WO2023188623A1

WO2023188623A1 - Linker and kit

Info

Publication number: WO2023188623A1
Application number: PCT/JP2022/047326
Authority: WO
Inventors: 宏紀安齋
Original assignee: 株式会社トクヤマ
Priority date: 2022-03-29
Filing date: 2022-12-22
Publication date: 2023-10-05

Abstract

Provided is a linker (100) including a first segment (10) and a second segment (20), wherein: a first hybridization site (11) included in the first segment (10) and a second hybridization site (21) included in the second segment (20) hybridize to form a double-stranded region; the first segment (10) includes a binding site (12) and a 5'-5' bond (13) between nucleotides; and the second segment (20) includes a polynucleotide single-stranded region (22).

Description

linkers and kits

The present invention relates to a linker and a kit.

As one of the techniques for searching for novel functional polypeptides, the cDNA display method, which is a technique that associates a polypeptide (phenotype) with a polynucleotide (genotype) that preserves the sequence information of the polypeptide, is known. There is. In the cDNA display method, a linker is used that connects a polypeptide (phenotype), mRNA encoding the polypeptide, and cDNA (genotype) obtained by reverse transcription of the mRNA.

As such a linker, for example, Patent Document 1 describes a main chain comprising a solid-phase binding site, a solid-phase cleavage site, a side chain linking site, a high-speed photocrosslinking site, and a reverse transcription initiation region, a fluorescent label, and a protein binding site. and a side chain with a linkage formation site.

International Publication No. 2016/159211

There is a high demand for linkers that connect proteins or peptides and polynucleotides that can be used to search for functional polypeptides, and new linkers are needed.

One aspect of the present invention aims to provide a novel linker that connects a protein or peptide and a polynucleotide, and its related technology.

In order to solve the above problems, a linker according to one aspect of the present invention is a linker comprising a first segment and a second segment, and has a polynucleotide sequence contained in the first segment. The first hybridization site and the second hybridization site having a polynucleotide sequence contained in the second segment hybridize to form a double-stranded region, and the first hybridization site is bonded to the 5' end side as viewed from the first hybridization site, and is located between a binding site that binds to a protein or peptide, and the first hybridization site and the binding site. , a 5'-5' bond between nucleotides, and the second segment includes a single-stranded polynucleotide region on the 3' end side as viewed from the second hybridization site.

A kit according to one aspect of the present invention is a kit for producing a linker including a first segment and a second segment, the kit including the first segment and the second segment. The first hybridization site having a polynucleotide sequence contained in the first segment and the second hybridization site having a polynucleotide sequence contained in the second segment are capable of hybridizing. the first segment has a sequence capable of forming a double-stranded region, the first segment is bound to the 5' end side as viewed from the first hybridization site, and a binding site that binds to a protein or peptide; a 5'-5' bond between nucleotides located between the first hybridization site and the binding site, and the second segment is located between the first hybridization site and the binding site; Contains a single-stranded polynucleotide region at the 3' end.

According to one aspect of the present invention, a novel linker that connects a protein or peptide and a polynucleotide, and its related technology can be provided.

FIG. 1 is a schematic diagram illustrating the configuration of a linker according to an embodiment of the present invention. 2 is a schematic diagram illustrating the configuration of a linker that is a modification of the linker shown in FIG. 1. FIG. FIG. 1 is a schematic diagram illustrating the configuration of a composite body according to an embodiment of the present invention. FIG. 2 is a schematic diagram illustrating the configuration of the linker manufacturing method shown in FIG. 1. FIG. FIG. 2 is a schematic diagram illustrating the configuration of a cDNA display method using the linker shown in FIG. 1. FIG. 1 is a schematic diagram showing the structure of the mRNA-linker complex of Example 1 of the present invention. 1 is a gel electrophoresis photograph showing the verification results of the formation of an mRNA-linker complex in Example 1 of the present invention. These are gel electrophoresis photographs showing the results of the cDNA display method when the linker of Reference Example 1 of the present invention was used (left figure) and when the linker of Example 1 of the present invention was used (right figure).

One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each configuration described below, and various changes can be made within the scope of the claims. Further, 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. Further, in this specification, "A to B" indicates that the number is greater than or equal to A and less than or equal to B, unless otherwise specified.

As used herein, "linker" refers to mRNA-linker complex, linker-protein/peptide complex, mRNA-linker-protein/peptide complex, mRNA-linker-cDNA complex, and mRNA-linker-protein/peptide complex. - refers to a compound capable of binding with mRNA, protein/peptide, cDNA, etc. to form at least one complex selected from the group consisting of: -cDNA complex.

As used herein, the term "polynucleotide sequence" refers to a base sequence of a constituent unit in a polynucleotide, and here, the term "polynucleotide" refers to a linear polymer containing nucleotides as constituent units. As used herein, expressions such as "X has a polynucleotide sequence" and "X has a polynucleotide sequence" are intended to mean that the structure of X includes a polynucleotide having a specific base sequence. Ru. In a polynucleotide sequence, each base may be a natural base such as A (adenine), T (thymine), G (guanine), C (cytosine) and U (uracil), or derivative bases derived from natural bases. It may also be an artificially synthesized synthetic base. In a polynucleotide sequence, each nucleotide is independently selected from ribonucleotides, deoxyribonucleotides, and derivatives thereof. Polynucleotides may include structural units derived from sources other than nucleotides. Examples of structural units derived from other than nucleotides include synthetic amino acids such as CNV-D phosphoramidites. The polynucleotide preferably contains 50% by mass or more, more preferably 70% by mass or more, and even more preferably 90% by mass or more of a portion having nucleotides as a constituent unit, based on the total mass of the polynucleotide. .

As used herein, "hybridization" means that the single-stranded regions of two polynucleotides form a double-stranded region through hydrogen bonding between complementary base pairs. The complementarity between single-stranded regions is preferably 20% or more, more preferably 40% or more, and even more preferably 45% or more.

As used herein, "protein/peptide" means a protein or a peptide. As used herein, each of protein and peptide has the meaning commonly understood in the art. Further, in this specification, any one or both of a protein and a peptide may be referred to as a "polypeptide."

[Linker]
One aspect of the present invention relates to a linker. A linker according to one aspect of the present invention is a linker comprising a first segment and a second segment, wherein a first hybridization site having a polynucleotide sequence contained in the first segment and a second The second hybridization site having a polynucleotide sequence contained in the segment hybridizes to form a double-stranded region, and the first segment is 5' as viewed from the first hybridization site. a binding site that is attached to the terminal side and that binds to a protein or peptide, and a 5'-5' bond between nucleotides that is located between the first hybridization site and the binding site, The second segment includes a single-stranded region of the polynucleotide at the 3' end as viewed from the second hybridization site. According to such a configuration, a novel linker that connects a protein or peptide and a polynucleotide can be provided.

A linker according to one aspect of the present invention includes a first segment and a second segment. In the linker, the same main chain may contain both the first segment and the second segment, and each of the two main chains may contain each of the first segment and the second segment. It's okay. When each of the two main chains contains the first segment and the second segment, each of the first segment and the second segment can be synthesized at low cost, and therefore the linker can be synthesized at low cost. can do. When both the first segment and the second segment are included in the same main chain, the 3' end of the first segment and the 5' end of the second segment are directly bonded. They may also be connected indirectly through other members.

(1st segment)
The first segment is a segment that has the property of forming a bond between the linker and the protein/peptide in a complex that the linker can form. In one aspect of the invention, the first segment includes a first hybridization site, a binding site, and a 5'-5' bond. Further, the first segment may further include at least one selected from the group consisting of a first spacer site, a second spacer site, and a fluorescent label site.

(First hybridization site)
The first hybridization site is a site located on the backbone of the first segment and has a polynucleotide sequence. The first hybridization site hybridizes with the second hybridization site described below in the linker to form a double-stranded region. The polynucleotide sequence possessed by the first hybridization site can be appropriately selected by a person skilled in the art, as long as the first hybridization site and the second hybridization site can hybridize to form a double-stranded region. can be done.

At the first hybridization site, the length of the polynucleotide sequence is preferably 5 nucleotides or more, more preferably 10 nucleotides or more, and even more preferably 15 nucleotides or more. When the length of the polynucleotide sequence is within such a numerical range, the efficiency of forming a double-stranded region by the first hybridization site and the second hybridization site is improved. Further, at the first hybridization site, the length of the polynucleotide sequence is preferably 90 nucleotides or less, more preferably 60 nucleotides or less, and even more preferably 40 nucleotides or less. When the length of the polynucleotide sequence is within such a numerical range, the linker can be manufactured at low cost.

The G/C content of the first hybridization site is preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and even more preferably 50% or more. Even more preferred. When the G/C content of the first hybridization site is within such a numerical range, the efficiency of forming a double-stranded region by the first hybridization site and the second hybridization site is improved. Further, the G/C content of the first hybridization site is preferably 99% or less, more preferably 98% or less, even more preferably 90% or less, and even more preferably 80% or less. Even more preferred. By having the G/C content of the first hybridization site within such a numerical range, nonspecific adsorption by the first hybridization site is reduced. In this specification, "G/C content" refers to the amount of G (guanine) bases, C (cytosine) bases, and these bases contained in a polynucleotide sequence relative to the total number of all bases contained in the polynucleotide sequence. means the percentage of the total number of derivative bases derived from.

(Binding site)
The binding site is bound to the 5' end side as viewed from the first hybridization site, and is a site that binds to a protein or peptide. The binding site is a site that has the property of binding to the C-terminus of a protein or peptide that is being elongated by translation of mRNA by ribosomes. Preferably, the binding site has the property of specifically binding to the protein/peptide, for example under conditions that allow ribosomes to translate mRNA.

The binding site can have any structure as long as it has the above properties. Examples of the binding site include puromycin, 3'-N-aminoacylpuromycin aminonucleoside (PANS-amino acid), 3'-N-aminoacyladenosine aminonucleoside (AANS-amino acid), ribocytidyl puromycin (rCpPur), Examples include aminoacyl-tRNA 3'-terminal analogs such as deoxydylpuromycin (dCpPur) and deoxyuridylpuromycin (dUpPur).

Examples of PANS-amino acids include PANS-Gly whose amino acid portion is glycine, PANS-Val whose amino acid portion is valine, PANS-Ala whose amino acid portion is alanine, and all amino acids. PANS-amino acid mixture. Examples of AANS-amino acids include AANS-Gly where the amino acid part is glycine, AANS-Val where the amino acid part is valine, AANS-Ala where the amino acid part is alanine, and the amino acid part corresponds to all amino acids. AANS-amino acid mixture.

The binding site is at least one selected from the group consisting of puromycin, 3'-N-aminoacylpuromycin aminonucleoside (PANS-amino acid), and 3'-N-aminoacyladenosine aminonucleoside (AANS-amino acid). is preferred. A binding site with such a configuration has a high binding capacity for proteins and peptides, and therefore the linker can form the desired complex with high efficiency.

The binding site may be located on the 5' end side in the first segment as viewed from the first hybridization site. The binding site may be located, for example, at the free end of the first segment and may be further bound to any other member.

(5'-5' bond)
A 5'-5' bond is a 5'-5' bond between nucleotides located between the first hybridization site and the binding site. When the linker according to one aspect of the present invention has such a configuration, the 3' end-(first hybridization site)-(5'-5' bond)- (Binding site) Three members are located linearly in the order of -3' end. Therefore, in one aspect of the present invention, the first hybridization site, 5'-5' bond and binding site included in the first segment have a single-stranded structure, so that the first segment and It is easy to manufacture a linker comprising this. Furthermore, since the binding site is located at the 3' end, an aminoacyl-tRNA 3' end analog containing puromycin, which has excellent binding ability to proteins/peptides, can be employed as the structure of the binding site. Therefore, the linker comprising the first segment can form a complex with the protein/peptide with high efficiency.

A 5'-5' bond is a bond between a 5' carbon atom of a nucleotide and a 5' carbon atom of another nucleotide through a phosphodiester bond. The two nucleotides forming the 5'-5' bond can be appropriately selected by those skilled in the art, and may be different types of nucleotides or the same type of nucleotides. The two nucleotides forming the 5'-5' bond are preferably a pair of T and T. According to such a configuration, non-specific hybridization within the linker can be reduced.

(First spacer part)
The first spacer site is located between the first hybridization site and the 5'-5' bond and has a polyA _m sequence (where m represents an integer of 2 or more and 50 or less). It is. As used herein, the term "polyA _m sequence" refers to a polynucleotide sequence consisting of m consecutive A's (adenylic acids). The inclusion of the first spacer site in the first segment favorably adjusts the distance between the binding site and the reactive site of the ribosome that translates the mRNA, allowing the linker to complex with the protein/peptide with high efficiency. can be formed.

In the first spacer portion, m is preferably 2 or more, more preferably 6 or more, and even more preferably 8 or more. By having m within such a numerical range, the linker can form a complex with the protein/peptide with high efficiency. In the first spacer region, m is preferably 40 or less, more preferably 30 or less, and even more preferably 20 or less. By having m within such a numerical range, the linker can form a complex with the protein/peptide with high efficiency.

Furthermore, the difference between m and the number of nucleotides contained in the polynucleotide of the single-stranded region described below is preferably 20 or less, more preferably 10 or less, and even more preferably 0. According to such a configuration, the positional relationship between the binding site and the ribosome that has reached the end point of mRNA translation is suitably adjusted, so that the linker forms a complex with the protein/peptide with high efficiency. be able to.

The first spacer site may be directly bonded to the first hybridization site, or may be indirectly bonded to the first hybridization site via any other member.

The first spacer site and the 5'-5' bond may or may not be adjacent to each other. In other words, the A at the 5' end of the first spacer site may or may not be one of the two nucleotides forming a 5'-5' bond.

In the linker according to one aspect of the present invention, the first spacer site is not limited to a site having a polyA _m sequence. The first spacer region may be, for example, a region having a poly G _m sequence, a poly C _m sequence, a poly T _m sequence, or a poly U _m sequence, and does not or hardly hybridizes with the single-stranded region described below. A site having a sequence that does not occur can be suitably used as the first spacer site.

(Second spacer part)
The second spacer moiety is located between the binding site and the 5'-5' bond and is (PEG) _n , where PEG represents polyethylene glycol (-[CH ₂ CH ₂ O]-) and n represents an integer from 1 to 20). The inclusion of the second spacer site in the first segment favorably adjusts the distance between the binding site and the reactive site of the ribosome that translates the mRNA, allowing the linker to complex with proteins/peptides with high efficiency. can be formed.

In the second spacer portion, n is preferably 1 or more, more preferably 2 or more, and even more preferably 3 or more. By having n within such a numerical range, the linker can form a complex with the protein/peptide with high efficiency. In the second spacer region, n is preferably 18 or less, more preferably 10 or less, and even more preferably 6 or less. By having n within such a numerical range, the linker can form a complex with the protein/peptide with high efficiency.

The second spacer region may further include other members in addition to (PEG) _n . Other members included in the second spacer site include, for example, arbitrary nucleotides. The second spacer portion may have other members at both ends of (PEG) _n , or may have other members at only one end of (PEG) _n . Other members include, for example, nucleotides and polynucleotides.

In the linker according to one aspect of the present invention, the second spacer moiety is not limited to a moiety having (PEG) _n . The second spacer moiety may be, for example, a moiety with a biocompatible polymer in place of (PEG) _n . Examples of biocompatible polymers include water-soluble polymers such as polypropylene glycol and polyvinylpyrrolidone, and polysaccharides.

(Total length of first spacer part and second spacer part)
In embodiments where the first segment includes at least one of a first spacer site and a second spacer site, by appropriately selecting the sum of the lengths of the first spacer site and the second spacer site, The efficiency of complex formation between linker and protein/peptide can be adjusted.

(fluorescent labeling site)
The fluorescent labeling site is a site that has properties as a probe for detecting the linker or the complex formed by the linker. The inclusion of the fluorescent label site in the first segment allows the linker and the linker-containing complex to be detected by fluorescence observation.

The fluorescent labeling site may have any structure as long as it has the above properties. Examples of fluorescent labeling sites include FAM (carboxyfluorescein), JOE (6-carboxy-4',5'-dichloro2',7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), and TET (tetrachlorofluorescein). , HEX (5'-hexachloro-fluorescein-CE phosphoramidite), Cy3, Cy5, Alexa568, Alexa647, phycobiliprotein, rare earth metal chelate, dansyl chloride, and tetramethylrhodamine isothiocyanate.

The position of the fluorescent labeling site can be appropriately selected by those skilled in the art as long as the linker can form a complex. The fluorescent label moiety may be located on a side chain attached to any position of the main chain of the first segment, including, for example, the first hybridization site, the 5'-5' bond, and the binding site. When the fluorescent label site is located on a side chain, the positions to which the side chain binds include, for example, the first hybridization site, the first spacer site, and the second spacer site.

(Second segment)
The second segment may be a segment that has the property of forming a bond between the linker and at least one of mRNA and cDNA in a complex that the linker is capable of forming. In one aspect of the invention, the second segment includes a second hybridization site and a single-stranded region. The second segment may also include a reverse transcription initiation site.

(Second hybridization site)
The second hybridization site is a site located on the backbone of the second segment and has a polynucleotide sequence. The second hybridization site hybridizes with the first hybridization site described above in the linker to form a double-stranded region. The polynucleotide sequence possessed by the second hybridization site can be appropriately selected by those skilled in the art, as long as the first hybridization site and the second hybridization site can hybridize to form a double-stranded region. can be done.

In the second hybridization site, the length of the polynucleotide sequence is preferably 5 nucleotides or more, more preferably 10 nucleotides or more, and even more preferably 15 nucleotides or more. When the length of the polynucleotide sequence is within such a numerical range, the efficiency of forming a double-stranded region by the first hybridization site and the second hybridization site is improved. Further, in the second hybridization site, the length of the polynucleotide sequence is preferably 90 nucleotides or less, more preferably 60 nucleotides or less, and even more preferably 40 nucleotides or less. When the length of the polynucleotide sequence is within such a numerical range, the linker can be manufactured at low cost.

(single-stranded region)
The single-stranded region is a polynucleotide located on the 3' end side when viewed from the second hybridization site.

The single-stranded region can be a region that hybridizes with mRNA. Here, mRNA that hybridizes with the single-stranded region may be subject to genetic engineering techniques such as screening, qualitative analysis, and quantitative analysis. The structure of the mRNA may be appropriately selected by the user of the linker depending on the polynucleotide sequence of the single-stranded region of the linker. Alternatively, the user may select the polynucleotide sequence of the single-stranded region depending on the polynucleotide sequence of the target mRNA.

In the single-stranded region, the length of the polynucleotide sequence is preferably 5 nucleotides or more, more preferably 6 nucleotides or more, and even more preferably 8 nucleotides or more. By having the length of the polynucleotide sequence within such a numerical range, the single-stranded region can hybridize with mRNA with high efficiency. Furthermore, in the single-stranded region, the length of the polynucleotide sequence is preferably 90 nucleotides or less, more preferably 60 nucleotides or less, and even more preferably 40 nucleotides or less. When the length of the polynucleotide sequence is within such a numerical range, the linker can be manufactured at low cost.

Although not limited to this, it is preferred that the polynucleotide in the single-stranded region does not match the polynucleotide contained in the second hybridization site. According to such a configuration, it is possible to reduce the occurrence of hybridization between the single-stranded region and the first hybridization site that is complementary to the second hybridization site.

The single-stranded region may be directly bound to the second hybridization site, or may be indirectly bound to the second hybridization site via any other member.

The single-stranded region may include a site that can form a crosslinked structure with a part of mRNA. According to such a configuration, in the mRNA-linker complex, the bond between the linker and mRNA is stabilized, so that a stable complex can be realized. Examples of sites that can form a cross-linked structure with a portion of mRNA include photoreactive nucleotides.

Note that when a linker is used, the single-stranded region can exhibit a desired function, such as the function of hybridizing with mRNA, and form a double-stranded region. Single-stranded regions may also be protected when no linker is used, by methods known to those skilled in the art.

(reverse transcription start site)
The reverse transcription initiation site is a site for initiating synthesis of cDNA complementary to mRNA hybridized to the single-stranded region. The reverse transcription initiation site has a polynucleotide sequence and is located within the single-stranded region, eg, at the 3' end of the single-stranded region.

The polynucleotide sequence of the reverse transcription initiation site can be appropriately selected by those skilled in the art. Examples of the polynucleotide sequence possessed by the reverse transcription initiation site include a 5'-TCCT-3' sequence.

(Configuration between first segment and second segment)
In one aspect of the present invention, the first hybridization site included in the first segment and the second hybridization site included in the second segment hybridize to form a double-stranded region. There is. According to such a configuration, the linker can form a complex with at least one selected from the group consisting of mRNA, cDNA, protein, and peptide.

In the linker, the first segment has the property of forming a bond between the linker and the protein or peptide, and the second segment has the property of forming a bond between the linker and at least one of mRNA and cDNA. have a property. Therefore, a user using a linker can design each of the first segment and the second segment according to conditions such as desired binding characteristics and detection method. Therefore, the linker can be applied to a wide range of applications by designing each of the first segment and the second segment according to the user's needs.

Further, in the linker, the conformation between the first segment and the second segment is stabilized by hybridization between at least the first hybridization site and the second hybridization site. . Therefore, compared to prior art linkers in which the conformation is stabilized by introducing side chains to the segment using a cross-linking agent, the linker according to one aspect of the present invention uses a cross-linking agent to stabilize the conformation. It can be produced without any treatment (for example, crosslinking reaction, reduction reaction of the crosslinking site, etc.). Therefore, the linker according to one aspect of the present invention can be produced in a shorter time.

Furthermore, the first segment and the second segment included in the linker may each be single-stranded. In such a case, both the first segment and the second segment are easy to manufacture. The linker itself is also easy to manufacture, since the linker can be manufactured by hybridizing the first segment and the second segment to each other.

In the double-stranded region, a part of the first hybridization site and a part of the second hybridization site may be linked to each other by a covalent bond. According to such a configuration, the double-stranded region is more stabilized, and therefore the distance between the binding site included in the first segment and the single-stranded region included in the second segment is more stabilized. Therefore, the ability of the linker to form a complex is further improved.

The mode of covalent bonding between the first hybridization site and the second hybridization site can be appropriately selected by those skilled in the art. Examples of the mode of covalent bonding include photocrosslinking formed by photoreactive bases, click chemistry, and crosslinking reactions such as Diels-Alder reactions.

Examples of photoreactive bases include 3-cyanovinylcarbazole (also referred to as ^cnv K). 3-cyanovinylcarbal is, for example, CNV-K phosphoramidite (5'-O-(4,4'-dimethoxytrityl)-1'-(3-cyanovinylcarbazol-9-yl)-2'- deoxy-β-D-ribofuranosyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) and CNV-D phosphoramidite (3-O-(4,4'-dimethoxy trityl)-2-N-(N-carboxy-3-cyanovinylcarbazole)-D-threonin-1-yl-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), etc. can be introduced into a polynucleotide sequence by using a 3-cyanovinylcarbazole compound. The photoreactive base may be contained in either the first hybridization site or the second hybridization site, or in both.

In the double-stranded region, the base adjacent to the 3' end of the A/G purine ring base complementary to 3-cyanovinylcarbazole is preferably a T/C/U pyrimidine ring base. According to such a configuration, 3-cyanovinylcarbazole not only hybridizes with the complementary purine ring base, but also cross-links with the adjacent base at the 3' end of the purine ring base with high efficiency. can be formed.

The photoreactive base is preferably located in the middle region of the double-stranded region. Although not limited to this, it is preferred that the photoreactive base is two or more bases away from each of the 3' and 5' ends of the first hybridization site or the second hybridization site. According to such a configuration, the efficiency of forming photocrosslinks by the photoreactive base is further improved.

The number of covalent bonds between the first hybridization site and the second hybridization site may be 0 or 1, or may be 2 or more, and may be appropriately selected by those skilled in the art.

(Other members in the linker)
In one aspect of the present invention, the linker may include other members than those described above. Other members that the linker may include include, for example, a third segment.

The third segment connects the 3' end of the first segment and the 5' end of the second segment so that the same single main chain contains both the first segment and the second segment. It can be a segment to be connected. The third segment may be a polynucleotide having a hairpin structure, and may include two cleavage sites positioned to sandwich the hairpin structure. The third segment may also include a solid phase binding site within the hairpin structure.

The two cleavage sites are sites that can be selectively cleaved in order to separate the third segment from the linker at a desired timing in the method of using the linker. The cleavage site may be at least one selected from the group consisting of deoxyinosine, riboG, and ribopyrimidine, but is not limited thereto.

The solid phase binding site is a site that binds to a solid phase in order to immobilize the linker on a solid phase, such as a substrate or a carrier, in the method of using the linker. The solid phase binding site can be appropriately selected by those skilled in the art depending on the solid phase used. As the solid phase binding site, for example, a compound selected from the group consisting of biotin, streptavidin, alkynes, azides by click chemistry, amino groups, N-hydroxysuccinimide esters (NHS), SH groups, and Au; Examples include a site consisting of polyA bound to.

(Linker according to one embodiment of the present invention)
A linker 100 according to an embodiment of the present invention will be described with reference to FIG. FIG. 1 is a schematic diagram illustrating the configuration of a linker 100 according to an embodiment of the present invention.

As shown in FIG. 1, the linker 100 includes a first segment 10 and a second segment 20. The linker 100 is a linker that can be used, for example, in a phenotype-genotype mapping method such as a cDNA display method.

In this embodiment, the first segment 10 includes a first hybridization site 11, a binding site 12, a 5'-5' bond 13, a first spacer site 14, and a second spacer site 15. , and a fluorescent labeling site 16. The first hybridization site 11 has the polynucleotide sequence set forth in SEQ ID NO:1. Binding site 12 is puromycin. The 5'-5' bond 13 is a 5'-5' bond between A at the 5' end of the first spacer site 14 and T at the 5' end of the second spacer site 15. The first spacer site 14 is a site having the _polyA12 sequence set forth in SEQ ID NO:2. The second spacer site 15 is a site having the sequence 3'-CC(PEG) ₃ CTCT ^* CT-5'. Fluorescent labeling moiety 16 is FITC bound to T ^* of second spacer moiety 15 as a side chain.

In this embodiment, the second segment 20 includes a second hybridization site 21 and a single-stranded region 22. The second segment 20 also includes a reverse transcription initiation site 23 at the 3' end of the single-stranded region 22. The second hybridization site 21 has the polynucleotide sequence set forth in SEQ ID NO:3. Single-stranded region 22 is a site having the polynucleotide sequence set forth in SEQ ID NO: 4. The reverse transcription start site 23 is TCCT at the 3' end of the single-stranded region 22.

The first hybridization site 11 and the second hybridization site 21 hybridize to form a double-stranded region. Furthermore, in the double-stranded region, ^cnv K contained in the first hybridization site 11 and T contained in the second hybridization site 21 are linked to each other by a covalent bond CB1.

A linker 101, which is a modification of the linker 100, will be described with reference to FIG. 2. FIG. 2 is a schematic diagram illustrating the configuration of a linker 101 that is a modification of the linker 100 shown in FIG.

As shown in FIG. 2, the linker 101 includes a first segment 10, a second segment 20, and a third segment 30. The linker 101 is a linker that can be used, for example, in a phenotype-genotype mapping method such as a cDNA display method, in particular for solid phase synthesis. The first segment 10 and the second segment 20 have the same functions and configurations as those included in the linker 100, so a detailed description thereof will be omitted.

In this embodiment, the third segment 30 includes

cleavage sites

31 and 32 and a solid phase binding site 33. Each of the

cleavage sites

31 and 32 is riboG. The solid phase binding site is biotin.

In this embodiment, the 3' end of the first segment 10 and the 5' end of the second segment 20 are indirectly coupled via the third segment 30, thereby The segment, the second segment, and the third segment share one main chain.

[First complex]
One aspect of the present invention relates to composites. A complex according to one aspect of the present invention is an mRNA that hybridizes with a linker according to one aspect of the present invention and a single-stranded region included in a second segment provided by the linker to form a double-stranded region. and. According to such a configuration, an mRNA-linker complex containing a novel linker can be realized. Hereinafter, in this specification, the complex according to this aspect may be referred to as a "first complex."

In the complex according to one embodiment of the present invention, part of the single-stranded region and part of the mRNA may be linked to each other by a covalent bond. According to such a configuration, in the mRNA-linker complex, the bond between the linker and mRNA is stabilized, so that a stable complex can be realized. Examples of the mode of covalent bonding include photocrosslinking formed by photoreactive nucleotides, click chemistry, and crosslinking reactions such as Diels-Alder reactions.

The complex according to one embodiment of the present invention may further include a protein or peptide bound to a binding site included in the first segment. The protein or peptide bound to the binding site can be a protein or peptide encoded by the mRNA included in the complex. According to such a configuration, it is possible to realize a complex that can be used to perform a method of associating a phenotype (protein or peptide) with a genotype (mRNA), such as an mRNA display method.

The complex according to one embodiment of the present invention may further include cDNA that is bound to the 3' end of the second segment and is complementary to the mRNA. cDNA can be synthesized by reverse transcribing mRNA hybridizing to single-stranded regions. According to such a configuration, it is possible to realize a complex that can be used for performing a method of associating a phenotype (protein or peptide) with a genotype (cDNA), such as a cDNA display method.

(Composite according to one embodiment of the present invention)
A composite body 200 according to an embodiment of the present invention will be described with reference to FIG. 3. FIG. 3 is a schematic diagram illustrating the configuration of a composite body 200 according to an embodiment of the present invention.

As shown in FIG. 3, the complex 200 includes a linker comprising a first segment 10 and a second segment 20, mRNA 40, polypeptide 50, and cDNA 60. A portion of the second segment 20 and a portion of the mRNA 40 are linked to each other by a covalent bond CB2.

[Method for manufacturing linker]
One aspect of the present invention relates to a method for manufacturing a linker. A method for producing a linker according to one embodiment of the present invention is a method for producing a linker according to one embodiment of the present invention, and includes a hybridization step. Moreover, the method for producing a linker may further include a purification step and a chemical modification step.

(Hybridization process)
In the hybridization step, the first segment and the second segment are subjected to an annealing treatment, whereby the first hybridization site contained in the first segment and the second hybridization site contained in the second segment are combined. This is the step of hybridizing with the target region to form a double-stranded region.

Each of the first segment and the second segment may be manufactured by manufacturing methods known to those skilled in the art. The production of each of the first segment and the second segment may be outsourced to, for example, a polynucleotide synthesis company.

The annealing treatment can be performed by methods known to those skilled in the art for annealing polynucleotides. In the annealing process, for example, a solution containing the first segment and the second segment is heated at 60° C. to 100° C. for 2 minutes to 60 minutes using a heating device such as a heat block, an aluminum block, or a water bath. This can be carried out by allowing the solution to stand at room temperature for 2 to 60 minutes to gently lower the temperature of the solution, and further cooling to -5°C to 10°C. In the annealing process, for example, the solution containing the first segment and the second segment is heated at 90°C for 5 minutes on an aluminum block, then at 70°C for 5 minutes on an aluminum block, and finally at room temperature. It is preferable to carry out the test by allowing the test to stand still for 10 minutes, and then cooling it by placing it on ice.

In the annealing treatment, the solution containing the first segment and the second segment may further contain at least one selected from the group consisting of an inorganic salt, a salt of an organic compound, and a pH buffer.

In the desired linker, if a portion of the first hybridization site and a portion of the second hybridization site are linked to each other by a covalent bond, the hybridization step forms the covalent bond. It may also include processing for If the covalent bond is photocrosslinking, the treatment to form the covalent bond may be UV irradiation. The duration of UV irradiation can be, for example, 0.5 minutes to 5 minutes. In the hybridization step, the annealing treatment and the treatment for forming a covalent bond may be performed simultaneously or sequentially.

(Method for manufacturing a linker according to an embodiment of the present invention)
A linker manufacturing method S1 according to an embodiment of the present invention will be described with reference to FIG. 4. FIG. 4 is a schematic diagram illustrating the configuration of the method S1 for manufacturing the linker 100 shown in FIG. 1.

As shown in FIG. 4, the method S1 for manufacturing the linker 100 includes a hybridization step S11. In the hybridization step, the first segment 10 and the second segment 20 are subjected to an annealing treatment and a UV irradiation treatment to produce a linker 100 containing a covalent bond CB1 in the double-stranded region.

[Uses of linker]
One aspect of the present invention relates to the use of linkers. Although not limited to, linkers according to one aspect of the present invention can be used in functional polypeptide search techniques known to those skilled in the art. Techniques for searching for functional polypeptides include, for example, phenotype-genotype mapping methods such as cDNA display methods and mRNA display methods.

By replacing a conventional linker with a linker according to one embodiment of the present invention, the linker can be applied to a wide range of applications without dedicated optimization. Therefore, users can use the linker without laborious optimization.

(Application of linker according to one embodiment of the present invention)
Applications of the linker according to one embodiment of the present invention will be described with reference to FIG. 5. FIG. 5 is a schematic diagram illustrating the configuration of cDNA display method S2 using the linker 100 shown in FIG. 1.

As shown in FIG. 5, the cDNA display method S2 includes a hybridization step S21, a translation step S22, and a reverse transcription step S23.

The hybridization step S21 is a step of forming an mRNA-linker complex 202 by hybridizing the single-stranded region 22 included in the linker 100 and the mRNA 40. In the hybridization step, linker 100 and mRNA 40 are subjected to annealing treatment and UV irradiation treatment to form mRNA-linker complex 202 containing covalent bond CB2.

The translation step S22 is a step of translating the mRNA 40 and binding the generated polypeptide 50 to the binding site 12 included in the linker 100 to form an mRNA-linker-polypeptide complex 203. In the translation step S22, translation of mRNA 40 can be performed by a method known to those skilled in the art. Although not limited to this, the generated polypeptide 50 may have its C-terminus bound to the binding site 12, thereby forming an mRNA-linker-polypeptide complex 203.

The reverse transcription step S23 is a step in which cDNA 60 complementary to mRNA 40 is synthesized by reverse transcription of mRNA 40, and an mRNA-linker-polypeptide-cDNA complex 200 is formed. In the reverse transcription step S23, reverse transcription of mRNA 40 can be performed by a method known to those skilled in the art. Further, the reverse transcription step S23 may include a purification treatment as appropriate.

〔kit〕
One aspect of the invention relates to a kit. A kit according to one aspect of the present invention is a kit for manufacturing a linker including a first segment and a second segment, the kit including the first segment and the second segment, and the kit includes the first segment and the second segment. A first hybridization site containing a polynucleotide sequence contained in one segment and a second hybridization site containing a polynucleotide sequence contained in a second segment hybridize to form a double-stranded region. The first segment has a sequence capable of forming a protein or peptide, and the first segment is bonded to the 5' end side when viewed from the first hybridization site, and has a binding site that binds to a protein or peptide and a first hybridization site. a 5'-5' bond between the nucleotides located between the second segment and the second hybridization site; Contains areas. According to such a configuration, a kit for producing a novel linker that connects a protein or peptide and a polynucleotide can be realized.

Each of the first segment and the second segment included in the kit according to an embodiment of the present invention has the same function and configuration as the first segment and the second segment included in the linker according to an embodiment of the present invention. Therefore, detailed explanation thereof will be omitted. In addition, in the kit, the first hybridization site included in the first segment and the second hybridization site included in the second segment have sequences that can hybridize to form a double-stranded region. It is enough if you have it. In other words, in the kit, the first hybridization site and the second hybridization site do not need to form a double-stranded region.

The kit may include a container in which both the first segment and the second segment are enclosed, or may include a container in which each is enclosed separately.

The kit may include a solvent for dissolving at least one of the first segment and the second segment, or materials for preparing the solvent. Examples of the solvent include: water; alcohols such as methanol, ethanol and mercaptoethanol; and dimethylacetamide and dimethylformamide. The solvent may be encapsulated in a container in which the first segment and the second segment are encapsulated, so that the first segment and the second segment are dissolved in a solution of the first segment and the second segment. The segment may be enclosed in a container.

The kit may include members for carrying out the uses of the linker described above. Examples of such members include ribosomes and enzymes such as reverse transcriptase, salts for adjusting ionic strength, pH buffers, preservatives, and the like.

The kit may include instructions that instruct the user on how to use the kit. The manual may be a paper medium on which instructions for the user are printed, or may be an electronic medium that stores instructions for the user. Further, instead of explaining how to use the kit, the instruction manual may be an instruction manual that explains how to access the method of using the kit (for example, a URL of a medium that can be viewed on the Internet).

〔summary〕
As understood from the above, the linker according to the first aspect of the present invention is a linker comprising a first segment and a second segment, and which includes a polynucleotide sequence contained in the first segment. The first hybridization site having a polynucleotide sequence and the second hybridization site having a polynucleotide sequence contained in the second segment hybridize to form a double-stranded region, and the first hybridization site has a polynucleotide sequence contained in the second segment. The segment is bonded to the 5' end side when viewed from the first hybridization site, and is located between a binding site that binds to a protein or a peptide, and the first hybridization site and the binding site. and a 5'-5' bond between nucleotides, and the second segment includes a single-stranded polynucleotide region on the 3' end side as viewed from the second hybridization site.

In the linker according to the second aspect of the present invention, in addition to the structure of the linker according to the first aspect, a part of the first hybridization site and a part of the second hybridization site share connected to each other by bonds.

In the linker according to the third aspect of the present invention, in addition to the structure of the linker according to the first or second aspect, the first segment connects the first hybridization site with the 5'-5' bond. and a first spacer region having a polyA _m sequence (where m represents an integer of 2 or more and 50 or less).

In the linker according to the fourth aspect of the present invention, in addition to the structure of the linker according to any one of the first to third aspects, the G/C content of the first hybridization site is 20%. The above is 99% or less.

In the linker according to the fifth aspect of the present invention, in addition to the structure of the linker according to any one of the first to fourth aspects, the single-stranded region of the second segment hybridizes with mRNA. The second segment includes a reverse transcription initiation site for initiating synthesis of cDNA complementary to the mRNA.

In addition to the structure of the linker according to any one of the first to fifth aspects, the linker according to the sixth aspect of the present invention has the structure that the binding site is puromycin, 3'-N-aminoacylpuromycin amino It is at least one selected from the group consisting of nucleosides (PANS-amino acids) and 3'-N-aminoacyladenosine aminonucleosides (AANS-amino acids).

In addition to the structure of the linker according to any one of the first to sixth aspects, the linker according to the seventh aspect of the present invention has the structure that the first segment is connected to the binding site and the 5'-5' A second spacer moiety having (PEG) _n (wherein PEG represents polyethylene glycol and n represents an integer of 1 or more and 20 or less) is included between the bond and the bond.

A kit according to an eighth aspect of the present invention is a kit for producing a linker comprising a first segment and a second segment, the kit comprising: the first segment and the second segment. A first hybridization site having a polynucleotide sequence contained in the first segment and a second hybridization site having a polynucleotide sequence contained in the second segment are hybridization sites. The first segment has a sequence capable of forming a double-stranded region by soybean, and the first segment is bound to the 5' end side when viewed from the first hybridization site, and has a binding site that binds to a protein or peptide. and an internucleotide 5'-5' bond located between the first hybridization site and the binding site, and the second segment is located between the second hybridization site and the binding site. It contains a single-stranded polynucleotide region at the 3' end when viewed from above.

[Additional notes]
The present invention is not limited to the embodiments described above, and various modifications can be made within the scope shown in the claims, and embodiments obtained by appropriately combining each technical means disclosed in the embodiments described above. are also included within the technical scope of the present invention.

An embodiment of the present invention will be described below.

[Example 1]
(Production of linker)
Linker L1 used in this example was synthesized. First, a compound FS1 having the following sequence was synthesized as a first segment. In addition, a compound SS1 having the following sequence was synthesized as a second segment. The synthesis was entrusted to Tsukuba Oligo Service Co., Ltd. and was carried out using an automatic nucleic acid synthesizer according to the phosphoramidite method.

Compound FS1 [Sequence: 3'-PCCZZZCTC(FT ^* )CT-5'-5'-(X2)-(X1)-3']
Compound SS1 [Sequence: 5'-(X3)-(X4)-3']
Here, P represents puromycin, Z represents PEG (-[CH ₂ CH ₂ O]-), and FT ^* represents FITC-dT.
X1 represents the following array.
5'-CGGTATAGGCA ^cnv KGCGTGGAAA-3' (SEQ ID NO: 1, 21mer)
X2 represents the following array.
5'-AAAAAAAAAAAAA-3' (SEQ ID NO: 2, 12mer)
X3 represents the following array.
5'-TTTCCACGCGTGCCCTATAACCG-3' (SEQ ID NO: 3, 21mer) X4 represents the following sequence.
5'-GC ^* C ^* A ^cnv KGC ^* C ^* TCCT-3' (SEQ ID NO: 4, 12mer)
Here, ^cnv K represents 3-cyanovinylcarbazole and C ^* represents 5-methyl-dC.

Compound FS1 (final concentration 500 μM) and compound SS1 (final concentration 500 μM) were added to a mixed solution of 4 μL of 0.25 M Tris-HCl buffer (pH 7.5) and 4 μL of 1 M NaCl aqueous solution. The resulting mixed solution was maintained at 90°C for 2 minutes, then cooled to 70°C for 1 minute, maintained at 70°C for 1 minute, and then cooled to 25°C for 15 minutes to perform an annealing treatment. Compound FS1 and compound SS1 were hybridized. Next, the mixed solution was irradiated with UV at a wavelength of 365 nm, an intensity of 500 mJ/ ^cm2 , and an irradiation time of 60 seconds to form a covalent bond between compound FS1 and compound SS1 by photocrosslinking, and linker L1 of Example 1 was formed. Obtained.

(Formation of mRNA-linker complex)
Linker L1 (final concentration 100 μM) and mRNA encoding interleukin-17A (IL-17A) (prepared by Tsukuba Oligo Service Co., Ltd.) were added to 25 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl. , SEQ ID NO: 5) (final concentration 100 μM) was added. The mixed solution was subjected to an annealing treatment by incubating at 90°C for 1 minute, then at 70°C for 1 minute, and decreasing the temperature to 25°C at a rate of 0.08°C/sec. The 3' end and the 3' end of the mRNA were hybridized. Next, the mixed solution is irradiated with UV at a wavelength of 365 nm, an intensity of 500 mJ/cm ² , and an irradiation time of 60 seconds to form a covalent bond between linker L1 and mRNA by photocrosslinking, thereby forming an mRNA-linker L1 complex. I got it. FIG. 6 shows the structure of the mRNA-linker L1 complex. FIG. 6 is a schematic diagram showing the structure of the mRNA-linker L1 complex of Example 1 of the present invention.

Formation of the mRNA-linker L1 complex was verified by SDS-PAGE of 4% stacking gel-6% separation gel containing 8M urea. The verification results are shown in FIG. FIG. 7 is a gel electrophoresis photograph showing the verification results of the formation of mRNA-linker complex L1 in Example 1 of the present invention. In Figure 7, lane 1 shows the 100bp DNA ladder, lane 2 shows the 10bp DNA ladder, lane 3 shows the linker L1 obtained by HPLC purification, lane 4 shows the mRNA, and lane 5 shows the annealing treatment and UV Figure 2 shows a mixed solution containing mRNA, linker L1 and mRNA-linker L1 complex that has been subjected to irradiation.

As shown in lane 5 of FIG. 7, it was confirmed that an mRNA-linker L1 complex was formed. Furthermore, the formation efficiency of the mRNA-linker L1 complex, calculated from the ratio of the fluorescence intensity of FITC contained in linker L1, was 78% to 80%. This value of the formation efficiency is comparable to the value of the formation efficiency of the mRNA-linker LR1 complex by the linker LR1 used in Reference Example 1 (60% to 90%, catalog value).

(Formation of mRNA-linker-polypeptide complex (mRNA display))
The mRNA-linker L1 complex obtained by HPLC purification was translated using a cell-free translation system to form an mRNA-linker-polypeptide complex (hereinafter sometimes referred to as mRNA display). The composition of the reaction solution used for cell-free translation was Translation Mix 0.5 μL, Retic Lysate 1 17.5 μL, RNase inhibitor 0.5 μL, mRNA-linker L1 complex 3 pmol, and ultrapure water (added to a final volume of 25 μL). Met. The tube containing the above translation reaction solution was incubated at 30°C for 20 minutes, then 12 μL of 3M KCl and 3 μL of 1M MgCl ₂ were added to the translation reaction solution, and the tube was further incubated at 37°C for 60 minutes. Next, 10 µL of ethylenediaminetetraacetic acid (pH 8.0) was added to the translation reaction solution, incubated at 37°C for 10 minutes, and then 50 µL of 2x binding buffer was added to transform the IL-1 synthesized using the free translation system. mRNA display L1 was obtained in which 17A was bound to the mRNA-linker L1 complex.

(Formation of mRNA-linker-polypeptide-cDNA complex (cDNA display))
The purified mRNA display L1 was reverse transcribed to form an mRNA-linker-polypeptide-cDNA complex (hereinafter sometimes referred to as cDNA display). The composition of the reaction solution used for reverse transcription was: 20 μL of 5x ReverTra Ace (manufactured by Toyobo) attached buffer, 10 μL of 10 mM dNTP mixed solution, 2 μL of Rever Tra Ace (100 U/μL), and 30 μL of the solution containing mRNA display L1 obtained above. , and ultrapure water (added to a final volume of 100 μL). The above reaction solution for reverse transcription was incubated at 42° C. for 90 minutes while stirring with a rotator to obtain cDNA display L1 in which cDNA complementary to mRNA was bound to mRNA display L1.

(CDNA display purification)
The solution containing cDNA display L1 contains an mRNA-linker L1-cDNA complex to which no IL17-A is bound, and an mRNA-linker L1-polypeptide-cDNA complex to which IL17-A is bound (i.e., cDNA display L1). including both. In order to isolate these two types of complexes, His-tag purification was performed. First, His Mag sepharose Ni was added to the solution containing the above cDNA display L1, and the solution was incubated at 25° C. for 1 hour while shaking using a shaker. The solution was centrifuged at 5000 rpm and the supernatant (His sup.) was removed. Next, 50 μL of His tag elution buffer (containing a high concentration of imidazole) was added to the precipitated residue, and the mixture was incubated at room temperature for 30 minutes with shaking using a shaker. Then, the solution was centrifuged at 5000 rpm, and the supernatant (His Elute) was collected to obtain IL-17A-bound cDNA display L1.

[Reference example 1]
Linker LR1 of Reference Example 1 of the present invention was prepared according to the description of Example 1 of WO 2016/159211. An mRNA-linker LR1 complex, an mRNA-linker LR1-polypeptide complex (mRNA display LR1), and an mRNA-linker LR1- Formation and purification of a polypeptide-cDNA complex (cDNA display LR1) was performed.

〔result〕
The efficiency of forming mRNA and cDNA display of linker L1 and linker LR1 was verified by SDS-PAGE of 4% stacking gel-6% separating gel containing 8M urea. The verification results are shown in FIG. Figure 8 is a gel showing the results of the cDNA display method when linker LR1 of Reference Example 1 of the present invention was used (left figure) and when linker L1 of Example 1 of the present invention was used (right figure). This is an electrophoresis photograph.

As shown in FIG. 8, by using linker L1, mRNA display L1 and cDNA display L1 could be formed. In addition, mRNA display, cDNA display (including complexes to which IL-17A is not bound), and cDNA display (complexes to which IL-17A is not bound) calculated from the ratio of the fluorescence intensity of FITC contained in linker L1. The formation efficiency of (not including) was as shown in Table 1 below.

As shown in Table 1, the formation efficiency of mRNA display and cDNA display when linker L1 of Example 1 of the present invention was used was comparable or higher than that of linker LR1 of Reference Example 1. Furthermore, the reverse transcription efficiency when linker L1 was used was also comparable or higher than that using linker LR1. From this, it is expected that the linker L1 of Example 1 of the present invention can be used as an excellent linker in functional polypeptide search techniques such as cDNA display methods.

The present invention can be utilized, for example, in searching for functional polypeptides.

10 First segment 11 First hybridization site 12 Binding site 13 5'-5' bond 14 First spacer site 15 Second spacer site 20 Second segment 21 Second hybridization site 22 Single strand Region 23 Reverse transcription start site 40 mRNA
50 polypeptide 60 cDNA
100 linker

Claims

A linker comprising a first segment and a second segment,
A first hybridization site having a polynucleotide sequence contained in the first segment and a second hybridization site having a polynucleotide sequence contained in the second segment hybridize to each other. It forms a main chain region,
The first segment is
A binding site that is bonded to the 5′ end side as viewed from the first hybridization site and that binds to a protein or peptide;
a 5'-5' internucleotide bond located between the first hybridization site and the binding site;
The second segment is
comprising a single-stranded polynucleotide region on the 3' end side as viewed from the second hybridization site;
linker.
A portion of the first hybridization site and a portion of the second hybridization site are linked to each other by a covalent bond,
A linker according to claim 1.
The first segment has a polyA m sequence (where m represents an integer of 2 or more and 50 or less) between the first hybridization site and the 5'-5' bond. 1 spacer portion,
The linker according to claim 1 or 2.
The G/C content of the first hybridization site is 20% or more and 99% or less,
The linker according to claim 1 or 2.
The single-stranded region of the second segment is a region that hybridizes with mRNA,
The second segment includes a reverse transcription initiation site for initiating synthesis of cDNA complementary to the mRNA.
The linker according to claim 1 or 2.
The binding site is at least one selected from the group consisting of puromycin, 3'-N-aminoacylpuromycin aminonucleoside (PANS-amino acid), and 3'-N-aminoacyladenosine aminonucleoside (AANS-amino acid). ,
The linker according to claim 1 or 2.
The first segment has (PEG) n (wherein, PEG represents polyethylene glycol, and n represents an integer of 1 or more and 20 or less) between the binding site and the 5'-5' bond. a second spacer portion having
The linker according to claim 1 or 2.
A kit for manufacturing a linker comprising a first segment and a second segment, the kit comprising:
The kit includes the first segment and the second segment,
A first hybridization site having a polynucleotide sequence contained in the first segment and a second hybridization site having a polynucleotide sequence contained in the second segment hybridize to each other. has a sequence capable of forming a full-stranded region,
The first segment is:
A binding site that is bonded to the 5′ end side as viewed from the first hybridization site and that binds to a protein or peptide;
a 5'-5' internucleotide bond located between the first hybridization site and the binding site;
The second segment is
comprising a single-stranded polynucleotide region on the 3' end side as viewed from the second hybridization site;
kit.