecules such as cDNA, and RNA/DNA hybrid molecules (complementary duplexes).

As used herein, "peptide" refers to a compound in which two or more amino acids are bound by peptide bonds. The number of amino acids is not limited, and may be, for example, 2 to 1000, preferably 3 to 200, more preferably 4 to 100, and even more preferably 5 to 50. Examples of the number of amino acids include 10 or more, 20 or more, 30 or less, 40 or less, and the like. The peptide may be, for example, a fragment or full-length protein. In one example, the peptide may be an antibody (such as scFv) or a fragment thereof.

The peptide of the nucleic acid molecule-peptide complex is a peptide encoded by the nucleic acid molecule, and is a peptide synthesized using a cell-free protein synthesis system. Examples of cell-free protein synthesis systems include cell-free protein synthesis systems that use extracts of E. coli, wheat germ, rabbit reticulocytes, etc., and reconstituted cell-free protein synthesis systems that use E. coli ribosomes and that mix together purified factors required for translation.

A nucleic acid molecule-peptide complex can be obtained by linking a nucleic acid molecule to a peptide via a peptide acceptor molecule such as puromycin. A nucleic acid molecule-peptide complex can also be obtained by linking a nucleic acid molecule to a peptide via a ribosome. Whether or not a peptide acceptor molecule is involved, the nucleic acid molecule-peptide complex can be a nucleic acid molecule-peptide-ribosome complex.

(Functional molecule)
Examples of the functional molecule include fluorescent molecules such as carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), tetraphenylethylene (TPE), and N,N-dimethylamino-1,8-naphthalimide (DMN); inhibitory molecules such as immune checkpoint inhibitors; and ligand molecules such as folic acid, methotrexate (MTX), and folate receptor binding ligands that bind to folate receptors. In a preferred example, the functional molecule is a bulky molecule that is difficult to directly incorporate into a peptide using a cell-free protein synthesis system (does not pass through the ribosome tunnel).

The method for producing the modified product of this embodiment includes a step of introducing a functional molecule into a reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent.

Examples of reactive groups include thiol groups, amino groups, and carboxyl groups. The reactive groups and functional molecules are bonded by a chemical synthesis reaction in a water-soluble polar organic solvent. An example of a thiol group is the thiol group of cysteine. An example of an amino group is the amino group of lysine or arginine. An example of a carboxyl group is the carboxyl group of glutamic acid or aspartic acid. An example of a functional group that reacts with a thiol group is a maleimide group. An example of a functional group that reacts with an amino group is a carboxyl group, an N-hydroxylsuccinimide ester group, an isothiocyanate group, and the like. An example of a functional group that reacts with a carboxyl group is an amino group.

Another example of a reactive group is a bioorthogonal reactive group. For example, one bioorthogonal reactive group linked to a functional molecule is reacted with the other bioorthogonal reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex by a bioorthogonal chemical click reaction or a strain-promoted inverse electron demand Diels-Alder reaction. By this reaction, the functional molecule can be introduced into the peptide portion of the nucleic acid molecule-peptide complex.

Examples of bioorthogonal reactive groups include azide groups such as azidophenyl group, N6-((prop-2-yn-1-yloxy)carbonyl) group, and tetrazine group; alkyne groups such as o-propargyloxy group, N6-((prop-2-yn-1-yloxy)carbonyl) group, bicyclo[6,1,0]nonyne group, and dibenzocyclooctyne group; cyclooctene groups such as transcyclooctene group and norbornene group; and the like. Note that non-natural amino acids having these bioorthogonal reactive groups that can be incorporated into peptides using a cell-free protein synthesis system are publicly known, and therefore by using these non-natural amino acids, it is possible to prepare a nucleic acid molecule-peptide complex incorporating an amino acid having a bioorthogonal reactive group.

The water-soluble polar organic solvent used in the step of introducing the functional molecule is preferably an aprotic polar organic solvent, since it can suppress precipitation of the nucleic acid molecule. Examples of aprotic polar organic solvents include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetone, and acetonitrile.

The solvent used in the step of introducing the functional molecule may be a mixed solvent of an aqueous solvent and a water-soluble polar organic solvent. Examples of aqueous solvents include water or a buffer solution. The concentration of the water-soluble polar organic solvent contained in the mixed solvent of an aqueous solvent and a water-soluble polar organic solvent is preferably 2% by mass or more, more preferably 5% by mass or more, and even more preferably 10% by mass or less, in terms of promoting chemical synthesis reactions. Furthermore, in terms of suppressing precipitation of nucleic acid molecules, it is preferably 50% by mass or less, more preferably 45% by mass or less, and even more preferably 40% by mass or less.

[Nucleic acid display method]
A nucleic acid display method according to one aspect of the present invention includes the method for producing a modified form of the present embodiment, and includes a step of selecting a desired modified form from the modified forms of the nucleic acid molecule-peptide complex. Examples of the nucleic acid display method include an mRNA display method and a cDNA display method.

Below, an example of a nucleic acid display method according to one embodiment of the present invention will be described with reference to Figure 4c. Figure 4c is a schematic diagram of a cDNA display according to one embodiment of the present invention.

(Step 1: Creating a library of candidate DNA molecules)
In step 1, a library of candidate DNA molecules is prepared, which includes a plurality of different candidate DNA molecules including a promoter region and a region downstream thereof that codes for a candidate peptide. Here, the candidate DNA refers to a DNA sequence that may ultimately be selected as a peptide aptamer. Similarly, when the target is an RNA or a peptide, the candidate DNA is called a candidate RNA or a candidate peptide.

In the present specification, the term "library" refers to a collection of multiple (two or more) different molecules (e.g., multiple different DNA molecules, multiple different RNA molecules, multiple different DNA-peptide complexes, or multiple different RNA-peptide complexes). For example, the term "library" refers to a collection of multiple different molecules classified into the same category (e.g., the category of DNA molecules, the category of RNA molecules, or the category of RNA-peptide complexes). In addition, the term may be referred to as a DNA library (a library of DNA molecules), an RNA library (a library of RNA molecules), a DNA-peptide complex library (a library of DNA-peptide complexes), an RNA-peptide complex library (a library of RNA-peptide complex molecules), or the like, in accordance with the name of the category of molecules. In the nucleic acid display method according to one aspect of the present invention, since selection starting from a large number of candidate molecules is facilitated as necessary, the "library" in this embodiment may preferably contain 10 ⁹ or more, more preferably 10 ¹⁰ or more, 10 ¹¹ or more, or 10 ¹² or more, and even more preferably 10 ¹³ or more different molecules.

As used herein, "selection" refers to the substantial separation of a molecule from other molecules in a population. For example, "selection" refers to the substantial separation of a molecule from a collection of multiple different molecules that fall into the same category. As used herein, "selection" can enrich desired molecules at least 2-fold, preferably 30-fold or more, more preferably 100-fold or more, and even more preferably 1000-fold or more, relative to non-desired molecules in the library after selection. As provided herein, the selection step can be repeated any number of times in a given manner, and different types of selection steps can be combined.

The candidate DNA molecule includes a region that codes for the candidate peptide. In addition, the candidate DNA molecule may, if necessary, include a region for performing transcription using the antisense strand as a template (transcription control region). An example of a transcription control region is a promoter region. These transcription control regions may be appropriately selected depending on the type of RNA polymerase used in the transcription reaction in step 2. In one example, the promoter region may be one that is recognized by T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase (T7 promoter, SP6 promoter, or T3 promoter).

The candidate DNA molecule, which includes the promoter region and the region downstream thereof that encodes the candidate peptide, may be single-stranded, double-stranded, or a mixture of both (partially double-stranded and the rest single-stranded), as long as a candidate RNA molecule that encodes the peptide is produced by a transcription reaction.

In detail, the promoter region in the candidate DNA molecule may be single-stranded, double-stranded, or a mixture of both, as long as a candidate RNA molecule encoding the candidate peptide is transcribed from a region encoding the candidate peptide located downstream. For example, a part of the promoter region may be single-stranded (sense strand or antisense strand) and the other part may be double-stranded. In this specification, a "promoter region" also includes a region partially composed of a single strand (sense strand or antisense strand). If the promoter region exhibits promoter activity, the candidate RNA molecule encoding the candidate peptide is transcribed. Promoter activity can be measured by a method known in the art. Furthermore, the transcription of the candidate RNA molecule encoding the candidate peptide can be confirmed by detecting the RNA molecule or detecting the peptide generated by translation by a method known in the art.

Furthermore, the region in the candidate DNA molecule that codes for the candidate peptide may be single-stranded, double-stranded, or a mixture of both, so long as a candidate RNA molecule that codes for the candidate peptide is transcribed by the action of the promoter region located upstream. For example, the entire coding region may be single-stranded (sense strand or antisense strand). Since the candidate RNA molecule is transcribed using the sequence of the antisense strand as a template, the region that codes for the candidate peptide preferably includes at least the antisense strand. Such regions that are partially or entirely single-stranded (sense strand or antisense strand) are also included in the "region that codes for a peptide" in this specification.

If the reactive group is a thiol group, an amino group, or a carboxyl group, a library of DNA molecules is created so that the candidate peptides contain amino acids that contain that reactive group.

(Step 2: Creating a library of candidate RNA molecules)
In step 2, the candidate DNA molecules are transcribed using them as templates to prepare a library of corresponding candidate RNA molecules.

Transcription may be carried out by known methods. Typically, transcription is carried out in situ or in vitro, preferably in vitro. When transcription is carried out in vitro, the type of RNA polymerase is not particularly limited, and preferred examples include RNA polymerases derived from bacteriophages such as T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase, with T7 RNA polymerase being more preferred.

(Step 3: Ligation of peptide acceptor molecule and candidate RNA molecule)
In step 3, a peptide acceptor molecule is linked to the 3' end of the candidate RNA molecule. The peptide acceptor molecule is not particularly limited as long as it can be linked to a translated peptide, and examples thereof include known molecules such as puromycin, puromycin derivatives, and oligo-RNA/amino acid complexes.

The 3' end of the candidate RNA molecule and puromycin are typically linked via a linker containing an oligonucleotide. There is no particular limit to the length of the oligonucleotide, and it may be, for example, about 10 to 30 nucleotides, preferably 15 to 20 nucleotides. Examples of nucleotides in the oligonucleotide include DNA, RNA, PNA, and LNA. The linker may further contain other substances (e.g., polyethylene glycol (PEG)) in addition to the oligonucleotide. There is no particular limit to the length of the PEG, but in one example, it is preferably a linkage of 3 to 10 PEGs with 6 to 18 atoms in the main chain. In one example, the linker may have a structure of 5'-(oligonucleotide)-(PEG)-(peptide acceptor molecule)-3'. Such linkers may be prepared by known methods.

The linker may contain biotin or the like as a molecule that forms a bond with the solid phase, which will be described later. In addition, the linker may contain a fluorescent group such as fluorescein isothiocyanate (FITC) so that the presence or absence of binding to the linker can be easily detected.

(Step 4: Translation)
In step 4, the candidate RNA molecules to which the peptide acceptor molecules obtained in step 3 are bound are translated in a cell-free protein synthesis system to prepare a library of first complexes of candidate RNA molecules and peptides, in which the RNA molecules and the peptides encoded by the RNA molecules are linked via the peptide acceptor molecules.

Step 5: Reverse Transcription
In step 5, a library of second complexes of candidate RNA molecules, cDNA molecules corresponding to the candidate RNA molecules, and peptides is prepared by reverse transcribing the candidate RNA molecules of the first complex obtained in step 4 using the other end of the linker as a reverse transcription initiation site. The candidate RNA molecules and the cDNA molecules corresponding to the candidate RNA molecules are complementary to each other, forming an RNA/DNA hybrid (double-stranded structure).

In the case of mRNA display, the reverse transcription step (creation of the library of the second complex) is not essential, but it is preferable to perform the reverse transcription step in order to stabilize the RNA molecule linked to the peptide acceptor molecule. In the case of mRNA display, the cDNA generated by reverse transcription in the second complex is not linked to the peptide acceptor molecule.

In cDNA display, the cDNA molecules generated by reverse transcription are linked to the peptide acceptor molecule in the second complex.

(Step 6: Preparation of modified nucleic acid molecule-peptide complex)
In step 6, a functional molecule (DMN in FIG. 4c) is introduced by a chemical synthesis reaction in a water-soluble polar organic solvent to a reactive group (thiol group of cysteine in FIG. 4c) of an amino acid residue constituting the peptide portion of the second complex obtained in step 5. Step 6 is carried out by the above-mentioned method for producing a modified product of this embodiment.

(Step 7: Binding of the modified nucleic acid molecule-peptide complex to a binding partner)
In step 7, the library of nucleic acid molecule-peptide complexes is contacted with a binding partner (Hsp90α in the case of FIG. 4c) to carry out a binding reaction.

The binding reaction may be based on, for example, the binding between an antigen and an antibody, the binding between a protein receptor and a ligand, the binding between an adhesion molecule and a partner molecule, the binding between an enzyme and a substrate, the binding between a nucleic acid and a protein that binds to it, the binding between proteins in a signal transduction system, the binding between a glycoprotein and a protein, or the binding between a sugar chain and a protein. The binding partner may be appropriately selected depending on the purpose of the selection. The binding partner may be, for example, immobilized on a solid phase, or may be labeled with a substance that is captured by the solid phase. The solid phase may be any one that can bind to the binding partner, and may have a shape such as a plate, a rod, a particle, or a bead. The solid phase may be a material that is insoluble in water or an organic solvent, which is a medium used for screening. For example, plastic, glass, resin such as polystyrene, or metal such as a gold thin film may be used as the solid phase. Magnetic beads, etc. may also be used. The binding partner contained in one reaction system may be one type or multiple types.

The modified nucleic acid molecule-peptide complex that has not bound to the binding partner can be removed, for example, by washing with a buffer solution.

(Step 8: Recovery)
In step 8, the modified nucleic acid molecule-peptide complex is dissociated from the binding partner to recover the modified nucleic acid molecule-peptide complex. The method for dissociation may be appropriately selected depending on the type of bond with the binding partner. For example, proteases such as ficin, papain, trypsin, or antibody sequence-specific proteases such as IdeS and IdeZ can be used. Alternatively, denaturation can be performed using heat or a denaturing agent (urea, guanidine).

The modified nucleic acid molecule-peptide complex can be recovered, for example, by eluting (separating from) the substance bound to the binding partner.

By carrying out the above steps, the desired nucleic acid molecule-peptide complex is selected from the library of nucleic acid molecule-peptide complexes.

The selected nucleic acid molecule-peptide complex may then be analyzed for the sequence of the nucleic acid molecule and/or peptide. The analysis may be performed using a conventional amino acid sequence sequencer, or by reverse transcribing DNA from the RNA bound to such peptides and analyzing the base sequence of the resulting cDNA. Purification or quantification may also be performed as appropriate.

The cDNA obtained here can also be used to carry out step 1 above. For example, a PCR reaction is carried out on the cDNA obtained here or its amplified product. Steps 2 to 8 are then carried out again using the DNA molecule obtained from this. By repeating the cycle of steps 1 to 8 multiple times in this way, nucleic acid molecule-peptide complexes with the desired properties are concentrated. These are then used for analysis.

[Ribosome display method]
A ribosome display method according to one aspect of the present invention includes the method for producing the modified form of the present embodiment, and includes a step of selecting a desired modified form from the modified forms of the nucleic acid molecule-peptide complex. Hereinafter, an example of the ribosome display method according to one aspect of the present invention will be described with reference to FIG.

(Step A: Creation of a library of candidate DNA molecules)
A library of candidate DNA molecules can be prepared in the same manner as in "Step 1: Preparation of a library of candidate DNA molecules" in the nucleic acid display method. In the ribosome display method, candidate DNA molecules lacking a codon that indicates a translation stop are used.

(Step B: Creation of a library of candidate RNA molecules)
A library of candidate RNA molecules can be prepared in the same manner as in "Step 2: Preparation of a library of candidate RNA molecules" of the nucleic acid display method.

(Step C: Translation)
In step C, the candidate RNA molecules are translated in a cell-free protein synthesis system in the presence of amino acids bound to reactive groups (bicyclo[6,1,0]nonyne (BCN) groups in Fig. 1) for introducing functional molecules into the library. Because the candidate RNA molecules do not have codons that stop translation and contain ribosome arrest sequences (SecM), a library of complexes of the candidate RNA molecules, peptides, and ribosomes (PRM complexes; peptide-ribosome-mRNA complexes) is produced by the translation.

(Step D: Linking Reactive Groups with Functional Molecules)
In step D, a reactive group (tetrazine in FIG. 1) that reacts with a reactive group (BCN group in FIG. 1) bonded to an amino acid is linked to a functional molecule. The linking may be performed by a known method via a linker.

(Step E: Preparation of modified nucleic acid molecule-peptide complex)
In step E, a reactive group bound to an amino acid (BCN group in FIG. 1) is reacted with a reactive group linked to a functional molecule (tetrazine in FIG. 1) by a chemical synthesis reaction in a water-soluble polar organic solvent. This reaction introduces the functional molecule into the peptide portion of the PRM complex, producing a modified nucleic acid molecule-peptide complex. Step E is performed by the above-described method for producing a modified product of this embodiment. In FIG. 1, the functional molecule is introduced into the peptide portion of the PRM complex by a click reaction between the BCN group and tetrazine.

(Step F: Selection)
In step F, the modified nucleic acid molecule-peptide complex obtained in step E is subjected to selection (e.g., affinity selection). From the selected modified complex, mRNA encoding the bound protein is recovered, and the target DNA is obtained by RT-PCR. Step F may be performed in the same manner as "Step 7: Binding of the modified nucleic acid molecule-peptide complex to a binding partner" and "Step 8: Recovery" in the nucleic acid display method.

By repeating steps A to F above, nucleic acid molecule-peptide complexes with the desired properties are concentrated.

The nucleic acid display method and ribosome display method according to one aspect of the present invention can be used to identify proteins that interact with target molecules in drug discovery, analyze drug target proteins, obtain or modify antibody molecules, identify antigenic proteins and their antigenic sites recognized by antibodies, modify various functional proteins and peptides, and confirm the activity of vaccines.

〔summary〕
The method according to aspect 1 of the present invention is a method for producing a modified nucleic acid molecule-peptide complex by introducing a functional molecule into the peptide portion of a nucleic acid molecule-peptide complex in which a nucleic acid molecule and a peptide encoded by the nucleic acid molecule are linked, the method comprising the step of introducing the functional molecule into a reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent.

In the method according to aspect 2 of the present invention, in aspect 1 of the present invention, the water-soluble polar organic solvent may be an aprotic polar organic solvent.

In the method according to aspect 3 of the present invention, in aspect 1 or 2 of the present invention, the functional molecule may be a fluorescent molecule, an inhibitor molecule, or a ligand molecule.

In the method according to aspect 4 of the present invention, in any one of aspects 1 to 3 of the present invention, the reactive group may be a thiol group, an amino group, or a carboxyl group.

In the method according to aspect 5 of the present invention, in any one of aspects 1 to 3 of the present invention, the reactive group may be a bioorthogonal reactive group.

The method according to aspect 6 of the present invention may be any of aspects 1 to 3 and 5 of the present invention, in which the nucleic acid molecule-peptide complex is a nucleic acid molecule-peptide-ribosome complex to which a ribosome is further linked.

The method according to aspect 7 of the present invention is a nucleic acid display method or a ribosome display method, which includes any of the methods according to aspects 1 to 6 of the present invention and includes a step of selecting a desired modification from the modifications of the nucleic acid molecule-peptide complex.

- A library composed of multiple types of modified nucleic acid molecule-peptide complexes produced by the method of any one of aspects 1 to 6 of the present invention, wherein the modified nucleic acid molecule-peptide complexes have the same introduced functional molecule but different amino acid sequences of the peptide portion.
The library of modifications may, for example, contain 10 or more, ¹⁰ or more, ¹⁰ or more, ^{10 or} more, more preferably 10 or more, ¹⁰ or more, or ¹⁰ or more, even more preferably ^{10 or} more.
The above library of modifications is subjected to, for example, the method of aspect 7 or 8 of the present invention, and a step of selecting the desired modification is carried out.
In one example, the modified forms of the nucleic acid molecule-peptide complexes constituting the library of modifications have substantially the same length of amino acid sequence of the peptide portion, where the difference in length of the amino acid sequence is 20% or less, 10% or less, 5% or less, or 0%.
In one example, the library of modified substances contains the water-soluble polar organic solvent described in Aspect 1, etc., and in another example, does not contain the water-soluble polar organic solvent described in Aspect 1, etc. In other words, after the library of modified substances is produced, the water-soluble polar organic solvent may be removed.

The following examples are presented to further explain the embodiments of the present invention. Of course, the present invention is not limited to the following examples, and various details are possible. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed. Furthermore, all of the documents described in this specification are incorporated by reference.

In the examples, unless otherwise specified, % refers to mass %.

[Evaluation Example 1] Stability of PRM complex First, we investigated whether the peptide-ribosome-mRNA (PRM) complex formed by the ribosome display method can exist stably in the presence of DMSO. First, we created a cell-free protein synthesis system (CellFree) from an extract of E. coli (an E. coli strain lacking Amber (Amb, UAG codon) in the ΔRF1 and 94 genes). Using this CellFree, we translated RNA that was a mixture of a FLAG gene that does not contain Amb downstream (FLAG mRNA, upper part of Figure 2) and a part of the GFP gene that does not contain Amb as a control (GFP mRNA, lower part of Figure 2) in a ratio of 1:9.

The stability of the translated sample was evaluated by passing it through a gel filtration column equilibrated with a liquid adjusted to a DMSO concentration of 0-100%. Specifically, the translated sample solution was subjected to a gel filtration column, and then the solution was converted to a DMSO solution. The PRM complex bound to the anti-FLAG antibody-immobilized beads was then recovered, and the mRNA was purified. The purified mRNA was reverse transcribed into cDNA, amplified by PCR, and the intensity of the FLAG and GFP bands was determined. As a result, even when the DMSO concentration was changed to 0-100% after the translation was subjected to gel filtration, only the band derived from FLAG (650 bp) was detected in the sample selected with the anti-FLAG antibody-immobilized beads (Table 1). On the other hand, in the results of RT-PCR of the pre-translated sample (mRNA), GFP (850 bp) was detected at a density approximately 10 times higher than FLAG, indicating that there was no bias in the enrichment of the FLAG sequence by RT-PCR. These results showed that the stability of the PRM complex formed during ribosome display is not affected by the DMSO concentration.

[Evaluation Example 2] Examination of click reaction In order to confirm whether a click reaction occurs in a cell-free translation solution, thioredoxin containing exoBCN was expressed in a cell-free translation system, reacted with a tetrazine compound, and the protein after the reaction was purified with a FLAG tag that had been added to thioredoxin separately, and subjected to MS analysis. Specifically, thioredoxin containing exoBCN was translated for 2 hours in a cell-free translation system based on 100 μL of Escherichia coli lysate (0.4 mg/ml aminoacyl-tRNA synthetase, 4 μg/μl template, 5 mM exoBCN, 0. 2 mg/ml tRNA), and then 25 μL of a solution obtained after translation was added to 25 μL of a tetrazine compound modified with BMS1166 (checkpoint inhibitor), methotrexate (MTX), or tetraphenylethylene (TPE) dissolved in DMSO to a final concentration of 5 mM. After incubation at 37°C for 5 minutes, thioredoxin was purified using the FLAG tag, eluted with TFA, and analyzed by MS. The MS peaks obtained showed the expected increase in mass upon modification with each small molecule, confirming that small molecules can be modified in a short time by the click reaction even in the presence of impurities related to the cell-free translation system.

[Evaluation Example 3] Examination of introduction of target substance in ribosome display The introduction of target substance was examined using the method shown in Figure 1. It has been clarified that bicyclo[6.1.0]non-4-yne (BCN) modified amino acids in Figure 1 can be introduced into a cell-free translation system. In this evaluation example, BCN-linked amino acids were used, and after preparing a peptide library, it was revealed that the target substance can be introduced into the library by click reaction using a tetrazine compound in a water-soluble polar organic solvent.

Specifically, the FLAG gene without Amb downstream in Evaluation Example 1 was replaced with a FLAG gene with Amb downstream, and exoBCN and BCNRS/tRNA(CUA) were added as unnatural amino acids during translation so that the Amb codon was translated as exoBCN. ExoBCN was incorporated into the peptide library as an unnatural amino acid, and a click reaction was carried out with a target inhibitor-modified tetrazine dissolved in DMSO to examine whether a target substance could be incorporated into the ribosome display method (Figure 2). In this evaluation example, the fluorescent substance FAM was used as a model compound instead of the target substance.

Next, a peptide containing exoBCN (PRM complex) was mixed with FAM-tetrazine at each DMSO concentration to cause a click reaction as shown in Figure 3, and then the peptide was recovered using anti-FAM antibody-immobilized beads to examine whether the desired FLAG sequence could be concentrated.

The translated sample was passed through a gel filtration column equilibrated with a liquid adjusted to a DMSO concentration of 0-100%, and then reacted with peptides containing FAM-tetrazine and exoBCN at each DMSO concentration. To recover only peptides that underwent a click reaction, they were recovered using beads immobilized with anti-FAM antibodies and RT-PCR was performed. The results are shown in Table 3. In Table 3, "+" indicates that the FLAG band was concentrated, and "-" indicates that the FLAG band was not concentrated.

As shown in Table 3, only the desired FLAG band was detected at DMSO concentrations between 2% and 50%. On the other hand, in the system using FAM-modified tetrazine, at DMSO concentrations above 50%, the desired FLAG band was not enriched, as was the case with the FLAG gene RNA that did not contain amber, which was used as a negative control.

Evaluation example 1 showed that even a DMSO concentration of 100% did not adversely affect the stability of the PRM complex. Furthermore, evaluation example 3 showed that selection using beads modified with FAM antibodies can be performed at DMSO concentrations of 2-50%, allowing for a wider range of options.

[Evaluation Example 4] Preparation of a library of target substances using cDNA display In this evaluation example, a peptide aptamer that specifically binds to the cancer marker Hsp90α and changes its fluorescence intensity upon binding was selected. In this selection, cDNA display technology was used. The cDNA display technology allows modification of the peptide backbone with a brominated compound (4-DMN-Br) of 4-DMN (4-N,N-dimethylamino-1, 8-naphthalimide), an environmentally responsive fluorescent substance, in an organic solvent before the selection round. Due to its sequence homology with Hsp90α, Hsp70 was used as a negative selection. In addition, human IgG and human albumin were also used as negative selection.

Hsp90α is a marker that oncologists are focusing on for early diagnosis. Some cancer markers are known to be specific for tissue types with calcitonin-thyroid cancer, human chorionic gonadotropin-germ cell tumors. Carcinoembryonic antigen (CEA) is expressed in various malignant tissues including breast, gastric and lung cancer. Molecular chaperones have been reported to be involved in tumor growth and metastasis, and Hsp90's potential as a non-specific tumor marker is being investigated. Hsp90 is known to play an important role in various cellular functions such as cell proliferation and differentiation, stress response, and protection of cellular proteins from stress such as hyperthermia.

In adopting the cDNA display technique, a DNA library containing 13 random amino acids with a cysteine at the 8th position was first designed. The thiol group of the cysteine can react with 4-DMN-Br as shown in Figure 4a to obtain fluorogenic peptide aptamer candidates. It has been reported that fluorogenic peptide aptamers have been selected for various target molecules. Many previous studies have employed 7-nitrobenzofurazan (NBD)-modified tRNA, a relatively small fluorescent dye, and ribosome display to prepare and select fluorogenic peptide aptamers. In this evaluation example, DMN, which is bulkier than NBD, was used instead of NBD because it is expected to obtain a higher signal-to-noise ratio than NBD, since its fluorescence intensity is lower than NBD in a hydrophilic environment. cDNA display technique was used to incorporate DMN into peptide aptamer candidates. The cDNA display used in this evaluation example prepares more stable cDNA-peptide ternary complexes and allows modification of peptides before selection.

　Previously, the inventors used ribosome display technology with tRNA-carrying environmentally sensitive fluorescent probes to select peptide aptamers whose fluorescence changed in response to various targets. The peptide aptamers were directly selected based on their binding ability and changed fluorescence in the presence of verotoxin.

After in vitro transcription, the mRNA was ligated to a short biotin segment puromycin-linker (SBS-Pu-linker). After in vitro translation, the peptide was transferred onto the puromycin site of the SBS-Pu-linker. Thus, the C-terminus of the peptide was fused to the cDNA encoding the peptide by covalent bonding, forming a cDNA display complex (Figure 4b).

The scheme for peptide selection using cDNA display used in this evaluation example is shown in Figure 4c.

　Below, we provide details of the cDNA display protocol used in this evaluation example.

Protocol 1: Ligation of mRNA and SBS linker Ligation of mRNA and SBS linker was carried out using the reaction system shown in Table 4 below. The reaction times for the reaction system shown in Table 4 were (1) 90°C for 2 minutes, (2) 70°C for 1 minute, (3) 50°C for 1 minute, and (4) stopped. After completion of the reaction, the mixture was kept at 25°C for 30 minutes and then on ice to obtain a ligation product of mRNA and SBS linker (SBS-linker-mRNA).

Protocol 2: Translation 15.5 μL of reconstituted cell-free translation solution (containing ΔRF1, ribosomes, and Rnase) was mixed with 10 μL of SBS-linker-mRNA and maintained at 37°C for 30 minutes. The reaction solution was then transferred to room temperature (20-26°C) for 12 minutes to enhance binding. A homemade pure system was used in this translation, which does not contain RF1.

Next, 0.6 μL of 0.5 M EDTA (pH 8.0, Rnase-free) was added to the reaction solution and incubated for 5 minutes.

Protocol 3: Preparation of DMN-modified peptide-cDNA Simultaneously with the translation in Protocol 2, 20 μL of Dynabeads Myone C1 (streptavidin, hereafter referred to as "beads C1") was washed twice with 200 μL of Solution A (100 mM NaOH, 50 mM NaCl) and then twice with 200 μL of Solution B (100 mM NaCl). After washing, 25 μL of the translation system prepared in Protocol 2 and 25 μL of 2× binding buffer (20 mM Tris-HCl (pH 8.0), 0.2 mM EDTA, 2 M NaCl, 0.2% Tween20) were added to the washed beads C1, and the mixture was rotated at 65 rpm at room temperature for 30 minutes. After that, the mixture was washed three times with 200 μL of 1× binding buffer and once with 100 μL of 1× ReverTraAce buffer (Toyobo).

Protocol 4: Reverse transcription reaction and binding with DMN 10 μL of 5x ReverTraAce buffer, 8 μL of 10 mM dnNTPs, 1 μL of ReverTraAce (100 U/μL), and 31 μL of water (RNase-free) were added to the prepared beads and mixed. Reverse transcription reaction was carried out by maintaining 25 μL of the mixture at 42°C for 30 minutes. Next, the beads were washed twice with 200 μL of 1x SBT (50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 0.5 M NaCl, 0.05% Tween20) and then twice with 200 μL of Wash buffer (50 mM Tris, 150 mM NaCl).

Next, 0.2μL of Tween20 (0.1%), 40μL of 5xDMN (1.5mM in DMF), 20μL of 10x reaction buffer (100mM Tris pH8, 1M NaCl, 2mM TCEP), and 140μL of Rnase, Dnase-free water were added to the beads and mixed. The mixture was divided into two tubes, and the tubes were rotated at 65 rpm for 1 hour at 25°C to carry out the DMN binding reaction. The beads were then washed twice with 200μL of Wash buffer and then twice with 200μL of 1xS.B.T.

Protocol 5: Affinity Selection After DMN binding, 31.5 μL of water (RNase-free), 10 μL of 5xSB (250 mM Tris-HCl (pH 7.6), 5 mM EDTA, 2.5 M NaCl), 5 μL of 0.1% BSA, 2.5 μL of Tween 20 (1%), and 1 μL of Rnase T1 (1000 U/μL) were added to the beads and mixed. The 50 μL mixture was kept at 37°C for 10 min. The supernatant was then collected.

5.1 Preparation of human Hsp70 and Hsp90 beads
The human Hsp70 and Hsp90 beads were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20) and then divided into four tubes.

(5.2 Preparation of human IgG beads)
The human IgG beads were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20) and then divided into four tubes.

5.3 Preparation of Human Albumin Beads
The human albumin beads were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20) and then divided into four tubes.

5.4 Addition of Hsp70 beads
5 μL of MyOne Carboxylic Acid beads, which are beads on which Hsp70 was immobilized in advance (Hsp70 beads), were added to the tube, and the tube was washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20), and then divided into five tubes. After adding the supernatant of 0096 (mRNA-peptide solution excised using RNAseT1) to one of the tubes, 85 μL of 1.33× selection buffer was added, and the mixture was incubated at 25°C for 5 minutes at 65 rpm, and the supernatant was collected. The same procedure was performed on the remaining four tubes containing Hsp70-immobilized beads.

The collected supernatant was exposed to IgG beads four times and then to human albumin beads four times to remove the DMN-modified peptide cDNA that bound to them. These steps correspond to negative selection, and the final supernatant was collected.

(5.5 Addition of HSP90α beads)
The beads on which HSP90α had been immobilized in advance (HSP90α-immobilized beads) were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20), and then mixed with the DMN-modified peptide cDNA that had undergone negative selection. The beads were then incubated at 60 rpm at 25°C for 30 minutes. The beads were then washed three times with 200 μL of 1× selection buffer (25°C).

Protocol 6: Elution After adding 40 μL of water to the HSP90α-immobilized beads, the beads were heated at 95° C. for 5 minutes to elute the DMN-modified peptide-cDNA. Next, 10 μL of 10×buffer (TAKARA), 8 μL of dNTP mix, 4 μL of Trap-Fwd-44 (5 μM), 4 μL of Rev-ScDNA3 (5 μM), 73 μL of water (RNase-free), and 1 μL of ExTaq enzyme (TAKARA) were added to 5 μL of the eluted template (i.e., DMN-modified peptide-cDNA) to prepare 100 μL of PCR solution, which was then subjected to PCR.

Of the 100 μL PCR solution, 2 μL was used for real-time PCR, and the remaining solution was used for PCR for the next round of selection.

Protocol 7: Purification of PCR products PCR products were purified using NucleoSpin® Gel and PCR Clean-up (Takara). RNA was synthesized using purified DNA. Specifically, 10 μL of purified DNA, 10 μL of RNase DNase-free water, 3.5 μL x 4 rNTPs, 10 μL of 5× buffer (Promega), 1 μL of RNase inhibitor, and 5 μL of Promega Enzyme Mix were mixed and kept at 37° C. overnight.

DNA was then removed by adding 2 μL of Turbo DNase (Thermo Fisher Scientific) and further incubating at 37°C for 30 min. RNA was then purified using an RNA purification kit (Zymo research) and protocols 1 to 7 above were repeated.

The conditions for each round are shown in Table 5.

The amino acid sequences selected by cDNA display are shown in Table 6. As a result of using cDNA display technology, three peptide aptamer candidates (peptides 1 to 3 in Table 6) with high affinity to Hsp90α were selected, and further research was conducted on the responsiveness of the change in fluorescence to increasing concentrations of Hsp90α.

[Evaluation Example 5] Fluorescence properties of peptide aptamers First, the fluorescence properties of peptides 1 to 3 selected in Evaluation Example 2 were examined. Peptide 2 showed a three-fold fluorescence enhancement in the presence of 6 μM of commercially available recombinant Hsp90α, while no significant enhancement was observed in Hsp70 (Figs. 5 and 6). However, peptide 1 showed similar fluorescence enhancement for Hsp70 and Hsp90α (Figs. 5 and 6). This is likely due to peptide 1 recognizing the common sequence portion of Hsp90α and Hsp70. Peptide 3 did not show fluorescence enhancement for Hsp90α and Hsp70 (Figs. 5 and 6). Since peptide 2 showed the desired properties, the specificity and selectivity of peptide 2 for Hsp90α were tested. SPR analysis of Hsp90α in the presence of various concentrations of peptide 2 was performed. As a result, the equilibrium dissociation constant (K _d ) was 3.13 ± 1.34 μM (Fig. 7).

To examine selectivity, Hsp90α was replaced with BSA, and peptide 2 showed no fluorescence enhancement against BSA even at a concentration of 8 μM, demonstrating the selectivity of peptide 2 for Hsp90α. Furthermore, the binding of peptide 2 to Hsp90α in the presence of 10% FBS was confirmed, and peptide 2 showed fluorescence enhancement against Hsp90α even in the presence of FBS. Because peptide 2 does not interact with FBS components, it can be added to cell culture media to test the behavior of Hsp90α within cells.

[Evaluation Example 6] Detection of Cellular Hsp90α Using Peptide 2 Next, the properties of peptide 2 at the cellular level were analyzed. Although peptide 2 can detect purified Hsp90α in solution, specific detection of cellular Hsp90α is a difficult task. To be suitable as an Hsp90α sensor, peptide 2 must specifically detect cellular Hsp90α. This requires high target selectivity and fluorescence that is stronger than the cellular autofluorescence. In this evaluation example, T98G cells, which are thermo-tolerant cells derived from glioma, were used. It has been reported that heat-shocked T98G cells produced a large amount of Hsp90α compared to other tumor cell lines (Kalamida et al.).

Figure 8 shows the results of confocal laser microscopy of HT-1080 cells and MCF-7 cells stained with peptide 2 and anti-Hsp90α antibody. The green fluorescence from DMN of peptide 2 colocalized with the red fluorescence from the anti-Hsp90α antibody around the cell membrane. This was thought to be because peptide 2 specifically recognizes Hsp90α. The bar in Figure 8 indicates 20 μm.

To investigate the behavior of Hsp90α in cells through comparison with anti-Hsp90α antibody, 98G cells were fixed and stained with anti-HSP90α antibody or peptide 2 under normal conditions and after 2 h of heat shock. Peptide 2 and anti-Hsp90α antibody were colocalized in the cytoplasm and peripheral regions of the cells. T98G under normal conditions showed higher fluorescence in the cytoplasm (Fig. 9A). However, heat-shocked T98G cells showed higher fluorescence intensity in the nuclear region than in the cytoplasm (Fig. 9A). Figure 9B shows the results of quantitative analysis of the total fluorescence of T98G cells stained with peptide 2 under heat-shocked (42°C) and normal (37°C) conditions. Quantitative analysis was performed by cytometric analysis.

Next, we tested the fluorescence generation of peptide 2 in T98G. Heat-shocked T98G cells showed higher fluorescence intensity than T98G cells under normal conditions (Figure 10, bar indicates 20 μm). And, similar to the monoclonal anti-Hsp90α antibody, the fluorescence intensity was high in the nuclear region. In T98G cells under normal conditions, fluorescence was observed throughout the cells. These results confirmed the specific target binding of peptide 2.

To quantitatively examine the difference in fluorescence intensity, cell images were analyzed by cytometry. Two-hour heat-shocked cells (42°C) and normal cells (37°C) were incubated with peptide 2, and the difference in fluorescence intensity was analyzed using image-based cell cytometry. As shown in Figure 9B, the fluorescence intensity was higher in heat-shocked T98G cells than in normal cells.

Hsp90α recognizes these functions together with various client proteins and participates in essential signal cascades in living cells. However, some client proteins of Hsp90α are also known to be relevant to cancer development by regulating tumor proliferation, adhesion, invasion, metastasis, angiogenesis and apoptosis (J. Wu, T. Liu, Z. Rios, Q. Mei, X. Lin, S. Cao, Heat shock proteins and cancer., Trends Pharmacol. Sci., 38, 226-256, 2017). Inhibition of Hsp90α has also been reported to suppress the expression of HIF-90α and NK-κB and induce epithelial-mesenchymal transition, inhibition of motility and invasiveness in colon cancer cells (G. P. Nagaraju, T. E. Long, W. Park, J. C. Landry, L. Taliaferro-Smith, A. B. Farris, R. Diaz, B. F. El-Rayes, Heat shock protein 90 promotes epithelial to mesenchymal transition, invasion, and migration in colorectal cancer., Mol. Carcinog. 54, 1147-11458, 2015). Overexpression of Hsp90α has also been reported in various cancers, including pancreatic, ovarian, breast, lung, and endometrial cancers. In addition, stress-induced isoforms of Hsp90α have been found primarily in the cytoplasm, and Hsp90α has also been reported to be secreted into the extracellular matrix via an unconventional exosome pathway. During stress, the production of Hsp90α is upregulated, and Hsp90α is translocated to the cell membrane and secreted or anchored to cell surface heparan sulfate proteoglycans. It has been reported that secreted Hsp90α plays an important role in activating matrix metalloproteinases, indirectly promoting the migration and invasion of cancer cells. It has also been reported that the plasma concentration of Hsp90α is higher in lung cancer patients than in normal individuals, and Hsp90α may be useful as a diagnostic biomarker for lung cancer. Therefore, a biosensor that can detect Hsp90α may be effective and useful in cancer prognosis or diagnosis.

Claims (7)

1. A method for producing a modified nucleic acid molecule-peptide complex, comprising introducing a functional molecule into a peptide portion of a nucleic acid molecule-peptide complex in which a nucleic acid molecule and a peptide encoded by the nucleic acid molecule are linked, the method comprising the steps of:
A method comprising a step of introducing a functional molecule into a reactive group of an amino acid residue constituting a peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent. The method according to claim 1, wherein the water-soluble polar organic solvent is an aprotic polar organic solvent. The method according to claim 1, wherein the functional molecule is a fluorescent molecule, an inhibitor molecule, or a ligand molecule. The method of claim 1, wherein the reactive group is a thiol group, an amino group, or a carboxyl group. The method of claim 1, wherein the reactive group is a bioorthogonal reactive group. The method according to claim 1, wherein the nucleic acid molecule-peptide complex is a nucleic acid molecule-peptide-ribosome complex to which a ribosome is further linked. The method according to any one of claims 1 to 6,
A nucleic acid display method or a ribosome display method, comprising a step of selecting a desired modified product from the modified products of the nucleic acid molecule-peptide complex.

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