Complex of circular DNA molecule and protein, and display method using the same

Abstract

To provide a novel method that forms a complex linking a peptide with DNA encoding the peptide to establish correspondence therebetween, and to provide a method for readily selecting DNA encoding a desired peptide from a complex library in which peptides correspond to DNA encoding the peptides.SOLUTION: A method for creating a complex in which a peptide is linked to a nucleotide sequence encoding the peptide, the method comprising: (1) preparing circular DNA having a nucleotide sequence encoding a peptide; and (2) transcribing and translating the nucleotide sequence from the circular DNA using a cell-free transcription-translation reaction to express a peptide, wherein the expressed peptide forms a complex linked to the circular DNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a complex of a peptide and DNA encoding the peptide, and a display method using the same. [Background technology]

[0002] In evolutionary molecular engineering, display methods have been developed as techniques for associating genotypes with phenotypes. For example, the phage display method (Non-Patent Document 1), which is widely used in the creation and selection of antibody libraries, associates the genotype within a phage with the phenotype displayed on the phage surface, and is useful in the search for novel highly functional molecules in a variety of fields. Known cell-free techniques include the ribosome display method (Non-Patent Document 2), which uses a complex of mRNA, ribosomes, and proteins, and the mRNA display method (Non-Patent Document 3), which associates mRNA with peptides via puromycin. In each case, a complex is formed between an mRNA molecule and a peptide expressed from the mRNA molecule, thereby associating a genotype with a phenotype.

[0003] CIS display has been developed as a DNA-based display method (Patent Document 1, Patent Document 2, Non-Patent Document 4). This method utilizes the property (called cis activity) of the DNA replication initiator protein (repA) to non-covalently bind only to the ori of the template DNA used for expression in the presence of CIS, which confers cis activity. When linear DNA in which DNA encoding repA is linked to a DNA library is transcribed and translated in a test tube, a protein-DNA complex library (pool of complexes) is formed, and each protein stably binds to the DNA that encodes it.

[0004] Meanwhile, the present inventors have developed the RCR (Replication Cycle Reaction) method as a technology for amplifying circular DNA in a cell-free system (Patent Documents 3 to 5, Non-Patent Document 5). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 1998 / 037186 [Patent Document 2] International Publication No. 2004 / 022746 [Patent Document 3] International Publication No. 2016 / 080424 [Patent Document 4] International Publication No. 2017 / 199991 [Patent Document 5] International Publication No. 2018 / 159669 [Non-patent literature]

[0006] [Non-Patent Document 1] Scott & Smith, Science (1990) Vol.249, No.4967, p.386-390. [Non-patent document 2] Hanes and Pluckthun, Proc. Natl. Acad. Sci. USA (1997) Vol.94, p.4937-4942. [Non-patent document 3] Roberts and Szostak, Proc. Natl. Acad. Sci. USA (1997) Vol. 94, p.12297-12302. [Non-patent document 4] Odegrip et al., PNAS (2004) Vol.101, No.9, p.2806-2810. [Non-patent document 5] Su'etsugu et al., Nucleic Acids Research (2017) Vol.45, No.20, p.11525-11534. Summary of the Invention [Problem to be solved by the invention]

[0007] Methods using mRNA as a genotype have the problem that RNA is more unstable and prone to degradation than DNA, and RNA cannot be directly amplified, requiring multiple steps such as reverse transcription and PCR, which reduces the efficiency of obtaining sequence information.

[0008] In the CIS display method using linear DNA, complexes that bind to the target are selected from a protein-DNA complex library, and then the DNA of the selected complex is amplified by PCR, followed by subsequent transcription and translation. This process of selecting and enriching DNA that encodes the target protein by binding of the complex to the target molecule is also called panning. In the CIS display method, the library is enriched by 100% of the total number of proteins in one round of panning based on the results of multiple rounds of panning. 3 It has been calculated that the concentration is 1:100 (Non-Patent Document 4). This is a lower concentration than conventional phage display, and as a result, many operations are required to obtain a protein with the desired function. Furthermore, the CIS display method requires the use of a cis-active protein such as repA, which limits flexibility. Furthermore, because linear DNA is used as a transcription template, when combined with a transcription-translation reaction using an E. coli extract, there is a problem that the template DNA is degraded by linear DNA-specific exonucleases such as RecBCD (ExoV).

[0009] Under these circumstances, the main object of the present invention is to provide a novel method for forming and associating a complex between a peptide and a DNA encoding the peptide, and a method for recovering a DNA encoding a desired peptide from a library of such associated complexes with fewer operations. [Means for solving the problem]

[0010] The present inventors first used circular DNA in the CIS display method, and performed DNA amplification after peptide binding to the target using the PCR method. In this attempt, they discovered favorable conditions, enabling selection of DNA after target binding with simpler procedures and higher enrichment rates than conventional CIS display methods. Further intensive research led to the discovery that a complex can be formed using circular DNA, without using the cis-activity of repA, in which a peptide is linked to a circular DNA containing DNA encoding the peptide. This finding led to the completion of the present invention.

[0011] That is, the present invention relates to the following. [1] A method for producing a complex in which a peptide and a base sequence encoding the peptide are linked, comprising: (1) providing a circular DNA having a base sequence encoding a peptide; (2) expressing a peptide by transcribing and translating the base sequence from the circular DNA using a cell-free transcription-translation reaction, and the expressed peptide forms a complex linked to the circular DNA. A method comprising: [2] A method for creating a complex library in which library-constituting peptides are linked to base sequences encoding the library-constituting peptides, the method comprising: (1) preparing a circular DNA library having nucleotide sequences encoding library constituent peptides; (2) expressing library-constituting peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, and forming a complex library in which the expressed peptides are linked to circular DNAs having base sequences encoding the peptides. A method comprising: [3] The method according to [1] or [2], wherein the circular DNA has a replication initiation sequence capable of binding to an enzyme having DnaA activity. [4] The method according to any one of [1] to [3], wherein the circular DNA does not have a binding sequence for a protein expressed from the circular DNA. [5] A method for recovering a base sequence encoding a target-binding peptide, comprising: (1) preparing a circular DNA library having base sequences encoding candidate peptides for target-binding peptides that bind to a target; (2) expressing candidate peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, thereby forming a complex library in which the expressed candidate peptides are linked to circular DNAs having base sequences encoding the peptides. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) isolating and recovering circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, obtained in (3); A method comprising: [6] (3) and (4) above, in the presence of a magnesium ion source in the range of 1 mM to 50 mM, and / or In the presence of an alkali metal ion source in the range of 150 mM to 1000 mM, The method according to [5], which is carried out by [7] (5) The method according to [5] or [6], further comprising subjecting the circular DNA having a nucleotide sequence encoding the target-binding peptide isolated and recovered in (4) above to a cell-free nucleic acid amplification reaction to amplify the nucleotide sequence encoding the target-binding peptide. [8] The circular DNA library has a replication initiation sequence capable of binding to an enzyme having DnaA activity, and further (5') (a) preparing a reaction mixture containing a reaction solution containing a first group of enzymes that catalyze the replication of circular DNA, (b) a second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and (c) a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs, and the circular DNA having a base sequence that encodes the target-binding peptide isolated and recovered in (4) above; and incubating the reaction mixture at a temperature ranging from 20°C to 80°C; The method according to [5] or [6], further comprising amplifying a circular DNA having a base sequence encoding the target-binding peptide by a PCR method comprising: [9] The method according to [8], wherein the circular DNA amplified in (5') is used as a circular DNA library in (2), and the steps (2), (3), and (4) are repeated.

[10] The method according to any one of [5] to [9], wherein the contact and binding in (3) above is carried out in the absence of RNase.

[11] A method for recovering a base sequence encoding a target-binding peptide, comprising: (1) Providing a circular DNA library having a base sequence encoding a candidate peptide of a target-binding peptide that binds to a target, a DNA target sequence, a base sequence encoding a base peptide that non-covalently binds to the DNA target sequence, and a DNA element that provides cis activity, wherein the candidate peptide is expressed linked to the base peptide; (2) expressing candidate peptides and base peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, and forming a complex library in which the base peptides linked to the expressed candidate peptides are non-covalently bound to a DNA target sequence on a circular DNA having a base sequence encoding the candidate peptides. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) isolating and recovering circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, obtained in (3); A method comprising:

[12] The above (3) and (4) in the presence of a magnesium ion source in the range of 1 mM to 50 mM, and / or In the presence of an alkali metal ion source in the range of 150 mM to 1000 mM, The method according to

[11] , which is carried out by

[13] The circular DNA library has a replication initiation sequence capable of binding to an enzyme having DnaA activity, and further (5') (a) preparing a reaction mixture containing a reaction solution containing a first group of enzymes that catalyze the replication of circular DNA, (b) a second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and (c) a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs, and the circular DNA having a base sequence that encodes the target-binding peptide isolated and recovered in (4) above; and incubating the reaction mixture at a temperature ranging from 20°C to 80°C; The method according to

[12] , further comprising amplifying circular DNA having a base sequence encoding the target-binding peptide by a PCR method comprising:

[14] The method according to

[13] , wherein the circular DNA amplified in (5') is used as a circular DNA library in (2), and the steps (2), (3), and (4) are repeated.

[15] A method for producing a target-binding peptide, comprising: amplifying a base sequence encoding the target-binding peptide from circular DNA containing the base sequence encoding the target-binding peptide, which is recovered by the method according to any one of [5] to

[14] above; and transcribing and translating the obtained base sequence to produce the target-binding peptide.

[16] A complex library in which library member peptides and base sequences encoding the library member peptides are linked together, (i) a circular DNA library having a base sequence encoding the library-constituting peptide and a replication initiation sequence capable of binding to an enzyme having DnaA activity; (ii) the library constituent peptides; wherein (i) and (ii) are connected via mRNA.

[17] A base sequence encoding a candidate peptide of a target-binding peptide that binds to the target; a DNA target sequence; a base sequence encoding a base peptide that non-covalently binds to the DNA target sequence; a DNA element that confers cis activity; a replication initiation sequence capable of binding to an enzyme having DnaA activity; A circular DNA having the formula: the DNA element that confers cis activity is located between the base sequence encoding the base peptide and the DNA target sequence; A circular DNA in which the candidate peptide is expressed linked to the platform peptide.

[18] A conjugate of a target and a target-binding peptide, the target-binding peptide is tethered to a circular DNA via an mRNA; A conjugate wherein the circular DNA has a base sequence encoding the target-binding peptide. [Effects of the Invention]

[0012] The present invention provides a novel method for forming and associating a complex in which a peptide is linked to DNA encoding the peptide, and also provides a method for simply selecting DNA encoding a desired peptide from a library of complexes in which peptides and DNA encoding the peptide are associated. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1(A) shows a schematic diagram of the experiment in Example 1. Figure 1(B) shows a schematic diagram of the three types of circular DNA used in Example 1. Figure 1(C) is a gel electrophoresis diagram showing the results of Example 1. [Figure 2] 2(A) shows a schematic diagram of two types of linear DNA and two types of circular DNA used in Example 2. FIG. 2(B) shows a gel electrophoresis diagram showing the results of Example 2. [Figure 3] 3(A) shows a schematic diagram of the experiment in Example 3. FIG. 3(B) shows a gel electrophoresis diagram showing the results of Example 3. [Figure 4]4(A) is a schematic diagram of a nicking endonuclease-type restriction enzyme recognition site in the circular DNA used in Example 4. FIG. 4(B) is a gel electrophoresis diagram showing the results of Example 4. [Figure 5] FIG. 5 is a gel electrophoresis diagram showing the results of Example 5. [Figure 6] 6(A) is a gel electrophoresis diagram showing the results of circular DNA display in Example 6. FIG. 6(B) is a gel electrophoresis diagram showing the results of CIS display using circular DNA in Example 6. [Figure 7] 7(A) shows a schematic diagram of the experiment in Example 7. FIG. 7(B) shows a gel electrophoresis diagram showing the results of Example 7. [Figure 8] FIG. 8 is a gel electrophoresis diagram showing the results of Example 8. [Figure 9] 9(A) shows a schematic diagram of the three types of circular DNA used in Example 9. FIG. 9(B) shows a gel electrophoresis diagram showing the results of Example 9. [Figure 10] 10(A) shows a schematic diagram of the experiment in Example 10. FIG. 10(B) is a gel electrophoresis diagram showing the results of Example 10. [Figure 11] 11(A) shows a schematic diagram of the circular DNA and sequence insertion site used in Example 11. FIG. 11(B) is a gel electrophoresis showing the results of Example 11. [Figure 12] FIG. 12 shows a schematic diagram of the plasmid pHis-RepA_CISoriR prepared in the preparation example. [Figure 13] FIG. 13 shows a comparison between the conventional method and the circular DNA display method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments of the present invention (also referred to as the present embodiments) will be described, but the present invention is not limited thereto. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

[0015] In one aspect, the present embodiment provides a method for producing a tethered complex of a peptide and a base sequence encoding the peptide, the method comprising: (1) providing a circular DNA having a base sequence encoding a peptide; (2) expressing a peptide by transcribing and translating the base sequence from the circular DNA using a cell-free transcription-translation reaction, and the expressed peptide forms a complex linked to the circular DNA. The present invention relates to a method, including:

[0016] The peptide used in the method of this embodiment is not particularly limited, and any peptide in which two or more amino acids, preferably ten or more amino acids, are linked by peptide bonds can be used. In one aspect, a part or all of a naturally occurring protein such as an enzyme, or a binding molecule such as a receptor or antibody or a fragment thereof can be used as the peptide.

[0017] In one aspect of this embodiment, the peptide is a target-binding peptide. The target-binding peptide is not particularly limited as long as it has the ability to bind to a target. The "target" can be selected from various receptors, enzymes, compounds, antigens, antibodies, immobilized metals, etc., depending on the target-binding peptide, but is not limited to these. In the examples described below, combinations of an antibody and a FLAG tag, and an immobilized metal, Ni, and a His tag, are used as model experiments as combinations of targets and target-binding peptides, but are not limited to these. For example, any peptide / proteinaceous binding factor can be used as a target.

[0018] In one embodiment, the target is an antigen, and the target-binding peptide is an antibody or a portion thereof, preferably a portion of an antibody. Antibodies have variable and constant regions, and it is sufficient that at least the antigen-binding site is contained in the target-binding peptide. For example, portions of antibodies such as the Fab region (the region of an antibody excluding the CH2 and CH3 regions), scFv (single-chain antibody in which VH and VL are linked via a linker), or nanobody (also called VHH) can also be used as the target-binding peptide.

[0019] In one aspect of this embodiment, an enzyme exhibiting peptide activity can be used. After preparing a complex containing a peptide linked to its encoding base sequence, the complex can be confined in microcompartments such as a water-in-oil emulsion, and the compartments exhibiting enzymatic activity can be selected and recovered to select a circular DNA having a base sequence encoding the desired peptide.

[0020] In this embodiment, the cyclic DNA may have one or more types of base sequences encoding peptides. One cyclic DNA may have two or more base sequences encoding a single peptide.

[0021] In this embodiment, the circular DNA has a DNA sequence for expressing the encoded peptide. More specifically, the base sequence encoding the peptide has a promoter region and a 5'UTR upstream thereof, and the 5'UTR contains a sequence encoding a ribosome binding site. This allows the base sequence encoding the peptide to be transcribed from the promoter into RNA in the presence of RNA polymerase, and then translated into the peptide.

[0022] The promoter and 5'UTR can be designed appropriately depending on the transcription / translation characteristics of the organism from which the cell-free transcription / translation system is used. In one embodiment, the promoter sequence preferably has a sequence capable of binding to σ factor in E. coli, and more preferably a sequence capable of binding to σ70 factor. Binding of the promoter to σ factor involves sequences in the -10 and -35 regions from the transcription initiation site. For example, the consensus sequence capable of binding to σ70 (s70) factor is preferably 5'-TATAAT-3' in the -10 region and 5'-TTGACA-3' in the -35 region. The origin of the promoter sequence can be appropriately selected depending on the transcription / translation system used. Examples of promoters include the nucleotide sequences and modified sequences of promoters such as the trp promoter, lac promoter, and araB promoter derived from E. coli; the T7 promoter, T3 promoter, T5 promoter, and SP6 promoter derived from bacteriophage; and synthetic promoters such as the Tac promoter. The modified promoter sequence refers to a base sequence in which a mutation has been introduced to substitute, delete, or insert one or more bases in the base sequence before modification while retaining the promoter function.

[0023] In this embodiment, the circular DNA may have multiple peptide-encoding sequences, one of which may be a base protein (base peptide) that is translated together with a sequence encoding any one peptide (e.g., a sequence encoding a candidate peptide for a target-binding peptide, as described below). Without being bound by theory, the presence of a sequence encoding such a base protein increases the size of the complex in which the expressed peptide and base protein are linked to the circular DNA, facilitating subsequent handling of the complex. For example, binding to the target is facilitated when forming a complex library (as described below), contacting the complex library with a target, and binding the target to the complex library via the candidate peptide linked to the complex library. The base protein is not particularly limited as long as it acts as a marker, and can be, for example, a known protein such as repA or GFP. In one embodiment, repA is preferred. In one embodiment, the base protein consists of 100 or more, 150 or more, e.g., 170 or more amino acids. When repA is used, a known repA such as that disclosed in Patent Document 1 can be used. The function of repA is not particularly limited, and may be, for example, a repA mutant that has lost the ability to bind to the on sequence that repA generally has. The sequence encoding the base protein has a DNA sequence for expressing the protein, as in the case of the above-mentioned peptides.

[0024] The base sequence encoding the base protein and the base sequence encoding the peptide are preferably adjacent on the circular DNA via a linker sequence. Preferably, in the circular DNA, the linker sequence is located upstream of the base sequence encoding the base protein, and the base sequence encoding the peptide is located upstream of that, and a DNA sequence for expressing the protein and peptide is located further upstream. This allows for the production of a base protein having the desired peptide at its terminus after transcription and translation.

[0025] In one aspect of this embodiment, the circular DNA used in the circular DNA display method preferably does not have a binding sequence for the peptide or protein to be expressed from the circular DNA. In one aspect of this embodiment, when CIS display is not used, the circular DNA preferably does not have a DNA element that confers cis activity. For example, when the circular DNA used in the circular DNA display method has a sequence encoding repA, the circular DNA does not have an oriR sequence or a CIS sequence.

[0026] In one aspect of this embodiment, the circular DNA does not have a Rho-independent strong terminator within 100 bases downstream of the peptide-encoding sequence (including, if present, the base sequence encoding the base protein). In this embodiment, a strong terminator sequence refers to a terminator that efficiently causes transcription termination. The transcription termination efficiency can be measured, for example, by the method described in Orosz et al., 1991, Eur. J. Biochem., and the transcription termination efficiency of rrnBT1 confirmed by this method is 87%. In one aspect, a strong terminator has a transcription termination efficiency of 60% or more, 70% or more, preferably 80% or more, and more preferably 85% or more when confirmed by this method. The transcription termination efficiency can also be calculated using an algorithm. Examples of terminator sequences that are strongly Rho-independent include the terminator sequence of the gene (fdhF) encoding formate dehydrogenase of Escherichia coli, and the T1 or T2 terminator (particularly the T1 terminator) sequence of a ribosomal RNA gene (rrnB, etc.).

[0027] In conventional CIS display methods, peptide display requires the presence of binding sequences for peptides and proteins expressed from DNA and DNA elements that confer cis activity. However, in the circular DNA display method of this embodiment, the translated peptide is linked to and associated with the circular DNA by a mechanism completely different from that of the CIS display method.

[0028] In this embodiment, the circular DNA can be prepared using techniques known to those skilled in the art. For example, the circular DNA of this embodiment can be obtained by ligating DNAs containing the above-described components together in a cell-free system. Known techniques for this purpose include the infusion method, the Gibson Assembly method, the Recombination Assembly method (RA method, WO2019 / 009361), and methods using commercially available kits such as USER® Cloning (NEB), and Golden Gate Assembly (NEBridge®, NEB). These known techniques can be used.

[0029] When using the RA method, a reaction solution containing two or more DNA fragments and a protein with RecA family recombinase activity is prepared. If at least one of the DNA fragments to be ligated is a linear double-stranded DNA fragment, the reaction solution further contains an exonuclease. Next, the two or more DNA fragments are ligated to each other in the reaction solution at regions where their base sequences are complementary to each other to obtain linear or circular DNA. For the components used in the RA method, see International Publication No. 2019 / 009361. For example, the exonuclease may be any exonuclease with any type or biological origin, as long as it has the enzymatic activity to sequentially hydrolyze linear DNA from the 3' or 5' end. For example, a linear double-stranded DNA-specific 3'→5' exonuclease is preferably used. The exonuclease used is preferably both a linear double-stranded DNA-specific 3'→5' exonuclease and a single-stranded DNA-specific 3'→5' exonuclease; for example, a combination of exonuclease III and exonuclease I, or a combination of exonuclease III, exonuclease I, and exonuclease T can be used.

[0030] The RecA family recombinase protein used in the RA method is not particularly limited as long as it polymerizes on single-stranded or double-stranded DNA to form filaments, has hydrolytic activity for nucleoside triphosphates such as ATP (adenosine triphosphate), and has the function of searching for homologous regions and performing homologous recombination (RecA family recombinase activity).Prokaryotic RecA homologs such as Escherichia coli RecA, bacteriophore RecA homologs such as T4 phage UvsX, archaeal RecA homologs, eukaryotic RecA homologs, and modified forms of these that retain RecA family recombinase activity can be used.

[0031] The circular DNA has the above sequence and is not particularly limited other than being circular, but is preferably double-stranded so that transcription and translation occur. Examples of circular DNA include circular DNA obtained by circularizing a non-circular DNA, circular DNA obtained by linking multiple non-circular DNAs, and artificially synthesized circular DNA. Examples of such non-circular DNA include natural circular DNA cleaved by enzyme treatment or the like, and artificially synthesized non-circular DNA. The circular DNA may or may not be supercoiled (it may be open circular). It may also have nicks or gaps.

[0032] The size of the circular DNA is not particularly limited as long as it can be translated, transcribed, and form a complex, and can be, for example, 300 bases or more and 100 kb or less. When the circular DNA is amplified in a cell-free nucleic acid amplification reaction, the size is not particularly limited as long as it can be amplified by the selected amplification method. In one embodiment, the circular DNA has a size of 300 bases or more and 100 kb or less, 500 bases or more and 50 kb or less, 1 kb or more and 15 kb or less, 1.5 kb or more and 5 kb or less, or 2 kb or more and 4 kb or less.

[0033] In one embodiment, when the circular DNA contains a replication initiation sequence capable of binding to an enzyme having DnaA activity (hereinafter, simply referred to as a "replication initiation sequence"), examples of the replication initiation sequence capable of binding to an enzyme having DnaA activity include known replication initiation sequences present in bacteria such as Escherichia coli and Bacillus subtilis, which can be obtained from public databases such as NCBI. Alternatively, a replication initiation sequence can be obtained by cloning a DNA fragment capable of binding to an enzyme having DnaA activity and analyzing its nucleotide sequence. The replication initiation sequence used in the present invention may also be a sequence in which one or more bases of a known replication initiation sequence have been mutated by substitution, deletion, or insertion, resulting in a modified sequence capable of binding to an enzyme having DnaA activity. The replication initiation sequence used in the present invention is preferably oriC or a modified sequence thereof, more preferably oriC derived from E. coli or a modified sequence thereof.

[0034] The circular DNA used as a template in this embodiment may be a circular DNA that originally contains a replication initiation sequence, or may be a circular DNA that does not originally contain a replication initiation sequence into which a replication initiation sequence has been introduced.

[0035] In one aspect, the circular DNA used in the method of this embodiment contains a replication initiation sequence capable of binding to an enzyme with DnaA activity and a strong gyrase-binding sequence (SGS). The SGS is a binding sequence to which DNA gyrase binds, introducing negative supercoiling into DNA. Examples of SGS include the consensus sequence RNNNRNR[T / G]GRYC[G / T]YNYN[G / T]NY (R = A or G, Y = C or T, N = A, G, C, or T) (SEQ ID NO: 1) or a sequence containing its complementary sequence (Lockshon and Morris, Journal of Molecular Biology, 1985, vol. 181, pp. 63-74). The SGS may be derived from any biological species as long as it contains the consensus sequence. For example, a phage-derived SGS or a plasmid-derived SGS can be used. The SGS may also contain sequences before and after the consensus sequence, or it may be a modified SGS derived from any biological species. The modified sequence refers to a base sequence into which one or more bases of the unmodified base sequence have been mutated by substitution, deletion, or insertion while retaining the function of increasing the amplification efficiency of the circular DNA containing it by the PCR method. In one aspect, from the viewpoint of fully obtaining the effect of increasing the amplification efficiency of the circular DNA in the replication initiation sequence, the SGS is preferably an SGS derived from bacteriophage Mu, more preferably an SGS derived from bacteriophage Mu (Mu-SGS) (Folarin et al., Bioengineering, 2019, vol.6(2):54.), and even more preferably SEQ ID NO: 2 or a sequence containing it.

[0036] The SGS may be located at any position on the circular DNA, and the distance from the replication initiation sequence is not particularly limited. In one embodiment, the SGS is adjacent to or close to the replication initiation sequence, and is located within 30 bases, 20 bases, 15 bases, 13 bases, or 10 bases from the end of the replication initiation sequence. In one embodiment, the SGS is adjacent to or close to the end of the double-strand cleavage region (DUE) of the replication initiation sequence, and is located within 30 bases, 20 bases, 15 bases, 13 bases, or 10 bases from the end of the DUE of the replication initiation sequence.

[0037] In one aspect, the circular DNA of this embodiment has a mutated double-strand breakthrough region (DUE) with a higher AT content than the wild-type, and preferably has 1 or more, 2 or more, preferably 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more mutations than the wild-type. The mutations are deletion of C or G, or substitution of C or G with A or T, with the latter being more preferred.

[0038] The DUE has three 13-base elements with a high AT content, referred to as the L, M, and R elements, respectively (Katayama et al., Frontiers in Microbiology (2017) Vol. 8:2496). The circular DNA of this embodiment preferably has a mutant DUE with a high AT content compared to the wild-type, and preferably at least one, preferably at least two, elements have a high AT content compared to the wild-type. Preferably, the mutant DUE is a mutant DUE in which the M or R element, more preferably the M and R elements, have a high AT content compared to the wild-type, for example, a sequence with an increased number of TA motifs (a sequence of consecutive T and A) compared to the wild-type. In one aspect, the DUE sequence is preferably TATATATTATATT (SEQ ID NO: 3) and TATTATTATTAG (SEQ ID NO: 4) or a sequence similar thereto. These similar sequences are sequences in which one or more, for example, 1, 2, 3, 4 or 5 bases, preferably 1 to 3 bases, more preferably 1 to 2 bases, and particularly preferably 1 base, of SEQ ID NO: 3 or 4 have been deleted, substituted or added.

[0039] In one aspect, the circular DNA of this embodiment comprises a replication initiator sequence capable of binding to an enzyme having DnaA activity and a gyrase binding sequence, wherein the replication initiator sequence has a mutant double-strand cleavage region (DUE) with a higher AT content than the wild-type. In one aspect, preferably, the replication initiator sequence has a mutant DUE in which the M and R elements have a higher AT content than the wild-type. In one aspect, the circular DNA of this embodiment has a sequence having SEQ ID NO: 63 or a modified version of the sequence of SEQ ID NO: 63.

[0040] The circular DNA of this embodiment may further have a ter sequence inserted facing outward relative to the replication initiation sequence. Examples of ter sequences include the sequence described in Patent Document 5 and mutant ter sequences. Examples of mutant ter sequences carried by the circular DNA of this embodiment include 5'-GN[A / G][T / A]GTTGTACC[T / G]A-3' (SEQ ID NO: 6) and 5'-GTATGTTGTAcCTA-3' (SEQ ID NO: 7), particularly 5'-AGTATGTTGTACCTAAAG-3' (SEQ ID NO: 8) having SEQ ID NO: 7.

[0041] With regard to a ter sequence, "inserted outward relative to the replication initiation sequence" means that the ter sequence is inserted in such a direction that, due to the action of a combination of proteins that bind to the ter sequence and inhibit replication, replication directed outward from the replication initiation sequence such as oriC is permitted, while replication directed inward toward the replication initiation sequence is not permitted and is terminated. With regard to ter sequences, "a pair of ter sequences inserted outward relative to the replication initiation sequence" means that one is inserted on the 5' side of the replication initiation sequence and the other is inserted on the 3' side of the replication initiation sequence. An example of a ter sequence inserted on the 3' side of the replication initiation sequence is a sequence containing a complementary sequence to the nucleotide sequence inserted on the 5' side of the replication initiation sequence.

[0042] In the method of this embodiment, the method for expressing the peptide by transcribing and translating the base sequence from the circular DNA using a cell-free transcription-translation reaction is not particularly limited, and any known method can be used. For example, the method can be carried out by adding the circular DNA to a reaction solution containing components necessary for the transcription reaction and components necessary for the translation reaction.

[0043] Components required for the transcription reaction include enzymes involved in transcription, such as RNA polymerase. For example, when T7 RNA polymerase is included, transcription proceeds from a template circular DNA containing a T7 promoter. Components required for the translation reaction include ribosomes, tRNA, amino acids, or derivatives thereof. Amino acids and amino acid derivatives can be selected appropriately depending on the DNA to be translated, and may be natural or unnatural amino acids. Preferred examples of amino acids include natural amino acids. Preferred examples of amino acid derivatives include homoalanine (the methyl group of alanine is replaced with an ethyl group), methylated lysine (the amino group of lysine is methylated), homoserine (the hydroxyl group of serine is replaced with an ethyl group), nitroanilide derivatives (the carboxyl group is replaced with a sodium group), and fluoroamino acids (the hydrogen atom is replaced with a fluorine atom). Components necessary for transcription reactions and components necessary for translation reactions include those described in known literature on in vitro transcription (IVT) and cell-free protein translation, such as Pavlov and Ehrenberg, Arch Biochem Biophys. (1996) Vol. 328, No. 1, pp. 9-16; Shimizu et al., Nature Biotechnology (2001) Vol. 19, pp. 751-755; Ohashi et al., Biochem Biophys Res Commun. (2007) Vol. 352, No. 1, pp. 270-276; Shimizu and Ueda, Methods Mol Biol. (2010) Vol. 607, pp. 11-21.

[0044] For example, commercially available kits and their components used for in vitro transcription (IVT) or cell-free protein synthesis can be used as these components. Examples of such kits include, but are not limited to, the protein synthesis kit "Mu-Cell-kun" (manufactured by Taiyo Nippon Sanso Corporation), the TNT® Cell-Free Expression System (manufactured by Promega), myTXTL® (manufactured by Arbor Biosciences), and the cell-free protein synthesis reagent kit (derived from wheat germ, manufactured by NU Protein). Cell-free protein synthesis systems are not limited to those derived from Escherichia coli. Kits suitable for transcription and translation from circular DNA are particularly preferred. For example, Mu-Cell-kun (N mini) contains Escherichia coli extract, creatine kinase, T7 RNA polymerase, potassium L-glutamate, sodium azide, NTPs, magnesium acetate, creatine phosphate, folinic acid, ammonium acetate, tRNA, DTT, amino acids, and buffer solutions. Some or all of these components can be used as components required for transcription and translation reactions.

[0045] When circular DNA is added to a reaction solution containing components necessary for the transcription reaction, including RNA polymerase, a transcription reaction occurs using the circular DNA as a template, and RNA derived from the circular DNA is obtained.

[0046] By adding the circular DNA to a reaction solution containing components necessary for the transcription reaction as well as components necessary for the translation reaction, such as ribosomes, tRNA, amino acids, or their derivatives, a translation reaction occurs using the RNA obtained from the transcription reaction as a template, resulting in the translation of the peptide encoded by the circular DNA. In this process, RNA polymerase transcribes on the circular DNA, not on the linear DNA, and further translation occurs, forming a complex in which the translated peptide is linked to the base sequence encoding this peptide. Without being bound by theory, as shown in Figure 2(A) (ΔCISoriR), it is believed that a complex is formed in which the translated peptide is linked to the base sequence encoding this peptide via the RNA polymerase on the circular DNA, the mRNA transcribed from it, and the ribosomes.

[0047] The reaction conditions for transcription and translation can be appropriately set by referring to the protocol of the above kit, etc. For example, the reaction can be carried out at about 30°C for 30 minutes or more, and the reaction may also be carried out for a longer period of time.

[0048] In one aspect, the present embodiment provides a method for creating a complex library in which library-member peptides are linked to base sequences encoding the library-member peptides, the method comprising: (1) preparing a circular DNA library having nucleotide sequences encoding library constituent peptides; (2) expressing library constituent peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, thereby forming a complex library in which the expressed peptides are linked to circular DNAs having base sequences encoding the peptides; The present invention also relates to a method comprising:

[0049] The method for creating a complex library can be carried out in the same manner as the above-mentioned method for forming complexes, except that a base sequence encoding the library-constituting peptides is used. The base sequences encoding the library-constituting peptides are not particularly limited as long as they are two or more base sequences encoding peptides, and may be base sequences encoding two or more peptides. In one embodiment, a base sequence obtained by mutating a portion of a base sequence encoding a desired peptide can be used as the base sequence encoding the library-constituting peptide. For example, two or more base sequences in which a portion of a base sequence encoding a desired peptide is randomized can be prepared and introduced into a circular DNA to prepare a circular DNA library. The type of base sequence is not particularly limited, but can be, for example, two or more types, 10 ... 4 more than species, 10 5 More than 10 species 6 The number of species can be 1 or more, and the maximum number of diversity can be the same as the number of molecules in the circular DNA library.

[0050] The circular DNA library thus obtained preferably contains at least 10 4 It is preferable that the number of circular DNA fragments contained in the DNA fragment is 10, for example, 10 6 pieces, 10 8 pieces, 10 10 pieces, 10 11 In addition, by increasing the volume, for example, 18 It may also contain one or more circular DNAs, which may contain two or more base sequences encoding peptides.

[0051] Similarly, the complex library formed from the circular DNA library thus obtained, in which translated peptides are linked to the circular DNA library, is preferably at least 10 4 Preferably, the complex contains 10 6 pieces, 10 8 pieces, 10 10 pieces, 10 11 It may contain one or more complexes, 18 In this complex library, peptides encoded by two or more different base sequences are associated with circular DNAs having base sequences encoding the respective peptides.

[0052] The present embodiment also provides a method for recovering a base sequence encoding a target-binding peptide, the method comprising: (1) preparing a circular DNA library having base sequences encoding candidate peptides for target-binding peptides that bind to a target; (2) expressing candidate peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, thereby forming a complex library in which the expressed candidate peptides are linked to circular DNAs having base sequences encoding the peptides. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) A method for detecting target-binding peptides comprising isolating and recovering circular DNA having a base sequence encoding the target-binding peptide from the target to which the complex library is bound, obtained in (3). Such a method is also referred to as a circular DNA display method.

[0053] (1) and (2) can be carried out using techniques similar to those for forming the complex library described above, except that a base sequence encoding a candidate peptide for a target-binding peptide that binds to a target is used as the base sequence encoding the peptide. The base sequence encoding the candidate peptide for a target-binding peptide that binds to a target can be, for example, a base sequence obtained by mutating a portion of a base sequence in a known base sequence encoding a target-binding peptide, such as a base sequence encoding a target-binding peptide with one or more bases deleted, substituted, or inserted. In one aspect, a circular DNA library can be prepared by preparing multiple types of base sequences in which a portion of the base sequence encoding the target-binding peptide has been randomized, and then incorporating the randomized base sequence into a circular DNA.

[0054] The complex library obtained in (2) is contacted with the target, and the target and complex library are bound via the candidate peptide linked to the complex library. This binding can be carried out using known techniques taking into account the properties of the target-binding peptide to be obtained, for example, in a buffer solution under conditions that allow binding. Suitable buffer solutions having a near-neutral pH, such as PBS and TBS, can be used.

[0055] The binding temperature and pH are not particularly limited, as long as they do not denature the candidate peptide linked to the complex library or cause it to be released from the complex library, and can be selected appropriately depending on the type of candidate peptide. In one embodiment, binding can be performed at a temperature of, for example, 0°C to room temperature, preferably 0 to 20°C, more preferably 0 to 10°C, and even more preferably around 0 to 5°C, and at a pH of 5 to 10, preferably 6 to 9, more preferably 7 to 8, and typically around pH 7.5. The binding time is also not particularly limited, and in one embodiment, binding can be performed for, for example, about 5 minutes to 12 hours, preferably 10 minutes to 6 hours, 20 minutes to 3 hours, or 30 minutes to 2 hours. The binding conditions can be adjusted appropriately depending on the target-binding molecule to be bound.

[0056] Binding conditions known in the art can be adopted depending on the type of candidate peptide. For example, binding conditions can include varying salt concentrations or adding surfactants.

[0057] When the target is insoluble, the target may be directly contacted with the complex library. However, from the viewpoint of facilitating isolation and recovery after binding, it is preferable to contact the target after immobilizing it on a carrier such as a solid phase or beads. Examples of carriers include beads such as plates made of various resins, magnetic beads, agarose beads, immunobeads, and hydrophilic beads. The target can be immobilized on the carrier by known methods, such as adding a molecule that has a binding interaction between the carrier surface and the target surface. Furthermore, when the circular DNA library contains candidate peptides for target-binding peptides for two or more different targets, binding to two or more different targets can also be performed.

[0058] The concentration of the complex library during the binding reaction is not particularly limited as long as it is a concentration that binds to the target. For example, 10 It can be about molecules / 10 μL.

[0059] In this manner, circular DNAs having a base sequence encoding the target-binding peptides are isolated and recovered from the target to which the complex library has been bound. The isolation and recovery method is not particularly limited, as long as it can isolate and recover circular DNAs having a base sequence encoding the target-binding peptides from circular DNAs having a base sequence encoding candidate peptides that did not bind to the target.

[0060] Conveniently, a complex linked to the target-binding peptide bound to the target can be eluted with an elution solution, either together with the target or together with the target-binding peptide, and the circular DNA having a nucleotide sequence encoding the target-binding peptide can be isolated and recovered together with the elution solution containing the circular DNA linked to the target-binding peptide. The elution solution is not particularly limited as long as it has a composition commonly used for elution purposes and can be selected appropriately depending on the type of candidate peptide. For example, a buffer solution with a pH of approximately 8, such as TE buffer, may be used. In one embodiment, from the standpoint of operability, if a cell-free nucleic acid amplification reaction is to be performed after elution, an elution solution that does not interfere with the progress of the reaction is preferred. However, after elution with any elution solution, the eluate may be washed and purified as necessary before being subjected to a cell-free nucleic acid amplification reaction.

[0061] The elution temperature and time are not particularly limited, and the elution can be performed, for example, at a temperature of 100°C or lower, 98°C or lower, or 95°C or lower, for about 1 to 10 minutes, preferably 3 to 10 minutes, and typically about 5 minutes.

[0062] In one embodiment, a washing step can be carried out prior to the elution to remove complexes that are not bound to the target. The washing solution can be phosphate-buffered saline (PBS), Tris-buffered saline (TBS), or a solution prepared by adding a small amount of surfactant to these buffers, such as PBST or TBST, as used under the binding and elution conditions described above. Washing can be carried out 1 to 20 times, for example, 1 to 10 times, preferably 1 to 5 times, more preferably 1 to 3 times, and even more preferably 2 or 1 times, for example, for 1 to 7 minutes, per wash. The temperature during washing is not particularly limited, but in one embodiment, free complexes can be efficiently removed by washing at 0 to 50°C, preferably about 10 to 40°C, for example, at room temperature.

[0063] The nucleotide sequence encoding the target-binding peptide can be recovered from the resulting circular DNA having the nucleotide sequence encoding the target-binding peptide. When the target and the complex library are bound using a target on a carrier such as beads, the carrier may be removed using a known method. For example, when magnetic beads are used, the magnetic beads can be removed using a magnetic separator or the like.

[0064] In a preferred embodiment, from the viewpoint of increasing the efficiency of isolation and recovery, the contacting and binding are carried out in the presence of a magnesium ion source in the range of 1 mM to 50 mM and / or an alkali metal ion source in the range of 150 mM to 1000 mM, and in one embodiment, preferably in the presence of at least a magnesium ion source in the range of 1 mM to 50 mM. In a further preferred embodiment, the contacting and binding, and isolation and recovery (and washing, if performed) are all carried out in the presence of a magnesium ion source in the range of 1 mM to 50 mM and / or an alkali metal ion source in the range of 150 mM to 1000 mM, and in one embodiment, preferably in the presence of a magnesium ion source in the range of at least 1 mM to 50 mM. In a further preferred embodiment, a series of operations from the complex obtained by transcription and translation to isolation and recovery and washing are all carried out in the presence of a magnesium ion source in the range of 1 mM to 50 mM.

[0065] The concentration of the magnesium ion source can be 1 mM or more and 50 mM or less, preferably 3 mM or more and 30 mM or less, more preferably 5 mM or more and 20 mM or less, for example, about 10 mM. The magnesium ion source is a magnesium ion (Mg 2+ ), such as Mg(OAc)2, MgCl2, and MgSO4. The preferred magnesium ion source is Mg(OAc)2.

[0066] The concentration of the alkali ion source can be 150 mM or more and 1000 mM or less, with the lower limit preferably being 200 mM or more or 250 mM or more, and the upper limit preferably being 800 mM or less, 600 mM or less, 500 mM or less, or 400 mM or less. In one embodiment, the concentration of the alkali ion source is preferably 200 mM or more and 500 mM or less, for example, about 300 mM. The alkali metal ion source is a substance that provides alkali metal ions in the reaction solution. Examples of alkali metal ions include sodium ions (Na + ), potassium ions (K +Examples of alkali metal ion sources include potassium glutamate, potassium aspartate, potassium chloride, potassium acetate, sodium glutamate, sodium aspartate, sodium chloride, and sodium acetate. A preferred alkali metal ion source is potassium glutamate or potassium acetate.

[0067] In a preferred embodiment, the contacting and binding are carried out in the absence of RNase. In a more preferred embodiment, the contacting and binding, as well as the isolation, recovery, and washing, are all carried out in the absence of RNase. Without being bound by theory, it is believed that this allows the formed complex to be maintained without degradation of the mRNA linking the expressed peptide to the nucleotide sequence encoding this peptide. RNases are enzymes that have the activity of degrading RNA, and examples include RNase A, RNase I, RNase E, RNase H, and RNase III. In one embodiment, the reaction solution preferably does not contain a double-stranded RNA-specific RNase. In one embodiment, the reaction solution preferably does not contain an RNase specific to an RNA-DNA hybrid strand. In one embodiment, the reaction solution preferably does not contain an RNase selected from RNase A, RNase H, and RNase III, and more preferably does not contain RNase A.

[0068] In one aspect of this embodiment, the isolated and recovered circular DNA having a base sequence encoding the target-binding peptide is subjected to a cell-free nucleic acid amplification reaction to amplify the base sequence encoding the target-binding peptide in the circular DNA, thereby making it possible to more reliably recover the base sequence encoding the target-binding peptide. The method used for the nucleic acid amplification reaction is not particularly limited, and examples thereof include PCR, helicase-dependent amplification (HDA) (Vincent, et al., EMBO Rep., 2004, vol.5 (8), pp.795-800), recombinase polymerase amplification (RPA) (Piepenburg, et al., PLoS. Biol., 2006, vol.4 (7), e204), rolling circle amplification (RCA) (Fire, et al., Proc. Natl. Acad. Sci., 1995, vol.92 (10), pp.4641-4645), ramification amplification (RAM) (Zhang, et al., Mol. Diagn., 2001, vol.6 (2), pp.141-150), and multiple displacement amplification (MDA) (Dean, et al., Genome Res., 2001, vol.11). (6), pp. 1095-1099, and Spits, et al., Nat. Protoc., 2006, vol. 1 (4), pp. 1965-1970), Loop-mediated isothermal amplification (LAMP) (Notomi, et al., Nucleic Acids Res., 2000, vol. 28 (12), E63), and the like can be used.

[0069] For example, when PCR is used, a primer pair can be designed to amplify a base sequence encoding a target-binding peptide, and a nucleic acid amplification reaction can be carried out.

[0070] When the circular DNA library is a library of circular DNAs having a replication initiation sequence capable of binding to an enzyme having DnaA activity, the isolated and recovered circular DNAs having a base sequence encoding the target-binding peptide can be amplified by a PCR method suitable for amplifying circular DNA, thereby easily obtaining a circular DNA library enriched for circular DNAs having a base sequence encoding the target-binding peptide. preparing a reaction mixture containing a reaction solution containing (a) a first group of enzymes that catalyze the replication of circular DNA, (b) a second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and (c) a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs, and the circular DNA having a base sequence that encodes the target-binding peptide obtained by the isolation and recovery; and incubating the reaction mixture at a temperature ranging from 20°C to 80°C; The method can be carried out by a method comprising:

[0071] The first group of enzymes that catalyze the replication of circular DNA can be, for example, the group of enzymes described in Kaguni JM & Kornberg A. Cell. 1984, 38:183-90. Specifically, the first group of enzymes can include the following: an enzyme having DnaA activity, one or more nucleoid proteins, an enzyme or enzymes having DNA gyrase activity, a single-strand binding protein (SSB), an enzyme having DnaB-type helicase activity, an enzyme having DNA helicase loader activity, an enzyme having DNA primase activity, an enzyme having DNA clamp activity, and DNA polymerase III. *Examples of the enzymes of the first group include one or more enzymes or enzymes selected from the group consisting of enzymes or enzymes having DnaA activity, single-stranded DNA binding protein (SSB), enzymes having DnaB-type helicase activity, enzymes having DNA helicase loader activity, enzymes having DNA primase activity, enzymes having DNA clamp activity, and DNA polymerase III. * It includes an enzyme or group of enzymes having the activity.

[0072] The biological origin of the enzyme having DnaA activity is not particularly limited, as long as it has initiator activity similar to that of DnaA, an initiator protein of E. coli. For example, DnaA derived from E. coli can be suitably used. DnaA derived from E. coli may be contained in the reaction solution as a monomer in a range of 1.0 nM to 10 μM, preferably 1.0 nM to 5.0 μM, 1.0 nM to 3 μM, 1 nM to 1.5 μM, 1.0 nM to 1.0 μM, 1.0 nM to 500 nM, 50 nM to 200 nM, or 50 nM to 150 nM, but is not limited thereto.

[0073] Nucleoid protein refers to a protein contained in a nucleoid. The one or more nucleoid proteins used in the present invention are not particularly limited in terms of their biological origin, as long as they are enzymes with activity similar to that of Escherichia coli nucleoid proteins. For example, Escherichia coli-derived IHF, i.e., a complex (heterodimer or homodimer) of IhfA and / or IhfB, or Escherichia coli-derived HU, i.e., a complex of hupA and hupB, can be suitably used. Escherichia coli-derived IHF may be contained in the reaction solution as a hetero / homodimer in a range of 5 nM to 400 nM, preferably 5 nM to 200 nM, 5 nM to 100 nM, 5 nM to 50 nM, 10 nM to 50 nM, 10 nM to 40 nM, or 10 nM to 30 nM, but is not limited thereto. HU derived from E. coli may be contained in the reaction solution in a range of 1 nM to 50 nM, preferably 5 nM to 50 nM, or 5 nM to 25 nM, but is not limited thereto.

[0074] The biological origin of the enzyme or enzymes having DNA gyrase activity is not particularly limited, as long as they have an activity similar to that of E. coli DNA gyrase. For example, a complex consisting of GyrA and GyrB derived from E. coli can be suitably used. The complex consisting of GyrA and GyrB derived from E. coli may be contained in the reaction solution as a heterotetramer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 50 nM to 200 nM, or 100 nM to 200 nM, but is not limited thereto.

[0075] The single-stranded DNA binding protein (SSB) is not particularly limited in terms of its biological origin, as long as it is an enzyme with activity similar to that of the single-stranded DNA binding protein of Escherichia coli. For example, SSB derived from Escherichia coli can be suitably used. SSB derived from Escherichia coli may be contained in the reaction solution as a homotetramer in a range of 20 nM to 1000 nM, preferably 20 nM to 500 nM, 20 nM to 300 nM, 20 nM to 200 nM, 50 nM to 500 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 50 nM to 150 nM, 100 nM to 500 nM, or 100 nM to 400 nM, but is not limited thereto.

[0076] The biological origin of the enzyme having DnaB-type helicase activity is not particularly limited, as long as it has activity similar to that of DnaB from Escherichia coli. For example, DnaB derived from Escherichia coli can be suitably used. DnaB derived from Escherichia coli may be contained in the reaction solution as a homohexamer in a range of 5 nM to 200 nM, preferably 5 nM to 100 nM, 5 nM to 50 nM, or 5 nM to 30 nM, but is not limited thereto.

[0077] The biological origin of the enzyme having DNA helicase loader activity is not particularly limited, as long as it has activity similar to that of E. coli DnaC. For example, E. coli-derived DnaC can be suitably used. E. coli-derived DnaC may be contained in the reaction solution as a homohexamer in a range of 5 nM to 200 nM, preferably 5 nM to 100 nM, 5 nM to 50 nM, or 5 nM to 30 nM, but is not limited thereto.

[0078] The biological origin of the enzyme having DNA primase activity is not particularly limited, as long as it has activity similar to that of E. coli DnaG. For example, E. coli-derived DnaG can be suitably used. E. coli-derived DnaG may be contained in the reaction solution as a monomer in a range of 20 nM to 1000 nM, preferably 20 nM to 800 nM, 50 nM to 800 nM, 100 nM to 800 nM, 200 nM to 800 nM, 250 nM to 800 nM, 250 nM to 500 nM, or 300 nM to 500 nM, but is not limited thereto.

[0079] The biological origin of the enzyme having DNA clamping activity is not particularly limited, as long as it has activity similar to that of E. coli DnaN. For example, E. coli-derived DnaN can be suitably used. E. coli-derived DnaN may be contained in the reaction solution as a homodimer in a range of 10 nM to 1000 nM, preferably 10 nM to 800 nM, 10 nM to 500 nM, 20 nM to 500 nM, 20 nM to 200 nM, 30 nM to 200 nM, or 30 nM to 100 nM, but is not limited thereto.

[0080] DNA polymerase III * As an enzyme or group of enzymes having this activity, DNA polymerase III from Escherichia coli *The biological origin of the enzyme or enzymes is not particularly limited, as long as they have the same activity as the complex. For example, an enzyme group containing any of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli, preferably an enzyme group containing a complex of DnaX, HolA, HolB, and DnaE derived from Escherichia coli, and more preferably an enzyme group containing a complex of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli, can be suitably used. The DNA polymerase III* complex derived from Escherichia coli may be contained in the reaction solution as a heteromultimer in a range of 2 nM to 50 nM, preferably 2 nM to 40 nM, 2 nM to 30 nM, 2 nM to 20 nM, 5 nM to 40 nM, 5 nM to 30 nM, or 5 nM to 20 nM, but is not limited thereto.

[0081] In the present invention, two sister circular DNAs forming a catenane refer to two circular DNAs that are synthesized by a DNA replication reaction and are in a linked state.

[0082] Examples of the second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes include one or more enzymes selected from the group consisting of enzymes with DNA polymerase I activity, enzymes with DNA ligase activity, and enzymes with RNase H activity, or a combination of such enzymes. In one embodiment, the second group of enzymes preferably includes an enzyme with DNA polymerase I activity and an enzyme with DNA ligase activity.

[0083] The biological origin of the enzyme having DNA polymerase I activity is not particularly limited, as long as it has activity similar to that of E. coli DNA polymerase I. For example, E. coli-derived DNA polymerase I can be suitably used. E. coli-derived DNA polymerase I may be contained in the reaction solution as a monomer in a range of 10 nM to 200 nM, preferably 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 40 nM to 150 nM, 40 nM to 100 nM, or 40 nM to 80 nM, but is not limited thereto.

[0084] The enzyme having DNA ligase activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DNA ligase. For example, E. coli-derived DNA ligase or T4 phage DNA ligase can be suitably used. E. coli-derived DNA ligase may be contained in the reaction solution as a monomer in a range of 10 nM to 200 nM, preferably 15 nM to 200 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, or 20 nM to 80 nM, but is not limited thereto.

[0085] The biological origin of the enzyme having RNase H activity is not particularly limited as long as it has the activity of degrading the RNA strand of an RNA:DNA hybrid. For example, RNase H derived from Escherichia coli can be suitably used. RNase H derived from Escherichia coli may be contained in the reaction solution as a monomer in a range of 0.2 nM to 200 nM, preferably 0.2 nM to 200 nM, 0.2 nM to 100 nM, 0.2 nM to 50 nM, 1 nM to 200 nM, 1 nM to 100 nM, 1 nM to 50 nM, or 10 nM to 50 nM, but is not limited thereto.

[0086] Examples of the third enzyme group that catalyzes the separation reaction of the two sister circular DNAs include the enzyme group described in Peng H & Marians KJ. PNAS. 1993, 90: 8571-8575. Specifically, the third enzyme group can include one or more enzymes selected from the group consisting of enzymes with topoisomerase IV activity, enzymes with topoisomerase III activity, and enzymes with RecQ-type helicase activity, or a combination of such enzymes. In one embodiment, the third enzyme group preferably includes an enzyme with topoisomerase IV activity and / or an enzyme with topoisomerase III activity.

[0087] The biological origin of the enzyme having topoisomerase III activity is not particularly limited, as long as it has activity similar to that of Escherichia coli topoisomerase III. For example, Escherichia coli-derived topoisomerase III can be suitably used. Escherichia coli-derived topoisomerase III may be contained in the reaction solution as a monomer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 20 nM to 100 nM, or 30 to 80 nM, but is not limited thereto.

[0088] The biological origin of the enzyme having RecQ helicase activity is not particularly limited, as long as it has activity similar to that of E. coli RecQ. For example, E. coli-derived RecQ can be suitably used. E. coli-derived RecQ may be contained in the reaction solution as a monomer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 20 nM to 100 nM, or 30 to 80 nM, but is not limited thereto.

[0089] The biological origin of the enzyme having topoisomerase IV activity is not particularly limited, as long as it has activity similar to that of Escherichia coli topoisomerase IV. For example, Escherichia coli-derived topoisomerase IV, which is a complex of ParC and ParE, can be suitably used. Escherichia coli-derived topoisomerase IV may be contained in the reaction solution as a heterotetramer in a range of 0.1 nM to 50 nM, preferably 0.1 nM to 40 nM, 0.1 nM to 30 nM, 0.1 nM to 20 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, 1 nM to 10 nM, or 1 nM to 5 nM, but is not limited thereto.

[0090] The reaction solution may further contain an enzyme. For example, when the circular DNA to be amplified by PCR has the above-mentioned ter sequence, the reaction solution may further contain a protein (e.g., Tus protein derived from Escherichia coli) that has the activity of binding to the ter sequence and inhibiting replication.

[0091] The reaction solution may further contain a linear DNA-specific exonuclease, which reduces the amount of linear DNA generated by double-strand breaks during the amplification reaction and improves the yield of the desired circular DNA.

[0092] A linear DNA-specific exonuclease is an enzyme that sequentially hydrolyzes linear DNA from the 5' or 3' end. There are no particular limitations on the type or biological origin of the linear DNA-specific exonuclease, as long as it has the activity of sequentially hydrolyzing linear DNA from the 5' or 3' end. Examples of usable exonucleases include RecBCD (exonuclease V), λ exonuclease, exonuclease III, exonuclease VIII, T5 exonuclease, T7 exonuclease, and Plasmid-Safe® ATP-Dependent DNase (epicentre). The linear DNA exonuclease may be contained in the reaction solution at a concentration of 0.001 to 1.0 U / μL, preferably 0.005 to 1.0 U / μL, 0.01 to 1.0 U / μL, 0.05 to 1.0 U / μL, or 0.1 to 1.0 U / μL, but is not limited thereto. The enzymatic activity unit (U) of the linear DNA exonuclease is defined as the amount of enzyme required to acid-soluble 1 nmol of deoxyribonucleotides in linear DNA in a reaction at 37°C for 30 minutes.

[0093] The reaction solution may further contain a single-stranded DNA-specific exonuclease, which has the effect of reducing the amount of low-molecular-weight by-products generated during the amplification reaction and improving the yield of the desired circular DNA.

[0094] A single-stranded DNA-specific exonuclease is an enzyme that sequentially hydrolyzes nucleotides at the 5' or 3' end of single-stranded DNA. There are no particular limitations on the type or biological origin of the single-stranded DNA-specific exonuclease, as long as it has the activity of sequentially hydrolyzing nucleotides at the 5' or 3' end of single-stranded DNA. For example, exonuclease I (exo I), RecJ, exonuclease T, etc. can be used. A preferred single-stranded DNA-specific exonuclease is exo I. The single-stranded DNA-specific exonuclease may be contained in the reaction solution in a range of 0.1 to 1.0 U / μL, preferably 0.15 to 1.0 U / μL, 0.2 to 1.0 U / μL, or 0.2 to 0.5 U / μL, but is not limited thereto. The enzyme activity unit (U) for exo I is defined as the amount of enzyme required to render 10 nmol of deoxyribonucleotides from single-stranded DNA acid-soluble in a 30-minute reaction at 37°C. The enzyme activity unit (U) for RecJ is defined as the amount of enzyme required to render 0.05 nmol of deoxyribonucleotides from single-stranded DNA acid-soluble in a 30-minute reaction at 37°C.

[0095] The enzymes contained in the reaction solution containing the first, second, and third enzyme groups may be commercially available enzymes, or may be extracted from microorganisms and purified as necessary. Extraction and purification of enzymes from microorganisms can be carried out appropriately using techniques available to those skilled in the art.

[0096] When enzymes other than those derived from Escherichia coli are used as the first, second, and third enzyme groups, they can be used in a concentration range corresponding in terms of enzyme activity units to the concentration range specified for the enzymes derived from Escherichia coli.

[0097] The reaction solution may contain a buffer, ATP, GTP, CTP, UTP, dNTP, a magnesium ion source, and an alkali metal ion source.

[0098] The buffer solution contained in the reaction solution is not particularly limited as long as it is suitable for use at pH 7 to 9, preferably pH 8. Examples include Tris-HCl, Tris-OAc, Hepes-KOH, phosphate buffer, MOPS-NaOH, and Tricine-HCl. Preferred buffers are Tris-HCl or Tris-OAc. The concentration of the buffer can be appropriately selected by those skilled in the art and is not particularly limited. In the case of Tris-HCl or Tris-OAc, for example, concentrations of 10 mM to 100 mM, 10 mM to 50 mM, or 20 mM can be selected.

[0099] ATP means adenosine triphosphate. The concentration of ATP contained in the reaction solution at the start of the reaction may be, for example, in the range of 0.1 mM to 3 mM, and preferably in the range of 0.1 mM to 2 mM, 0.1 mM to 1.5 mM, or 0.5 mM to 1.5 mM.

[0100] GTP, CTP, and UTP mean guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, respectively. The concentrations of GTP, CTP, and UTP contained in the reaction solution at the start of the reaction may each independently be in the range of, for example, 0.1 mM to 3.0 mM, preferably 0.5 mM to 3.0 mM, and 0.5 mM to 2.0 mM.

[0101] dNTP is a collective term for deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP). The concentration of dNTP contained in the reaction solution at the start of the reaction may be, for example, in the range of 0.01 to 1 mM, preferably 0.05 to 1 mM, or 0.1 to 1 mM.

[0102] The magnesium ion source is a magnesium ion (Mg 2+) is a substance that provides magnesium ions. Examples include Mg(OAc)2, MgCl2, and MgSO4. A preferred magnesium ion source is Mg(OAc)2. The concentration of the magnesium ion source contained in the reaction solution at the start of the reaction may be, for example, a concentration that provides magnesium ions in the range of 5 to 50 mM in the reaction solution.

[0103] The alkali metal ion source is a substance that provides alkali metal ions to the reaction solution. Examples of alkali metal ions include sodium ions (Na + ), potassium ions (K + ) Examples of alkali metal ion sources include potassium glutamate, potassium aspartate, potassium chloride, potassium acetate, sodium glutamate, sodium aspartate, sodium chloride, and sodium acetate. A preferred alkali metal ion source is potassium glutamate or potassium acetate. The concentration of the alkali metal ion source contained in the reaction solution at the start of the reaction may be a concentration that provides alkali metal ions in the reaction solution at 100 mM or more, preferably in the range of 100 mM to 300 mM, but is not limited thereto.

[0104] The reaction solution may further contain inhibitors of non-specific adsorption of proteins (bovine serum albumin, lysozyme, gelatin, heparin, casein, etc.), inhibitors of non-specific adsorption of nucleic acids (transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), glycogen, heparin, oligo-DNA, poly(IC) (polyinosine-polycytidine), poly(dI-dC) (polydeoxyinosine-polydeoxycytidine), poly(A) (polyadenine), and poly(dA) (polydeoxyadenine), etc.), linear DNA-specific exonucleases (RecBCD, λ exonuclease, exonuclease III, exonuclease VIII, T5 exonuclease, T7 exonuclease, Plasmid-Safe® ATP-Dependent The reaction mixture may contain DNase (epicentre), RecG-type helicase (RecG derived from Escherichia coli, etc.), ammonium salts (ammonium sulfate, ammonium chloride, ammonium acetate, etc.), and reducing agents (DTT, β-mercaptoethanol, glutathione, etc.). If the reaction mixture contains DNA ligase derived from Escherichia coli, it may also contain its cofactor, NAD (nicotinamide adenine dinucleotide).

[0105] The reaction solution is mixed with the circular DNA to form a reaction mixture, and the reaction mixture is incubated at a temperature in the range of 80° C. or less to amplify the circular DNA. The amplification may be isothermal or non-isothermal.

[0106] In the case of isothermal amplification, the isothermal conditions are not particularly limited as long as they allow the DNA amplification reaction or DNA replication reaction to proceed. For example, a certain temperature within the optimal temperature range of the DNA polymerase can be used. Examples of isothermal conditions include a certain temperature of 15°C or higher, 16°C or higher, 18°C ​​or higher, 20°C or higher, 23°C or higher, 24°C or higher, 25°C or higher, 26°C or higher, 27°C or higher, or 30°C or higher, and a certain temperature of 80°C or lower, 75°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, 50°C or lower, 45°C or lower, 40°C or lower, 35°C or lower, or 33°C or lower. Furthermore, the isothermal conditions may be, for example, a constant temperature within the range of 15°C to 80°C, 16°C to 80°C, 18°C ​​to 80°C, 20°C to 80°C, or 25°C to 80°C; a constant temperature within the range of 15°C to 75°C, 16°C to 75°C, 18°C ​​to 75°C, 20°C to 75°C, or 25°C to 75°C; a constant temperature within the range of 15°C to 70°C, 16°C to 70°C, 18°C ​​to 70°C, 20°C to 70°C, or 25°C to 70°C; a constant temperature within the range of 15°C to 65°C, 16°C to 65°C, 18°C ​​to 65°C, 20°C to 65°C, or 25°C to 65°C; a constant temperature within the range of 25°C to 50°C; a constant temperature within the range of 25°C to 40°C; a constant temperature within the range of 30°C to 33°C; or about 30°C. As used herein, "isothermal" in isothermal amplification means maintaining the temperature within a range of ±7°C, ±5°C, ±3°C, or ±1°C relative to the temperature set during the reaction. The reaction time for isothermal amplification can be appropriately set depending on the amount of the target circular DNA amplification product, and can be, for example, 1 hour to 30 hours, preferably 2 hours to 25 hours, more preferably 3 hours to 20 hours, and even more preferably 5 hours to 15 hours. In one aspect, the circular DNA of this embodiment can be amplified to a high concentration even in a short reaction time.

[0107] Non-isothermal amplification of circular DNA can be performed under temperature cycle conditions, in which incubation is repeated at two temperatures below 80°C, preferably below 65°C. The first temperature in the temperature cycle is a temperature at which replication initiation of double-stranded DNA can be initiated, and the second temperature is a temperature at which replication initiation is suppressed and DNA extension reaction proceeds. The first temperature can be 30°C or higher, for example, 30°C to 80°C, 30°C to 50°C, 30°C to 40°C, or 37°C. The incubation time at the first temperature is not particularly limited, but may be 10 seconds to 10 minutes per cycle, preferably 1 minute. The second temperature can be 27°C or lower, for example, 10°C to 27°C, 16°C to 25°C, or 24°C. The incubation time at the second temperature is not particularly limited, but is preferably set according to the length of the circular DNA to be amplified, and may be, for example, 1 second to 10 seconds per 1,000 bases per cycle. The number of temperature cycles is not particularly limited, but may be 10 to 50 cycles, 20 to 45 cycles, 25 to 45 cycles, or 40 cycles.

[0108] The method of this embodiment may further include, after the step of amplifying the circular DNA by incubation at a temperature in the range of 80°C or less, a step of diluting the reaction solution by at least 2-fold, preferably 3-fold, 4-fold, or 5-fold, with a reaction solution not containing the first to third enzyme groups, and then maintaining (re-incubating) the solution. While dilution of the enzyme groups suppresses new replication initiation, ongoing replication extension, catenation, and separation reactions continue due to the effects of the remaining enzymes. Furthermore, by-products generated by nicks or other defects during the reaction can be repaired during this process by the effects of residual ligase, etc. Therefore, the transition from amplification intermediates and by-products to the final product is specifically guided, and an improved yield of the desired circular DNA can be expected.

[0109] The dilution can be maintained under conditions in which the second enzyme group and the third enzyme group can act, for example, at 15 to 50°C, preferably 20 to 40°C, more preferably 25 to 35°C, for example, 10 minutes to 3 hours, preferably 15 minutes to 2 hours, more preferably 20 minutes to 1 hour.

[0110] The present embodiment also relates to a method for recovering a base sequence encoding a target-binding peptide, wherein the recovered eluate in the method for recovering a base sequence encoding a target-binding peptide is used to further carry out the following steps: That is, in this embodiment, after isolating and recovering a circular DNA having a base sequence encoding a target-binding peptide, (5) subjecting the isolated and recovered circular DNA having a base sequence encoding the target-binding peptide to a cell-free nucleic acid amplification reaction to amplify the base sequence encoding the target-binding peptide; The circular DNA containing the amplified base sequence is used as a circular DNA library. (2) expressing candidate peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, and forming a complex library in which the expressed candidate peptides are linked to the circular DNA library. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) isolating and recovering circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, obtained in (3). The present invention also relates to a method for recovering a nucleic acid sequence encoding a target-binding peptide.

[0111] The circular DNA having a nucleotide sequence encoding the target-binding peptide thus obtained may be subjected again to the cell-free nucleic acid amplification reaction of (5) above to amplify the circular DNA having a nucleotide sequence encoding the target-binding peptide, and the above steps (2), (3), and (4) may be repeated.

[0112] The present embodiment also relates to a method for recovering a base sequence encoding a target-binding peptide, wherein the circular DNA library has a replication initiation sequence capable of binding to an enzyme having DnaA activity, and the recovered eluate is used to further carry out the following steps: That is, in this embodiment, after collecting an eluate containing a circular DNA having a base sequence encoding a target-binding peptide, (5') preparing a reaction mixture containing a reaction solution containing (a) a first group of enzymes that catalyze the replication of circular DNA, (b) a second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and (c) a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs, and the isolated and recovered circular DNA having a base sequence that encodes the target-binding peptide; and incubating the reaction mixture at a temperature ranging from 20°C to 80°C; amplifying circular DNA having a base sequence encoding the target-binding peptide by a PCR method comprising the steps of: (2) expressing candidate peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, and forming a complex library in which the expressed candidate peptides are linked to the circular DNA library. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) isolating and recovering circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, obtained in (3). The present invention also relates to a method for recovering a nucleic acid sequence encoding a target-binding peptide.

[0113] The circular DNA having a nucleotide sequence encoding the target-binding peptide thus obtained may be subjected again to the cell-free nucleic acid amplification reaction of (5') above to amplify the circular DNA having a nucleotide sequence encoding the target-binding peptide, and the above steps (2), (3), and (4) may be repeated.

[0114] In this way, circular DNA having a base sequence encoding a target-binding peptide that binds to the target is recovered and amplified, and the above steps (2) to (4) are repeated to select the target-binding peptide again, thereby concentrating and selecting DNA encoding the target-binding peptide. In one aspect, according to the recovery method of this embodiment, 10 4 More than double, say 10 6 This allows for twice the enrichment of nucleotide sequences encoding target-binding peptides. Figure 13 shows a comparison of the method of this embodiment with conventional display methods such as phage display, mRNA display, and CIS display using linear DNA.

[0115] Compared to conventional CIS display methods, the cyclic display method of this embodiment allows for repeated selection of target-binding peptides with great ease. For example, in conventional CIS display methods, a library is created by linking sequences encoding candidate target-binding peptides with sequences required for CIS display (typically repA-CIS-oriR). After transcription-translation reactions, target binding, and post-binding isolation and purification, the library for the next round must be prepared by partially amplifying only the sequences encoding the candidate target-binding peptides and then linking them to the sequences required for CIS display. In contrast, the cyclic display method of this embodiment and the CIS display method using circular DNA eliminate the need for such linking steps for each round. Furthermore, even if washing steps are required after binding to the target, the number of steps can be significantly reduced. As shown in the examples below, the method of this embodiment requires only three washes, whereas conventional CIS display methods require six to twelve washes.

[0116] Furthermore, according to the circular display method of this embodiment and the CIS display method using circular DNA, it is possible to introduce mutations into the sequence encoding the target-binding peptide after each round of amplification, thereby further increasing the library size. For example, referring to International Publication No. 2020 / 027110, circular DNA and single-stranded DNA for mutagenesis can be subjected to a cell-free amplification reaction, such as the PCR method, to amplify the DNA editing product and increase the library size.

[0117] The present embodiment also provides a method for recovering a base sequence encoding a target-binding peptide, the method comprising: (1) Providing a circular DNA library having a base sequence encoding a candidate peptide of a target-binding peptide that binds to a target, a DNA target sequence, a base sequence encoding a base peptide that non-covalently binds to the DNA target sequence, and a DNA element that provides cis activity, wherein the candidate peptide is expressed linked to the base peptide; (2) expressing candidate peptides and base peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, and forming a complex library in which the base peptides linked to the expressed candidate peptides are non-covalently bound to a DNA target sequence on a circular DNA having a base sequence encoding the candidate peptides. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) isolating and recovering circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, obtained in (3); This method is also called a CIS display method using circular DNA.

[0118] In a preferred aspect of this embodiment, the steps (3) and (4) in the CIS display method using circular DNA are: in the presence of a magnesium ion source in the range of 1 mM to 50 mM, and / or In the presence of an alkali metal ion source in the range of 150 mM to 1000 mM, More preferably, the steps (3) and (4) in the CIS display method using circular DNA are carried out in the presence of an alkali metal ion source in the range of 150 mM to 1000 mM.

[0119] The circular DNA library (1) in the CIS display method using circular DNA differs from the circular display method of this embodiment in that it has the following (a) to (d). Regarding (b) to (d), reference can be made to known CIS display methods (Patent Document 1, Patent Document 2, and Non-Patent Document 4). (a) Nucleotide sequence encoding a candidate peptide of a target-binding peptide that binds to a target: See the description of the cyclic display method above. (b) DNA target sequence: The presence of a DNA element that confers cis-activity (d) results in a non-covalent bond between the expression peptide and the DNA target sequence from the sequence encoding the platform peptide (c). Such a DNA target sequence can be an ori sequence, such as the oriR sequence. (c) A base sequence encoding a platform peptide that non-covalently binds to the DNA target sequence: a typical example of such a platform peptide is repA. When the candidate peptide (a) is linked to this platform peptide and expressed, the candidate peptide is displayed on the circular DNA at one end of the platform peptide that is non-covalently bound to the DNA target sequence. (d) DNA element that confers cis activity: has cis activity that establishes a non-covalent bond between the DNA target sequence (b) and the sequence encoding the base peptide (c), and an example thereof is CIS.

[0120] The circular DNA library may have a replication initiation sequence capable of binding to an enzyme having DnaA activity. In one embodiment, the DNA element conferring cis activity is preferably located between the base sequence encoding the base peptide and the DNA target sequence in the circular DNA library.

[0121] (2) in the CIS display method using circular DNA can be carried out by expressing peptides using a cell-free transcription-translation reaction, with reference to the circular DNA display method described above. Among the expressed peptides, the platform peptide non-covalently binds to the DNA target sequence to form a complex library.

[0122] (3) in the CIS display method using circular DNA can be carried out by contacting the complex library with the target and binding the target to the complex library via the candidate peptide linked to the complex library, with reference to the above-mentioned circular DNA display method. Preferably, this contact and binding is carried out in the presence of a magnesium ion source in the range of 1 mM to 50 mM and / or an alkali metal ion source in the range of 150 mM to 1000 mM, and in one embodiment, preferably in the presence of an alkali metal ion source in the range of at least 150 mM to 1000 mM.

[0123] (4) in the CIS display method using circular DNA can be carried out by isolating and recovering circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, with reference to the above-mentioned circular DNA display method. This isolation and recovery is preferably carried out in the presence of a magnesium ion source in the range of 1 mM to 50 mM and / or an alkali metal ion source in the range of 150 mM to 1000 mM, and in one embodiment, preferably in the presence of an alkali metal ion source in the range of at least 150 mM to 1000 mM.

[0124] The concentration and type of magnesium ion source in preferred embodiments of (3) and (4) are as explained in the circular DNA display method above. Without being bound by theory, it is believed that by performing (3) and (4) in the CIS display method using circular DNA in the presence of a magnesium ion source at a predetermined concentration, the complex library formed in the CIS display method using circular DNA becomes more stable, and the base sequences encoding the target-binding peptides can be recovered even when the concentration of the base sequences encoding the target-binding peptides is low.

[0125] The concentration and type of alkali metal ion source in the preferred embodiments of (3) and (4) are as explained in the circular DNA display method above. Without being bound by theory, it is believed that by performing (3) and (4) in the CIS display method using circular DNA in the presence of a predetermined concentration of an alkali metal ion source, the complex library formed in the CIS display method using circular DNA becomes more stable, and the base sequences encoding the target-binding peptides can be recovered even when the concentration of the base sequences encoding the target-binding peptides is low.

[0126] In one aspect, this embodiment is a CIS display method using the above-mentioned circular DNA, wherein the circular DNA library has a replication initiation sequence capable of binding to an enzyme having DnaA activity, and further (5') (a) preparing a reaction mixture containing a reaction solution containing a first group of enzymes that catalyze the replication of circular DNA, (b) a second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and (c) a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs, and the circular DNA having a base sequence that encodes the target-binding peptide isolated and recovered in (4) above; and incubating the reaction mixture at a temperature ranging from 20°C to 80°C; and amplifying the circular DNA having the base sequence encoding the target-binding peptide by a PCR method comprising the steps of:

[0127] This embodiment also relates to a method for recovering a nucleotide sequence encoding a target-binding peptide, which involves repeating steps (2), (3), and (4) above by using the circular DNA amplified in step (5') above as a circular DNA library. In this way, by combining the CIS display method using circular DNA with the PCR method, circular DNA containing a nucleotide sequence encoding a target-binding peptide of interest can be recovered effectively with even fewer operations.

[0128] This embodiment also relates to a method for producing a target-binding peptide, which comprises amplifying a base sequence encoding the target-binding peptide from circular DNA containing the base sequence encoding the target-binding peptide, recovered by any of the methods described above, and then transcribing and translating the obtained base sequence to produce the target-binding peptide. The methods for amplifying, transcribing, and translating the base sequence are not particularly limited, and can be carried out with reference to the above descriptions.

[0129] This embodiment also relates to DNA recovered by the method of this embodiment and target-binding peptides produced by the method of this embodiment. The base sequence of these DNAs or the amino acid sequence of these peptides may be further modified.

[0130] This embodiment also provides a complex library in which library-constituting peptides and base sequences encoding the library-constituting peptides are linked together, comprising: (i) a circular DNA library having a base sequence encoding the library-constituting peptide and a replication initiation sequence capable of binding to an enzyme having DnaA activity; (ii) the library constituent peptides; wherein (i) and (ii) are connected via mRNA.

[0131] Such a complex library can be obtained by the method for preparing a complex library described above. In one embodiment, the (i) circular DNA library and the (ii) library constituent peptides are linked to mRNA via ribosomes, and the mRNA is linked to the circular DNA library via RNA polymerase. Such a complex library is useful for searching for novel highly functional molecules, since the peptides correspond to the base sequences encoding the peptides.

[0132] This embodiment also provides a method for detecting a target-binding peptide comprising: a DNA target sequence; a base sequence encoding a base peptide that non-covalently binds to the DNA target sequence; a DNA element that confers cis activity; a replication initiation sequence capable of binding to an enzyme having DnaA activity; A circular DNA having the formula: the DNA element that confers cis activity is located between the base sequence encoding the base peptide and the DNA target sequence; The present invention also relates to circular DNA in which the candidate peptide is expressed in conjunction with the platform peptide.

[0133] For each component, please refer to the above-mentioned descriptions of the CIS display method and the RCR method performed using circular DNA. By using such circular DNA as a circular DNA library, it is possible to efficiently perform a method for recovering base sequences encoding target-binding peptides by combining the CIS display method and the RCR method performed using circular DNA, which is useful for searching for novel highly functional molecules, etc.

[0134] This embodiment also relates to a conjugate of a target and a target-binding peptide, the target-binding peptide is tethered to a circular DNA via an mRNA; The present invention also relates to a conjugate wherein the circular DNA has a base sequence encoding the target-binding peptide.

[0135] Such conjugates can be obtained by referring to (1) to (3) in the method for recovering a base sequence encoding a target-binding peptide described above. In one embodiment, the target-binding peptide is associated with a circular DNA via mRNA linked to an RNA polymerase on the circular DNA. The base sequence encoding the target-binding peptide can be recovered from such conjugates. Because the target-binding peptide corresponds to the base sequence encoding the peptide, such conjugates are useful for, for example, discovering novel highly functional molecules. [Example]

[0136] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. The method for producing the plasmids used in the examples is shown in Preparation Examples.

[0137] [Example 1] In this example, it was demonstrated that protein display can be achieved independently of CISoriR by using circular DNA.

[0138] (Example 1-1: CIS display method using circular DNA and PCR method) RepA, the replication initiator protein of the R1 plasmid, binds to the downstream oriR in cis after transcription and translation. The CIS mechanism requires a CIS sequence located between the RepA gene and oriR. A technique (CIS display) has been reported in which a peptide library is displayed on linear DNA by fusing the peptide library to RepA in advance using this CIS mechanism (Non-Patent Document 4). Here, we constructed an evolutionary molecular engineering system that combines amplification detection with the RCR method by using circular DNA encoding oriC, which is necessary for the RCR method, in addition to RepA, CIS, and oriR used in the CIS display method (In vitro display-RCR method described below, Figure 1(A)).

[0139] First, we constructed two types of supercoiled circular DNAs (pHis-RepA_CISoriR (4.1 kb) and pFLAG-RepA_CISoriR (3.8 kb)) in which a His-tag or a FLAG-tag was fused to the N-terminus of RepA as a display peptide.

[0140] These circular DNAs were divided into pFLAG-RepA_CISoriR / pHis-RepA_CISoriR (pFLAG / pHis) molecules in a ratio of 10 -2 The mixture was mixed to obtain a final concentration, and added to the in vitro transcription / translation system, Mu-Cell-kun (manufactured by Taiyo Nippon Sanso Corporation), for protein expression (Figure 1(A), in vitro transcription translation (TXTL)). The promoter encoded in the circular DNA is the sigma70 (s70) promoter, which is acted upon by E. coli RNA polymerase, and transcription is initiated by the E. coli RNA polymerase contained in Mu-Cell-kun.

[0141] Subsequently, immunoprecipitation was performed using anti-FLAG antibodies (Fig. 1(A): selection), and the recovered product was added to the PCR reaction mixture for amplification (Fig. 1(A): PCR amplification). The reaction mixture (0.33 μL) was analyzed by 1.25% agarose gel electrophoresis and SYBR Green staining.

[0142] The results are shown in Figure 1(C) (CISoriR lane). A band for pFLAG-RepA_CISoriR (3.8 kb) was detected, while pHis-RepA_CISoriR, which was 100 times more abundant, was barely detected. This confirmed that the RepA CIS mechanism was responsible for the display of FLAG-tagged RepA specifically expressed on its own template circular DNA, and that the circular DNA was co-recovered (i.e., CIS display using circular DNA).

[0143] (Example 1-2: Circular DNA lacking the Rho-dependent transcription terminator in the CIS sequence) Next, the Rho-dependent transcription terminator present within the CIS sequence (Fig. 1(B), T Rho) was considered.

[0144] T Rho The FLAG-expressing circular DNA (pFLAG-RepA_ΔT) was prepared by replacing the rrnBT1 terminator with the Rho-independent strong terminator, rrnBT1 (Orosz et al., (1991) Eur. J. Biochem. Vol. 201, pp. 653-659). Rho ::rrnBT1, 3.8 kb) was constructed (Figure 1(B), ΔT Rho ::rrnBT1).

[0145] Instead of pFLAG-RepA_CISoriR in Example 1-1, this pFLAG-RepA_ΔT Rho Except for using ::rrnBT1, it was mixed with pHis-RepA_CISoriR and a CIS display-RCR experiment was carried out in the same manner as described in Example 1-1.

[0146] The results are shown in Figure 1(C) (ΔT Rho ::rrnBT1 lane). Unlike the results in Example 1-1, pFLAG-RepA_ΔT Rho The ::rrnBT1 (3.8 kb) band was not detected, but the pHis-RepA_CISoriR (4.1 kb) band was. This indicates that the Rho-dependent transcription terminator within the CIS sequence is important for CIS display. The presence of such a strong terminator may have led to the dissociation of RNA polymerase from DNA, preventing the display of expressed proteins on DNA.

[0147] (Example 1-3: Circular DNA in which the full-length CIS and the full-length oriR have been deleted) A circular DNA (pFLAG-RepA_ΔCISoriR, 3.5 kb) lacking the full-length CIS and the full-length oriR was constructed (Figure 1(B), ΔCISoriR). Except for using this pFLAG-RepA_ΔCISoriR instead of pFLAG-RepA_CISoriR in Example 1-1, the method described in Example 1-1 was followed. pHis-RepA_CISoriR was mixed with it, and the CIS display-RCR experiment was performed.

[0148] The results are shown in Figure 1(C) (lane of ΔCISoriR). The band of pFLAG-RepA_ΔCISoriR (3.5 kb) was detected, and almost no pHis-RepA_CISoriR, which is 100 times that amount, was detected. Similar to the results described in Example 1-1, selective recovery of pFLAG-RepA_ΔCISoriR was observed.

[0149] From these results, it was shown that even when the CISoriR region, which is an essential region in the conventional CIS display, is removed, by using circular DNA as a template, a display mechanism different from the CIS display functions, and it is possible to present the expressed protein. By using circular DNA as a template, it was considered that even without the CIS mechanism, RNA polymerase was retained on the DNA, and the expressed protein could be presented on the DNA.

[0150] The circular DNA used in this example is as follows. · pHis-RepA_CISoriR (4.1 kb) · pFLAG-RepA_CISoriR (3.8 kb) · pFLAG-RepA_ΔT Rho ::rrnBT1 (3.8 kb) · pFLAG-RepA_ΔCISoriR (3.5 kb)

[0151] <in vitro display-RCR method> The template DNA solution was subjected to cell-free protein expression as described below, followed by immunoprecipitation of the expressed protein and PCR amplification of the co-recovered circular DNA.

[0152] <Cell-free protein expression> Transcription-translation reactions were performed using Mu-Cell-Kun N (manufactured by Taiyo Nippon Sanso Corporation) according to the manufacturer's instructions. The reaction was performed in 5 μL, and 1 μL of a circular DNA solution mixed at an arbitrary ratio was added to 4 μL (0.8x volume) of Mu-Cell-Kun premix to prepare the reaction solution. The DNA solution was added to a final concentration of 0.05 nM. The transcription-translation reaction was performed at 30°C for 30 minutes and then stopped by cooling on ice for at least 5 minutes.

[0153] <Immunoprecipitation of expressed proteins> Experiments were performed using anti-DYKDDDDK tag antibody magnetic beads (Fujifilm Wako Pure Chemical Industries, Ltd.). First, 5 μL of 1x PBS buffer (1.47 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 2.68 mM KCl) was added to the total transcription / translation product (5 μL) and mixed thoroughly (2-fold dilution). 15 μL of the bead suspension was mixed with 300 μL of 1x PBS, and the supernatant was removed using a magnet separator. 10 μL of the prepared transcription / translation reaction dilution was added to the beads and mixed thoroughly using a vortex. The mixture was then shaken at 1000 rpm at 4°C for 1 hour in an Eppendorf ThermoMixer. The supernatant was removed from the beads using a magnet separator, and the beads were washed three times with 500 μL of 1x PBST (1x PBS, 0.05% (v / v) Tween 20). Then, 25 μL of TE (pH 8.0) (Nippon Gene) was added, mixed well, and then incubated at 95° C. for 5 minutes to elute the DNA. The beads were removed using a magnet separator, and the supernatant was used as an immunoprecipitated DNA sample.

[0154] <Detection of co-recovered circular DNA by PCR amplification> PCR amplification was carried out in 5 μL, and the mixture contained 200 mU / μL ExoI (NEB) and 200 mU / μL ExoV (NEB), but did not contain Tus, essentially following the method described in Non-Patent Document 5. 0.5 μL of the immunoprecipitated DNA sample was added (5 μL) to a PCR reaction solution that had been preincubated at 33°C for 30 minutes, and amplification was carried out at 33°C for 2 hours. This product was diluted 5-fold with the PCR reaction buffer and incubated at 30°C for 30 minutes (finalization) to obtain the final amplified product. 0.33 μL of this final amplified product was used for 1.25% agarose gel electrophoresis and detection by SYBR Green staining.

[0155] The composition of the PCR reaction solution other than ExoI and ExoV is shown in Table 1 below.

[0156] [Table 1]

[0157] In Table 1, SSB is SSB from E. coli, IHF is a complex of IhfA and IhfB from E. coli, DnaG is DnaG from E. coli, DnaN is DnaN from E. coli, Pol III* is a DNA polymerase III* complex consisting of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE from E. coli, DnaB is DnaB from E. coli, DnaC is DnaC from E. coli, DnaA is DnaA from E. coli, RNaseH is RNaseH from E. coli, Ligase is DNA ligase from E. coli, Pol I is DNA polymerase I from E. coli, GyrA is GyrA from E. coli, GyrB is GyrB from E. coli, Topo IV is a complex of ParC and ParE from E. coli, Topo III represents Escherichia coli-derived topoisomerase III, and RecQ represents Escherichia coli-derived RecQ.

[0158] SSB was prepared by purifying it from an Escherichia coli expression strain of SSB through a process including ammonium sulfate precipitation and ion exchange column chromatography. IHF was prepared from an Escherichia coli strain co-expressing IhfA and IhfB by purification using a process including ammonium sulfate precipitation and affinity column chromatography. DnaG was purified and prepared from an E. coli expression strain of DnaG by a process including ammonium sulfate precipitation, anion exchange column chromatography, and gel filtration column chromatography. DnaN was purified and prepared from an E. coli expression strain of DnaN by a process including ammonium sulfate precipitation and anion exchange column chromatography. Pol III* was purified and prepared from an E. coli strain co-expressing DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE by a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaB and DnaC were purified and prepared from an E. coli strain co-expressing DnaB and DnaC by a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaA was purified and prepared from an E. coli expression strain of DnaA through a process including ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography. GyrA and GyrB were purified and prepared from a mixture of E. coli expression strains of GyrA and GyrB by a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo IV was prepared by purifying a mixture of Escherichia coli strains expressing ParC and ParE through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo III was prepared by purifying it from an E. coli expression strain of Topo III through a process including ammonium sulfate precipitation and affinity column chromatography. RecQ was purified and prepared from an E. coli expression strain by a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. RNase H, Ligase, and Pol I were commercially available enzymes derived from Escherichia coli (manufactured by Takara Bio Inc.).

[0159] [Example 2] In this example, it was demonstrated that a circular DNA structure is required for CISoriR-independent display.

[0160] It was confirmed whether the circular structure of DNA was necessary for the CISoriR-independent display in Examples 1-3. As shown in Figure 2(A), the full length of the circular DNAs (pFLAG-RepA_CISoriR and pFLAG-RepA_ΔCISoriR) used in the experiments of Examples 1-1 and 1-3 was amplified by PCR to obtain linear DNAs (L_FLAG-RepA_CISoriR and L_FLAG-RepA_ΔCISoriR).

[0161] For these two linear DNAs and two circular DNAs, the ratio of pFLAG / pHis was 10 -2 The resulting mixture was mixed with the circular pHis-RepA_CISoriR so that the total amount of the chromatogram was 1000 ppm, and protein expression and immunoprecipitation were carried out in the same manner as described in Example 1.

[0162] The DNA eluted with 10 μL of TE (pH 8.0) was subjected to PCR amplification using the primer pair shown in Figure 2(A) (see Preparation Example for sequences), and the recovery of DNA encoding FLAG and His was detected.

[0163] The results are shown in Figure 2(B). DNA containing CISoriR was shown to selectively recover DNA encoding FLAG, regardless of whether it was linear or circular. On the other hand, in the case of DNA lacking CISoriR (ΔCISoriR), recovery of FLAG-encoding DNA was not observed with linear DNA, but was observed only with circular DNA.

[0164] These results indicate that the circular structure of DNA is necessary for CISoriR-independent display. As shown in Figure 2(A), in linear DNA, RNA polymerase falls off from the DNA end, preventing the display of the expressed protein corresponding to the DNA. In contrast, in circular DNA, RNA polymerase remains tethered to the DNA, and the expressed protein is displayed via mRNA and ribosomes.

[0165] The DNA used in this example is as follows: pHis-RepA_CISoriR (4.1 kb) L_FLAG-RepA_CISoriR (3.8kb) pFLAG-RepA_CISoriR (3.8 kb) ·L_FLAG-RepA_ΔCISoriR(3.5kb) pFLAG-RepA_ΔCISoriR (3.5 kb)

[0166] <pcr> In this example, a PCR reaction (10 μL) was carried out on 1 μL of the immunoprecipitated DNA sample using KOD One Master Mix (manufactured by TOYOBO CORPORATION) and the primer pair shown in Table 2 below (SUE10802 and SUE13144, both at a final concentration of 0.3 μM).

[0167] [Table 2]

[0168] [Example 3] In this example, it was demonstrated that proteins are displayed via mRNA in CISoriR-independent display.

[0169] In the CISoriR-independent display observed in Example 1, it was confirmed whether the expressed protein was displayed via mRNA.

[0170] pHis-RepA_CISoriR5.6ATmin (4.1 kb) and pFLAG-RepA_CISoriR5.6ATmin (3.8 kb) have CISoriR and oriC5.6ATmin as the replication origin of PCR. On the other hand, pHis-RepA_ΔCISoriR5.6ATmin (3.7 kb) and pFLAG-RepA_ΔCISoriR5.6ATmin (3.4 kb) have the same sequences as the above two types of plasmids except that they do not contain CISoriR.

[0171] Among these circular DNAs, pairs of circular DNAs with the same expressed proteins other than His or FLAG were selected as pHis / pFLAG pairs. -3 The mixture was mixed to a ratio of 0.5 nM to 10 μL, and protein expression was carried out in accordance with the method described in Example 1. The changes from Example 1 were that the reaction volume was doubled (10 μL), the final concentration of the circular DNA was increased to 0.5 nM, and Tween 20 was added to a final concentration of 0.5%.

[0172] Next, RNase A was added directly to the protein expression reaction solution, and the RNA degradation reaction described below was carried out (Figure 3(A)). Immunoprecipitation was carried out in the same manner as described in Example 1, except that the entire RNA degradation reaction solution was mixed with antibody beads without dilution with 1x PBS.

[0173] The recovered product was detected by PCR amplification. PCR amplification was performed using 1 μL of immunoprecipitated DNA sample, with the reaction volume doubled (10 μL). Furthermore, the PCR enzymes used were all reduced to half their concentrations, and Tween 20 was added to the buffer to a final concentration of 0.05%. Furthermore, the amplification reaction time was set to 1 hour. Other conditions were the same as those described in Example 1.

[0174] The results are shown in Figure 3(B). When DNA carrying CISoriR was used, selective recovery of DNA encoding FLAG was observed regardless of the concentration of RNase A (Fig. 3(B), CISoriR+). This suggests that CIS display is functional and that RepA directly binds to DNA, making it unaffected by RNase A (Fig. 3(A)).

[0175] On the other hand, with DNA lacking CISoriR, the pFLAG band was faint at an RNaseA concentration of 1 nM, and was not detected at RNaseA concentrations of 10 and 100 nM (Fig. 3(B), CISoriR-). It was thought that with DNA lacking CISoriR, the mRNA that connected the expressed protein to the circular DNA was degraded by RNaseA, preventing the co-recovery of the circular DNA encoding FLAG (Fig. 3(A)).

[0176] These results indicate that CISoriR-independent display is achieved through mRNA-mediated protein presentation.

[0177] The DNA used in this example is as follows: ·pHis-RepA_CISoriR5.6ATmin(4.1kb) ·pHis-RepA_ΔCISoriR5.6ATmin (3.7 kb) ·pFLAG-RepA_CISoriR5.6ATmin (3.8 kb) ·pFLAG-RepA_ΔCISoriR5.6ATmin (3.4 kb)

[0178] <RNA degradation reaction> In this example, a RNA degradation reaction solution (11 μL) was prepared by adding 1 μL of RNase A (manufactured by QIAGEN) diluted with 1x PBS to a cell-free protein expression reaction solution (10 μL). The reaction was carried out at 30 °C for 30 minutes to degrade the RNA.

[0179] [Example 4] In this example, it was shown that CISoriR-independent display is possible even with open circular DNA. The circular DNAs used in Examples 1 and 2 are supercoiled. It was examined whether CISoriR-independent protein presentation is possible with open circular DNA.

[0180] Both pFLAG-RepA (3.4 kb) and pHis-RepA (3.7 kb) do not have CISoriR and are plasmids having oriC5.6ATmin, a modified form of oriC, as the replication origin of RCR. Within oriC5.6ATmin, recognition sequences of nicking enzymes (Nt.BspQI and Nb.BbvCI) exist as single recognition sites in circular DNA (Figure 4(A)).

[0181] The ratio of pFLAG / pHis is 10 -4 pFLAG-RepA and pHis-RepA were mixed so as to be, and a cleavage reaction was carried out with a nicking enzyme (Nt.BspQI or Nb.BbvCI) described below. Thereafter, protein expression, immunoprecipitation, and amplification detection by RCR were carried out in the same manner as the method described in Example 1. Note that the protein expression and immunoprecipitation reactions were carried out using in vitro display-RCR ver2 described below.

[0182] The results are shown in Figure 4(B). Analysis of the PCR amplification products (lane "RCR products" in Figure 4(B)) confirmed that pFLAG-RepA was specifically recovered in samples that underwent protein synthesis and immunoprecipitation (+TXTL & IP), regardless of whether or not nickase restriction enzyme reaction was performed. On the other hand, in samples where PCR amplification of mixed and restriction enzyme-treated template DNA was performed directly without protein expression and immunoprecipitation (-TXTL & IP), pHis-RepA was predominantly present.

[0183] Furthermore, when the two types of DNA were mixed and the template DNA digested with restriction enzymes was directly electrophoresed (lane "template DNA" in Figure 4(B)), the circular DNA remained supercoiled in the sample incubated at 37°C without restriction enzymes (- restriction enzyme) and in the sample incubated at 37°C without restriction enzymes (- restriction enzyme incubation). On the other hand, in the sample incubated at 37°C with restriction enzymes (+ restriction enzyme incubation), the circular DNA was confirmed to be open-circular, regardless of whether Nt.BspQI (Nt) or Nb.BbvCI (Nb) was used.

[0184] These results demonstrate that circular DNA, whether supercoiled or open-circular, can display expressed proteins.

[0185] The DNA used in this example is as follows: pHis-RepA (3.7 kb) pFLAG-RepA (3.4 kb)

[0186] <Cleavage reaction with nickase restriction enzyme> In this example, Nt.BspQI (NEB) or Nb.BbvCI (NEB) (both at a final concentration of 0.2 Units / μL) was mixed with a DNA solution at a final concentration of 5 nM in NEBuffer r3.1 or 1xrCutsmartBuffer (10 μL reaction), and the reaction was carried out at 37°C for 5 hours.

[0187] <in vitro display-RCR ver2> The changes from the in vitro display-RCR described in Example 1 are as follows.

[0188] <Cell-free protein expression> The reaction volume was increased twice (10 μL), the final concentration of circular DNA was increased to 0.5 nM, and Tween 20 was added to a final concentration of 0.5%.

[0189] <Immunoprecipitation> The dilution ratio of the transcription / translation product was increased 5-fold, the bead volume was reduced to 5 μL, and the washing buffer volume was reduced to 100 μL. Furthermore, 1x PBS Buffer was replaced with 1x PBS_300NaCl_Mg Buffer (1.47 mM KH2PO4, 2.68 mM KCl, 8.1 mM Na2HPO4, 300 mM NaCl, 10 mM Mg(OAc)2) for all steps. Furthermore, the DNA elution conditions were changed to 70°C for 5 minutes. <<RCR>> The reaction volume was doubled (10 μL) using 1 μL of immunoprecipitated DNA sample. Furthermore, the PCR enzymes were reduced to half their concentrations, and Tween 20 was added to the buffer to a final concentration of 0.05%. Furthermore, the preincubation time was changed to 37°C for 10 minutes, the amplification time to 37°C for 1 hour, and the finalization time to 30°C for 15 minutes.

[0190] [Example 5] In this example, the selection efficiency for template DNA recovery in CISoriR-independent display was evaluated. We named this display method, which does not rely on the CIS mechanism, "circular DNA display," and quantitatively evaluated its selection efficiency for target DNA.

[0191] As shown in Table 3 below, pFLAG-RepA (3.4 kb) and pHis-RepA (3.7 kb) were used in the same manner, with a pFLAG / pHis ratio of 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , or 10 -8 The mixture was mixed so that the concentration was 100% and in vitro display-RCR ver2 was carried out in the same manner as in Example 4. -3 A control experiment in which protein synthesis did not occur (4°C, 30 minutes) was also carried out for the mixture.

[0192] [Table 3]

[0193] The results are shown in Figure 5. The ratio of pFLAG / pHis is 10 -3 , 10 -4 , 10 -5 and 10 -6 pFLAG-RepA (3.4 kb) was recovered from the sample of pFLAG / pHis=10, which did not undergo protein synthesis. -3 In the sample (reaction temperature of TXTL 4°C in Figure 5), no recovery of pFLAG-RepA was observed.

[0194] These results suggest that the ratio of pFLAG / pHis in circular DNA display is 10 -6 , i.e. 10 9 10 for molecule pHis-RepA 3 It was found that DNA could be selectively recovered from pFLAG-RepA, which is a molecule, via the displayed protein. Furthermore, the enrichment rate of the target circular DNA in this experiment was approximately 10 6 It was double.

[0195] The DNA used in this example is as follows: pHis-RepA (3.7 kb) pFLAG-RepA (3.4 kb)

[0196] [Example 6] In this example, the buffer composition was examined in circular DNA display and CIS display using the same circular DNA and RCR method as in Example 1-1.

[0197] As shown in Table 4 below, for pFLAG-RepA (3.4 kb) and pHis-RepA (3.7 kb) used in Example 5, which do not have CIS and oriR, the ratio of pFLAG / pHis was 10 -4 , 10 -5 and 10 -6 The plasmids were mixed so that the total volume was 1000 kJ / ml.

[0198] [Table 4]

[0199] In addition, pFLAG-RepA_CISoriR5.6ATmin (3.8 kb) and pHis-RepA_CISoriR5.6ATmin (4.1 kb), which were obtained by adding CIS and oriR to each plasmid, also showed a pFLAG / pHis ratio of 10 -4 , 10 -5 and 10 -6 The respective plasmids were mixed so that the following was obtained: In vitro display-RCRver2 was performed for each mixture in the same manner as described in Example 4.

[0200] Separately, a similar mixture of pFLAG-RepA_CISoriR5.6ATmin (3.8 kb) and pHis-RepA_CISoriR5.6ATmin (4.1 kb) plasmids was used to perform in vitro display-RCR ver1. * ver1 * In this case, the dilution ratio of the transcription / translation product in the immunoprecipitation was 2x. Furthermore, the buffer used was changed from 1x PBS_300NaCl_Mg buffer to 1x PBS buffer. Other than that, the experiment was carried out in the same manner as in in vitro display-RCRver2.

[0201] The results are shown in Figure 6. As shown in Figure 6(A), similar to the results described in Example 4, recovery of pFLAG-RepA (3.4 kb) was detected in all samples of circular DNA display (ΔCISoriR / ver2). In circular DNA display, a high salt concentration and / or the inclusion of a magnesium ion source is thought to enable efficient recovery of target DNA even at low concentrations.

[0202] As shown in Figure 6(B), in version 2 of CIS display using circular DNA and PCR, recovery of pFLAG-RepA_CISoriRmin5.6AT (3.8 kb) was detected in all samples, and the ratio of pFLAG / pHis was 10 -6 Comparing the results of the samples, the enrichment efficiency was higher than that of circular DNA display (CISoriR / ver2). In addition, ver1 returned to 1xPBS buffer * In the case of CISoriR / ver1, pFLAG was not detected in any of the samples. * ).

[0203] From the results of Figure 6(B), the buffer for in vitro display-RCR using CIS display was ver1. * The difference between ver. 1 and ver. 2 is that the salt concentration is high and / or the magnesium ion source is included. Additional experiments have shown that the high salt concentration is particularly important (data not shown). Furthermore, a comparison with Example 1-1 shows that the concentration in Example 1-1 (10 -2 ), the concentration of these components in the buffer is less important, but as in this example, low concentrations (10 -4 In the case of ( ), the concentrations of these components in the buffer were thought to be important in CIS display using circular DNA and RCR.

[0204] The DNA used in this example is as follows: ·pHis-RepA_CISoriR5.6ATmin(4.1kb) pHis-RepA (3.7 kb) ·pFLAG-RepA_CISoriR5.6ATmin(3.8kb) pFLAG-RepA (3.4 kb)

[0205] [Example 7] In this example, it was confirmed that the selection efficiency was improved by repeating the selection rounds of circular DNA display. In Example 5, 10 -6 Therefore, we investigated whether the selection efficiency of the target circular DNA could be further increased by repeating the in vitro display-RCR rounds of the selected and amplified DNA products (Figure 7(A)).

[0206] pFLAG-RepA (3.4 kb) and pHis-RepA (3.7 kb) were cloned into pFLAG / pHis = 10 -8 The mixture was mixed at a ratio of 0.01 to 0.1, and the final concentration of circular DNA was 5 nM, and 100 μL of in vitro transcription / translation reaction was performed. 10 μL of TE (pH 8.0) was used for elution. A total of 20 μL of PCR amplification reaction was performed using the entire eluate. The other conditions were the same as those for in vitro display-RCR ver. 2, which targeted the FLAG-tag, as described in Example 4.

[0207] The entire amount of the first-round amplified product was purified by ethanol precipitation, and the entire amount of the obtained product was used in the second-round in vitro transcription-translation reaction. In vitro display-RCR was performed in the same manner as in the first round to obtain the second-round amplified product. In addition, as a control, pFLAG / pHis=10 -8 Simultaneously, a direct PCR amplification experiment was carried out on a DNA mixture containing 100% ribonucleotides.

[0208] The results are shown in Figure 7(B). In the control (0R) without protein synthesis and immunoprecipitation and the first round (1R) sample, only pHis was detected, and no pFLAG recovery was detected, whereas in the second round (2R) sample, pFLAG recovery was observed.

[0209] These results demonstrate that the selection efficiency for recovering target circular DNA can be improved by repeating the selection rounds in circular DNA display. In addition, in this example, the enrichment rate in two rounds of circular DNA display was 10 8 times, and on average, the enrichment rate per round is 10 4 It was double.

[0210] The DNA used in this example is as follows: pHis-RepA (3.7 kb) pFLAG-RepA (3.4 kb)

[0211] [Example 8] This example concerns circular DNA display using different targets. In the circular DNA display of Examples 1 to 7, selection was performed using a FLAG-tag peptide as a target, but we also investigated whether selective template DNA could be recovered using circular DNA display targeting a 6xHis-tag peptide.

[0212] pHis-RepA (3.7 kb) and pFLAG-RepA (3.4 kb) were synthesized as shown in Table 5 below, with pHis / pFLAG at 10 -2 , 10 -3 , 10 -4 or 10 -5 The mixture was mixed in a ratio of

[0213] [Table 5]

[0214] In vitro display-RCR ver2 was performed in the same manner as described in Example 4. In this case, Ni beads were used instead of anti-FLAG antibody beads, and a pull-down assay targeting the His-tag was performed (Figure 8, + TXTL & Ni-pull down). In addition, pHis / pFLAG was used at 10 -2 The DNA solution mixed at a ratio of 100:1 was directly amplified by PCR, and a control experiment was performed without protein synthesis or pull-down assay (Figure 8, - TXTL & Ni-pull down).

[0215] The results are shown in Figure 8. Through transcription-translation reaction and pull-down assay, the pHis / pFLAG ratio was 10 -2 and 10 -3 Selective recovery of pHis-RepA (3.7 kb) was detected in the case of . Selective recovery of template DNA by circular DNA display was possible even when peptides other than the FLAG-tag were used as targets.

[0216] The DNA used in this example is as follows: pHis-RepA (3.7 kb) pFLAG-RepA (3.4 kb)

[0217] [Example 9] In this example, proteins expressed in circular DNA display were compared. In the circular DNA display of Examples 1 to 8, RepA was expressed cell-free and displayed on template DNA. Here, because circular DNA display does not depend on the CIS mechanism, we investigated whether proteins other than RepA could be displayed on template DNA.

[0218] Circular DNAs expressing E. coli SecM or Superfolder GFP (sfGFP) instead of RepA were constructed for pFLAG-RepA (3.4 kb) and pHis-RepA (3.7 kb), respectively (Figure 9(A)). These DNAs were named pFLAG-GFP (3.3 kb), pHis-GFP (3.6 kb), pFLAG-SecM (3.1 kb), and pHis-SecM (3.4 kb).

[0219] Among these circular DNAs, pairs of circular DNAs with the same expressed proteins other than His or FLAG were selected as pHis / pFLAG pairs. -5 The ExoI and ExoV were mixed at a ratio of 1:1. In vitro display-RCR ver. 2 targeting the FLAG-tag was performed in the same manner as described in Example 4 (Figure 9(B), + TXTL & IP). The RCR reaction was performed at a concentration of 100 mU / µL for ExoI and ExoV in the RCR reaction solution.

[0220] Also, pHis / pFLAG was 10 -5 The DNA solution mixed at a ratio of 1:1 was directly amplified by PCR, and a control experiment was carried out in which protein expression and immunoprecipitation were not performed (Figure 9(B), -TXTL & IP).

[0221] The results are shown in Figure 9(B). When circular DNAs engineered to express SecM or sfGFP were used instead of RepA, selective recovery of circular DNAs encoding the FLAG tag was detected via cell-free protein expression and immunoprecipitation. Circular DNA display was also possible when general proteins other than RepA were expressed.

[0222] The DNA used in this example is as follows: pHis-RepA (3.7 kb) pFLAG-RepA (3.4 kb) pHis-sfGFP (3.6 kb) pFLAG-sfGFP (3.3 kb) pHis-SecM (3.4 kb) pFLAG-SecM (3.1 kb)

[0223] [Example 10] This example demonstrated that circular DNA display is also possible using transcription by T7 RNAP or transcription-translation by a reconstituted system. In Examples 1 to 9, protein expression was carried out using E. coli RNA polymerase and translation factors contained in an E. coli extract fraction using the sigma70 promoter. In these Examples, we investigated whether the expressed protein could be presented on a circular DNA template even when transcription by T7 RNA polymerase from the T7 promoter and a transcription-translation system reconstituted in vitro using purified proteins were used.

[0224] For pFLAG-RepA (3.4 kb) and pHis-RepA (3.7 kb), the sigma70 promoter (P s70 ) to the T7 promoter (P T7 pFLAG-RepA_T7 (3.4 kb) and pHis-RepA_T7 (3.7 kb) were constructed in which the nucleotide sequence of the FLAG-RepA_T7 gene was replaced with the nucleotide sequence of the nucleotide sequence of the FLAG-RepA_T7 gene.

[0225] Among the circular DNAs, pairs of circular DNAs that are identical except for His or FLAG are identified by pHis / pFLAG. -4 The circular DNA was added to Cell-Free Kun (M-kun) or the PURE system (Gene Frontier) (described below) (10 μL each) to a final concentration of 0.5 nM, and protein expression was carried out (Figure 10(A)).

[0226] The Cell-Free System contains both E. coli RNA polymerase and T7 RNA polymerase. Since the PURE system contains only T7 RNA polymerase, experiments were performed using circular DNA encoding the sigma70 promoter by adding E. coli RNA polymerase to the PURE system. Other conditions were the same as those for in vitro display-RCR ver. 2 targeting the FLAG-tag in Example 4. A control experiment was also performed in which the mixed DNA solution was directly amplified by RCR without protein expression or immunoprecipitation (Figure 10(B), - TXTL & IP).

[0227] The results are shown in Figure 10(B). When transcription was performed using T7 RNA polymerase or when a transcription-translation reaction was reconstituted in vitro using purified proteins, the target circular DNA encoding the FLAG tag could be selectively recovered, similar to the results described in Examples 1 to 9.

[0228] These results indicate that circular DNA display can be achieved using a reconstituted transcription-translation system, and that it can be performed without relying on a specific transcription mechanism. Furthermore, when using the sigma70 promoter and Cell-Free-Kun, the pHis (3.7 kb) band was barely detected, suggesting that the combination of transcription by E. coli RNA polymerase and transcription-translation using E. coli extract fractions was the most efficient system tested.

[0229] The DNA used in this example is as follows: pHis-RepA (3.7 kb) pFLAG-RepA (3.4 kb) pHis-RepA_T7 (3.7 kb) pFLAG-RepA_T7 (3.4 kb)

[0230] <PURE system> In this example, PUREfrex 2.0 (Gene Frontier) was used as the PURE system. 10 μL of reaction solution was prepared by adding 1 μL of DNA solution to 9 μL of premix. The compositions of the sample with T7 promoter DNA and the sample with s70 promoter DNA were as follows: T7: Solution I (1 / 2 vol), Solution II (1 / 20 vol), Solution III (1 / 10 vol) (as per the manual). s70: SolutionI (1 / 2 vol), SolutionII (1 / 20 vol), SolutionIII (1 / 10 vol), 65 nM E.coli RNA Polymerase Holoenzyme (manufactured by NEB). The circular DNA solution was added so that the final concentration of the circular DNA was 0.5 nM, and the transcription-translation reaction was carried out at 37°C for 30 minutes.

[0231] [Example 11] In this example, the influence of transcription terminators on the selection efficiency of target circular DNA in circular DNA display was compared.

[0232] (Example 11-1) Circular DNA encoding His-GFP as an expression protein (pHis-GFPv2, 3.3 kb) and circular DNA encoding FLAG-SUMO-GFP as an expression protein (pFLAG-GFPv2, 3.6 kb) were mixed at a pFLAG / pHis ratio of 10 -4 The mixture was mixed so that the total amount of the target circular DNA was 1.5 kb, and in vitro display-RCR ver. 2 was performed in the same manner as described in Example 4. As a result, the band of the target circular DNA pFLAG (3.6 kb) was detected more strongly than the band of pHis (3.3 kb) (Figure 11(B), ΔCISoriR).

[0233] (Example 11-2) Each of the circular DNAs used in Example 11-1 contained a Rho-dependent terminator T immediately downstream of the sfGFP gene. Rho pFLAG-GFPv2_T, a circular DNA containing Rho (3.7kb) and pHis-GFPv2_T Rho A 3.4 kb circular DNA fragment was prepared (Fig. 11(A)). These circular DNA fragments were mixed and subjected to in vitro display-PCR in the same manner as described in Example 11-1. As a result, pFLAG was recovered by selection with an efficiency comparable to that of ΔCISoriR in Example 11-1 (Fig. 11(B), TRho).

[0234] (Example 11-3) For each of the circular DNAs used in Example 11-1, a CIS sequence was further inserted immediately downstream of the sfGFP gene to construct circular DNAs pFLAG-GFPv2_CIS (3.8 kb) and pHis-GFPv2_CIS (3.5 kb) (Figure 11(A)). Rho In addition, it is known to contain two Rho-independent weak terminators (Masai & Arai, NAR, 1988, Vol. 16(14A), p. 6493-6514). These circular DNAs were mixed and subjected to in vitro display-PCR in the same manner as described in Example 11-1. As a result, pFLAG was recovered by selection with an efficiency similar to that of ΔCISoriR in Example 11-1 (Figure 11(B), CIS).

[0235] (Example 11-4) For each of the circular DNAs used in Example 11-1, the Rho-dependent terminator λtR1 (Chen, CYA & Richardson, JP, JBC, 1987) was further inserted immediately downstream of the sfGFP gene to construct circular DNAs pFLAG-GFPv2_λtR1 (3.8 kb) and pHis-GFPv2_λtR1 (3.5 kb) (Figure 11(A)). These circular DNAs were mixed and subjected to in vitro display-PCR in the same manner as described in Example 11-1. As a result, pFLAG was recovered by selection with an efficiency similar to that of ΔCISoriR in Example 11-1 (Figure 11(B), λtR1).

[0236] The results of Examples 11-1 to 11-4 demonstrated that circular DNA display is possible even when a Rho-independent weak terminator (Example 11-3) or a Rho-dependent terminator (Examples 11-1 and 11-4) is encoded.

[0237] (Example 11-5) For each of the circular DNAs used in Example 11-1, circular DNAs were constructed by inserting rrnBT1, a terminator with strong Rho-independence. For each of the circular DNAs, two types ((1) and (2) below) with different insertion sites were constructed (Figure 11(A)). (1) pFLAG-GFPv2_0-rrnBT1 3.7 kb and pHis-GFPv2_0-rrnBT1 3.4 kb inserted immediately downstream of the sfGFP gene (2) pFLAG-GFPv2_2454-rrnBT1 3.7 kb and pHis-GFPv2_2454-rrnBT1 3.4 kb inserted downstream, 2454 bp away from the sfGFP gene.

[0238] The above (1) and (2) were mixed in the same manner as described in Example 11-1, and in vitro display-PCR was performed. When inserted immediately downstream of sfGFP, pHis was predominantly amplified, resulting in a reduced selective recovery efficiency for pFLAG, i.e., the target circular DNA (Figure 11(B), 0 bp / rrnBT1). On the other hand, when inserted at a position distant from sfGFP, pFLAG was selectively recovered with an efficiency comparable to that of ΔCISoriR (Figure 11(B), 2454 bp / rrnBT1).

[0239] These results indicate that a strong Rho-independent terminator located immediately downstream of the gene for a protein to be expressed inhibits circular DNA display, but not when it is located at a distance. The absence of a strong Rho-independent terminator located immediately downstream (within approximately 100 bp) of the gene for a protein to be expressed is considered to be important for circular DNA display.

[0240] The DNA used in this example is as follows: pFLAG-GFPv2 (3.6kb) pHis-GFPv2 (3.3kb) pFLAG-GFPv2_T Rho (3.7kb) pHis-GFPv2_T Rho (3.4kb) pFLAG-GFPv2_CIS (3.8kb) pHis-GFPv2_CIS (3.5kb) pFLAG-GFPv2_λtR1 (3.8 kb) pHis-GFPv2_λtR1(3.5kb) pFLAG-GFPv2_0-rrnBT1 (3.7 kb) pHis-GFPv2_0-rrnBT1 (3.4 kb) ·pFLAG-GFPv2_2454-rrnBT1(3.7kb) ·pHis-GFPv2_2454-rrnBT1(3.4kb)

[0241] [Preparation example] The plasmids and DNA preparation methods used in the above examples are shown below.

[0242] <Plasmid1:pHis-RepA_CISoriR(4.1kb)> The plasmid map is shown in FIG. The following DNA fragments were ligated using the Recombination Assembly (RA) method (International Publication No. 2019 / 009361). Each DNA fragment was mixed at an equal molecular weight ratio and added to RA V Buffer to a final concentration of 4 ng / μL to prepare 5 μL of RA reaction solution (1x RA V Buffer). This reaction solution was subjected to a DNA ligation reaction at 42°C for 30 minutes, incubated at 65°C for 2 minutes, and then transferred to ice for rapid cooling.

[0243] The RA reaction solution (1x RA V Buffer) contained 1 μM wild-type RecA (purified from a RecA-expressing E. coli strain using a process including polyethyleneimine precipitation, ammonium sulfate precipitation, and affinity column chromatography), 80 mU / μL exonuclease III (2170A, TaKaRa Bio), 1 U / μL exonuclease I (M0293, New England Biolabs), 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM tetramethylammonium chloride (TMAC), 5% by mass PEG8000, 10% by volume DMSO, 20 ng / μL creatine kinase (Product No. 10127566001, Sigma-Aldrich), and 4 mM creatine phosphate. The concentration of each component in the RA reaction solution is the concentration relative to the total volume of the RA ligation reaction solution.

[0244] A total of 5 μL of the resulting ligation product was added to prepare 50 μL of PCR reaction solution (composition shown in Table 1 + 200 mU / μL Exonuclease I (NEB) and 200 mU / μL Exonuclease V (NEB)). The PCR reaction solution was warmed at 33°C for 30 minutes before adding the ligation product. The ligation product was added and mixed thoroughly, and then amplification was carried out at 33°C for 5 hours. The entire amplification product was then diluted 5-fold with the reaction buffer shown in Table 1, and finalization was carried out at 30°C for 30 minutes. The entire finalization product (250 μL) was purified using a QIAprep® Spin Miniprep Kit (QIAGEN).

[0245] <Fragments used for linking> The fragments used for ligation were each provided with 20-40 base pair homologous ends to the adjacent fragments. The order of the fragments listed corresponds to the order of ligation. Fragment 1 (RepA-CIS-oriR fragment) The cloning plasmid carrying linker, repA, CIS, oriR, LacZα (219-379), terB, oriC WT, terB, and lacZ was used as a template and PCR amplification was performed using the following primers.

[0246] [Table 6]

[0247] [Table 7]

[0248] Fragment 2 (lacZ-s70 promoter fragment) The cloning plasmid containing LacZ and the s70 promoter was used as a template and PCR amplification was performed using the following primers.

[0249] [Table 8]

[0250] [Table 9]

[0251] Plasmid 1 (pHis-RepA_CISoriR) contains the following five fragments (1-i) to (1-v).

[0252] (1-i) s70 promoter-His cassette

[0253] [Table 10]

[0254] (1-ii)GGGGS-RepA-CIS-oriR The sequence is amplified by PCR using the following primers and the chromosome MG::71CW-Δ (Maisnier-Patin et al., Molecular Microbiology (1998) Vol. 30, No. 5, pp. 1067-1079) as a template.

[0255] [Table 11]

[0256] (1-iii)lacZα The sequence was amplified by PCR using the following primers and pBeloBAC11 (GenBank Accession #: U51113) as a template.

[0257] [Table 12]

[0258] (1-iv) terB-oriC-terB cassette

[0259] [Table 13]

[0260] (1-v)lacZ-2292 The sequence was amplified by PCR using the following primers and the E. coli MG1655 strain genome (NCBI Accession #: NZ_AKBV01000001) as a template.

[0261] [Table 14]

[0262] <Plasmid2:pFLAG-RepA_CISoriR(3.8kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is composed of the following three fragments (2-i) to (2-iii).

[0263] (2-i) s70 promoter cassette

[0264] [Table 15]

[0265] (2-ii) Tag cassette

[0266] [Table 16]

[0267] (2-iii) RepA-LacZ cassette The sequence is amplified by PCR using the following primers and plasmid 1 as a template. The lowercase letters indicate the overlapping portion with the s70 promoter cassette.

[0268] [Table 17]

[0269] <Plasmid3:pFLAG-RepA_ΔT Rho ::rrnBT1(3.8kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (3-i) and (3-ii).

[0270] (3-i)Ps70-rrnBT1 The sequence is amplified by PCR using the following primers and plasmid2 as a template. The lowercase letters indicate the overlapping portion with rrnBT1-LacZ.

[0271] [Table 18]

[0272] (3-ii)rrnBT1-LacZ The sequence is amplified by PCR using the following primers and plasmid2 as a template. The lowercase letters indicate the overlapping portion with Ps70-rrnBT1.

[0273] [Table 19]

[0274] <Plasmid4:pFLAG-RepA_ΔCISoriR(3.5kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (4-i) and (4-ii).

[0275] (4-i)Ps70-repA The sequence is amplified by PCR using the following primers and plasmid2 as a template. The lowercase letters indicate the overlapping portion with rrnBT1-LacZ.

[0276] [Table 20]

[0277] (4-ii) LacZα-LacZ The sequence is amplified by PCR using the following primers and plasmid2 as a template. The lowercase letters indicate the overlapping portion with Ps70-rrnBT1.

[0278] [Table 21]

[0279] <LinearDNA1:L_FLAG-RepA_CISoriR(3.8kb)> The fragment was prepared by PCR amplification using the following primers and plasmid 2 as a template, using the KOD one kit (manufactured by TOYOBO). The amplified product was purified using NucleoSpin Gel and PCR Clean-up (TaKaRa) according to the manual.

[0280] [Table 22]

[0281] <LinearDNA2:L_FLAG-RepA_ΔCISoriR(3.5kb)> It was prepared by PCR amplification using the same primers and kit as for LinearDNA1 and plasmid4 as a template. The amplification product was purified using the same kit as LinearDNA1 according to the manual.

[0282] <Plasmid5:pHis-RepA(3.7kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following five fragments (5-i) to (5-v).

[0283] (5-i) promoter-SUMO-His cassette

[0284] [Table 23]

[0285] (5-ii)GSx3-RepA The sequence was amplified by PCR using the following primers and the chromosome MG::71CW-Δ mentioned in (1-ii) as a template.

[0286] [Table 24]

[0287] (5-iii)lacZα The same fragment as that described in plasmid 1.

[0288] (5-iv)oriC5.6ATmin cassette In the examples, a cassette containing a cleavage enzyme recognition site is used (SEQ ID NO: 5). The boxed portion is the DUE of oriC. Capital letters or hyphens indicate mutation sites from the wild type. The double underlined portion is the gyrase binding sequence (Mu-SGS) derived from bacteriophage Mu (SEQ ID NO: 2). The single underlined portion contains the cleavage enzyme recognition site. SEQ ID NO: 63 is obtained by replacing the underlined portion with the wild type sequence (aactcaaaaactgaacaa), excluding the non-bold portions at the 5' and 3' ends.

[0289] [Table 25]

[0290] (5-v)lacZ-1801 The sequence was amplified by PCR using the following primers and the E. coli MG1655 genome as a template.

[0291] [Table 26]

[0292] <Plasmid6:pFLAG-RepA(3.4kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (6-i) and (6-ii).

[0293] (6-i) s70 promoter-FLAG cassette

[0294] [Table 27]

[0295] (6-ii)repA-LacZ The sequence is amplified by PCR using the following primers and plasmid 5 as a template.

[0296] [Table 28]

[0297] <Plasmid7:pHis-sfGFP(3.6kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (7-i) and (7-ii).

[0298] (7-i)sfGFP

[0299] [Table 29]

[0300] (7-ii) His vector fragment The sequence is amplified by PCR using the following primers and plasmid 5 as a template.

[0301] [Table 30]

[0302] <Plasmid8:pFLAG-sfGFP(3.3kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (8-i) and (8-ii).

[0303] (8-i)sfGFP The same fragment as described in Plasmid 7.

[0304] (8-ii)FLAG vector fragment The sequence is amplified by PCR using the following primers and plasmid 5 as a template.

[0305] [Table 31]

[0306] <Plasmid9:pHis-SecM(3.4kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (9-i) and (9-ii).

[0307] (9-i)His-SecM The sequence was amplified by PCR using the following primers and the E. coli MG1655 genome as a template. The lowercase letters indicate the overlapping portion with the His vector fragment.

[0308] [Table 32]

[0309] (9-ii)His vector fragment The same fragment as described in Plasmid 7.

[0310] <Plasmid10:pFLAG-SecM(3.1kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (10-i) and (10-ii).

[0311] (10-i)FLAG-SecM The sequence was amplified by PCR using the following primers and the E. coli MG1655 genome as a template. The lowercase letters indicate the overlapping portion with the FLAG vector fragment.

[0312] [Table 33]

[0313] (10-ii)FLAG vector fragment The same fragment as described in Plasmid 8.

[0314] <Plasmid11:pHis-RepA_T7(3.7kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (11-i) and (11-ii).

[0315] (11-i) T7 cassette

[0316] [Table 34]

[0317] (11-ii)His vector fragment_T7 The sequence is amplified by PCR using the following primers and plasmid 5 as a template. The lowercase letters indicate the overlapping portion with the T7 cassette.

[0318] [Table 35]

[0319] <Plasmid12:pFLAG-RepA_T7(3.4kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (12-i) and (12-ii).

[0320] (12-i) T7 cassette The same fragment as described in Plasmid 11.

[0321] (12-ii)FLAG vector fragment_T7 The sequence is amplified by PCR using the following primers and plasmid 6 as a template. The lowercase letters indicate the overlapping portion with the T7 cassette.

[0322] [Table 36]

[0323] <Plasmid13:pHis-RepA_CISoriR5.6ATmin(4.1kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following five fragments (13-i) to (13-v).

[0324] (13-i)s70 promoter-SUMO-His cassette (SKIK-)

[0325] [Table 37]

[0326] (13-ii)GSx3-RepA-CIS-oriR The sequence is amplified by PCR using the following primers and R1 Plasmid as a template.

[0327] [Table 38]

[0328] (13-iii)lacZα The same fragment as that described in plasmid 1.

[0329] (13-iv)oriC5.6ATmin cassette The same fragment as that described in plasmid 5.

[0330] (13-v)lacZ-1801 The same fragment as that described in plasmid 5.

[0331] <Plasmid14:pHis-RepA_ΔCISoriR5.6AT(3.7kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (14-i) and (14-ii).

[0332] (14-i)s70-SUMO-His-RepA The sequence is amplified by PCR using the following primers and Plasmid 13 as a template. The lowercase letters indicate the overlapping portion with Lac&oriC5.6ATmin.

[0333] [Table 39]

[0334] (14-ii)Lac&oriC5.6ATmin The sequence is amplified by PCR using the following primers and plasmid 13 as a template. The lowercase letters indicate the overlapping portion with s70-SUMO-RepA.

[0335] [Table 40]

[0336] <Plasmid15:pFLAG-RepA_CISoriR5.6ATmin(3.8kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments (15-i) and (15-ii).

[0337] (15-i)GSx3-RepA-CIS-oriR The same fragment as described in Plasmid 13.

[0338] (15-ii)Lac&oriC5.6ATmin-FLAG The sequence is amplified by PCR using the following primers and plasmid 13 as a template. The lowercase letters indicate the overlapping portion with GSx3-RepA-CIS-oriR.

[0339] [Table 41]

[0340] <Plasmid16:pFLAG-RepA_ΔCISoriR5.6ATmin(3.4kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following two fragments:

[0341] (16-i)s70-FLAG-RepA The sequence is amplified by PCR using the following primers and plasmid 15 as a template. The lowercase letters indicate the overlapping portion with Lac&oriC5.6ATmin.

[0342] [Table 42]

[0343] (16-ii)Lac&oriC5.6ATmin The same fragment as described in Plasmid 14.

[0344] <Plasmid17:pFLAG-GFPv2(3.6kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following six fragments:

[0345] (17-i)s70 promoter-FLAG cassette v2

[0346] [Table 43]

[0347] (17-ii)GS-SUMO-GS

[0348] [Table 44]

[0349] (17-iii)sfGFP v2

[0350] [Table 45]

[0351] (17-iv)lacZα The same fragment as that described in plasmid 1.

[0352] (17-v)oriC5.6ATmin cassette The same fragment as that described in plasmid 5.

[0353] (17-vi)lacZ-1801 The same fragment as that described in plasmid 5.

[0354] <Plasmid18:pHis-GFPv2(3.3kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid consists of the following five fragments:

[0355] (18-i)s70 promoter-His cassette v2

[0356] [Table 46]

[0357] (18-ii)sfGFP v2 The same fragment as described in Plasmid 17.

[0358] (18-iii)lacZα The same fragment as described in Plasmid 1.

[0359] (18-iv)oriC5.6ATmin cassette The same fragment as that described in plasmid 5.

[0360] (18-v)lacZ-1801 The same fragment as that described in plasmid 5.

[0361] <Plasmid19:pFLAG-GFPv2_T Rho (3.7kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the same as that of the full-length plasmid 17, except that the following fragment was inserted between sfGFP v2 and lacZα:

[0362] [Table 47]

[0363] <Plasmid20:pHis-GFPv2_T Rho (3.4kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the full-length plasmid 18 with the following fragment inserted between sfGFP v2 and lacZα: T Rho :Similar fragment to that described in Plasmid 19.

[0364] <Plasmid21:pFLAG-GFPv2_CIS(3.8kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the same as that of the full-length Plasmid 17, except that the following fragments were inserted between sfGFP v2 and lacZα:

[0365] [Table 48]

[0366] <Plasmid22:pHis-GFPv2_CIS(3.5kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the full-length plasmid 18 with the following fragment inserted between sfGFP v2 and lacZα: CIS: a fragment similar to that described in Plasmid21.

[0367] <Plasmid23:pFLAG-GFPv2_λtR1(3.8kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the same as that of the full-length Plasmid 17, except that the following fragments were inserted between sfGFP v2 and lacZα:

[0368] [Table 49]

[0369] <Plasmid24:pHis-GFPv2_λtR1(3.5kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the full-length plasmid 18 with the following fragment inserted between sfGFP v2 and lacZα: λtR1: the same fragment as described in lasermid23.

[0370] <Plasmid25:pFLAG-GFPv2_0-rrnBT1(3.7kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the same as that of the full-length Plasmid 17, except that the following fragment was inserted between sfGFP v2 and lacZα:

[0371] [Table 50]

[0372] <Plasmid26:pHis-GFPv2_0-rrnBT1(3.4kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the full-length plasmid 18 with the following fragment inserted between sfGFP v2 and lacZα: rrnBT1: the same fragment as described in Plasmid25.

[0373] <Plasmid27:pFLAG-GFPv2_2454-rrnBT1(3.7kb)> This plasmid was constructed in a similar manner to that described for plasmid 1. The sequence of this plasmid is the same as that of the full-length Plasmid 17, except that the following fragments were inserted between lacZ-1801 and s70 promoter-FLAG cassette v2: rrnBT1: the same fragment as described in Plasmid25.

[0374] <Plasmid28:pHis-GFPv2_2454-rrnBT1(3.4kb)> This plasmid was constructed in a manner similar to that described for plasmid 1 above. The sequence of this plasmid is the full-length plasmid 18 with the following fragment inserted between lacZ-1801 and s70 promoter-His cassette v2: rrnBT1: the same fragment as described in Plasmid25.< / pcr>

Claims

1. A method for producing a complex in which a peptide is linked to a base sequence encoding the peptide, the method comprising: (1) providing a circular DNA having a base sequence encoding a peptide; (2) expressing a peptide by transcribing and translating the base sequence from the circular DNA using a cell-free transcription-translation reaction, and the expressed peptide forms a complex linked to the circular DNA. A method comprising:

2. A method for preparing a complex library in which library-constituting peptides are linked to base sequences encoding the library-constituting peptides, comprising: (1) preparing a circular DNA library having base sequences encoding library constituent peptides; (2) expressing library-constituting peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, thereby forming a complex library in which the expressed peptides are linked to circular DNA having a base sequence encoding the peptides. A method comprising:

3. The method according to claim 1 or 2, wherein the circular DNA has a replication initiation sequence capable of binding to an enzyme having DnaA activity.

4. The method of claim 1 or 2, wherein the circular DNA does not have a binding sequence for a protein expressed from the circular DNA.

5. A method for recovering a base sequence encoding a target-binding peptide, comprising: (1) preparing a circular DNA library having base sequences encoding candidate peptides for target-binding peptides that bind to a target; (2) expressing candidate peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, thereby forming a complex library in which the expressed candidate peptides are linked to circular DNAs having base sequences encoding the peptides. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) isolating and recovering a circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, obtained in (3); A method comprising:

6. The above (3) and (4) in the presence of a magnesium ion source in the range of 1 mM to 50 mM, and / or In the presence of an alkali metal ion source in the range of 150 mM to 1000 mM, The method of claim 5 , wherein the method is carried out by

7. (5) The method according to claim 5 or 6, further comprising: subjecting the circular DNA having a base sequence encoding the target-binding peptide isolated and recovered in (4) to a cell-free nucleic acid amplification reaction, thereby amplifying the base sequence encoding the target-binding peptide.

8. the circular DNA library has a replication initiation sequence capable of binding to an enzyme having DnaA activity, and （５’） preparing a reaction mixture containing a reaction solution containing (a) a first group of enzymes that catalyze the replication of circular DNA, (b) a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and (c) a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and the circular DNA having a base sequence that encodes the target-binding peptide isolated and recovered in (4); and incubating the reaction mixture at a temperature ranging from 20°C to 80°C; The method of claim 5 or 6, further comprising amplifying a circular DNA having a base sequence encoding the target-binding peptide by a PCR method comprising:

9. The method according to claim 8, wherein the steps (2), (3) and (4) are repeated by using the circular DNA amplified in the step (5') as a circular DNA library in the step (2).

10. The method according to claim 5 or 6, wherein the contacting and binding in (3) is carried out in the absence of RNase.

11. A method for recovering a base sequence encoding a target-binding peptide, comprising: (1) Providing a circular DNA library having a base sequence encoding a candidate peptide of a target-binding peptide that binds to a target, a DNA target sequence, a base sequence encoding a base peptide that non-covalently binds to the DNA target sequence, and a DNA element that provides cis activity, wherein the candidate peptide is expressed linked to the base peptide; (2) expressing candidate peptides and base peptides from the circular DNA library by transcribing and translating the base sequences using a cell-free transcription-translation reaction, and forming a complex library in which the base peptides linked to the expressed candidate peptides are non-covalently bound to a DNA target sequence on a circular DNA having a base sequence encoding the candidate peptides. (3) contacting the complex library with the target to bind the target to the complex library via the candidate peptide linked to the complex library; (4) isolating and recovering a circular DNA having a base sequence encoding a target-binding peptide from the target to which the complex library has been bound, obtained in (3); A method comprising:

12. The above (3) and (4) in the presence of a magnesium ion source in the range of 1 mM to 50 mM, and / or In the presence of an alkali metal ion source in the range of 150 mM to 1000 mM, The method of claim 11 , wherein the method is carried out by

13. the circular DNA library has a replication initiation sequence capable of binding to an enzyme having DnaA activity, and （５’） preparing a reaction mixture containing a reaction solution containing (a) a first group of enzymes that catalyze the replication of circular DNA, (b) a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and (c) a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and the circular DNA having a base sequence that encodes the target-binding peptide isolated and recovered in (4); and incubating the reaction mixture at a temperature ranging from 20°C to 80°C; The method of claim 12, further comprising amplifying circular DNA having a base sequence encoding the target-binding peptide by a PCR method comprising:

14. The method according to claim 13, wherein the circular DNA amplified in the step (5') is used as a circular DNA library in the step (2), and the steps (2), (3), and (4) are repeated.

15. A method for producing a target-binding peptide, comprising: amplifying a base sequence encoding the target-binding peptide from circular DNA containing the base sequence encoding the target-binding peptide, which is recovered by the method of claim 5 or 11; and transcribing and translating the obtained base sequence to produce the target-binding peptide.

16. A complex library in which library-constituting peptides and base sequences encoding the library-constituting peptides are linked together, (i) a circular DNA library having a base sequence encoding the library-constituting peptide and a replication initiation sequence capable of binding to an enzyme having DnaA activity; (ii) the library constituent peptides; wherein (i) and (ii) are connected via mRNA.

17. a base sequence encoding a candidate peptide of a target-binding peptide that binds to the target; a DNA target sequence; a base sequence encoding a base peptide that non-covalently binds to the DNA target sequence; a DNA element that confers cis activity; a replication initiation sequence capable of binding to an enzyme having DnaA activity; A circular DNA having the formula: the DNA element that confers cis activity is located between the base sequence encoding the base peptide and the DNA target sequence; A circular DNA in which the candidate peptide is expressed linked to the platform peptide.

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