JP6619983B2 - MRNA display method using head-to-head cross-linking - Google Patents

Description

  The present invention relates to a novel mRNA display method that does not use the tail-to-tail of the 3 ′ end of mRNA and the C terminus of protein, among mRNA display methods, which are genotype-phenotype association technologies. . More specifically, the present invention relates to an mRNA display method using a head-to-head bridge capable of rotating a ribosome using the property that a demethylase and a substrate thereof are covalently bonded.

  In evolutionary engineering, various techniques are used to select a peptide that specifically binds to a specific molecule from a vast number of libraries of peptides or proteins having a random sequence. Among these technologies, display technology which is a kind of “genotype-phenotype association technology” is important.

Display technologies used for drug discovery using proteins and peptides include, for example, cell display and phage display as in vivo display technologies, ribosome display as in vitro display technologies, mRNA display and cDNA display, etc. (See Non-Patent Document 1). The library sizes that can be handled by these display technologies are 10 ⁷ for in vivo display technology and 10 ¹² for in vitro display technology, as shown in FIG.

  Such display technology is described as follows: “In order to select functional peptide molecules, sequence information (DNA, RNA) corresponding to peptide molecules is linked to these molecules, the screened molecules are sorted by function, and their sequences are selected. It is defined as a technique of “amplifying an information part by PCR and then reading by sequencing”.

  In the mRNA display method and the cDNA display method described above, a linker (a molecule for intermolecular linking) is used. The linker used here has an mRNA linking site for linking to mRNA encoding a gene and a protein linking site for linking a protein synthesized from this mRNA. As a protein linking site, puromycin Aminoacyl tRNA analogs such as

  The mRNA display method described above is generally performed in the following steps using mRNA. First, a DNA library is prepared, and transcription from this DNA library to mRNA and linking to a linker are performed. Here, the 5 'end of the linker is linked to the 3' end of the mRNA (mRNA tail). Next, the mRNA linked to the linker is translated to produce a protein (peptide), and the produced protein is linked to the linker (refer to Patent Documents 1 and 2, hereinafter, in order, “Conventional Technology 1” and "Prior art 2"). In this case, since the C-terminal tail of the protein and the tail of the mRNA are bound via a linker, hereinafter, the binding of the prior art 1 or 2 is referred to as tail-to-tail binding.

  Here, several ribosomes are bound on the mRNA bound to the linker, and the ribosome synthesizes proteins and peptides while sequentially reading the mRNA. Then, when the full length of a certain protein or peptide is synthesized, the protein or peptide is displayed at the protein linking site of the linker.

WO2006 / 072773 Japanese Patent No. 4318721

Patrick Amstutz, Patrik Forrer, Christian Zahnd and Andreas Plueckthun, Current Opinion in Biotechnology 2001, 12: 400-405

Prior art 1 is an excellent technique in that an mRNA-linker-peptide conjugate displaying the target protein can be obtained. The mRNA used in the prior art 1 has a time for linking its C-terminal to puromycin at the end of the linker when protein synthesis is completed, so that the ribosome after synthesis is not dissociated from the mRNA. Excludes stop codon. For this reason, the ribosome does not spontaneously dissociate and continues to bind to the mRNA indefinitely, and the subsequent ribosome appears in a polysome state in which the subsequent train is connected to the back of the train just stuck at the station (see FIG. 2). .
For this reason, the ribosome bound to the mRNA must be finally dissociated forcibly, and this translation reaction has a problem that the ribosome must be used at a high concentration.

  And the ribosome in the middle of the protein synthesis stays on the mRNA and cannot turn over and reuse. For this reason, there is a problem that expensive ribosomes cannot be fully utilized, and there has been a strong social and economic demand for reusing ribosomes.

  In addition, in vitro synthesis systems use rabbit reticulocytes and wheat germ extracts, but since these contain RNases, there is a problem that mRNA is degraded and yield decreases. In order to solve these problems, Pure System (manufactured by Gene Frontier Co., Ltd.) was developed. Since Pure System does not contain nuclease, the nucleotide chain used is not degraded, but it is expensive. In order to maintain the yield using this Pure system, it is necessary to use a turnover system.

By the way, the mRNA display method can handle a library having a large size of 10 ¹² . In order to obtain molecules with high affinity, it can be finally narrowed down to several types of molecules only by the usual mRNA display method. While this achieves the objective, functional assays of individual candidate molecules are needed to perform sputum in function (generally various functions other than affinity, such as enzyme inhibition and receptor agonists). Necessary.

Peptide and protein functions are not detected by a single molecule and must be amplified. In this case, it is impossible to amplify an amount of protein or peptide that can be detected by the conventional mRNA display method type construct having no stop codon, and the construct having the stop codon of the present invention is used. Only then can such amplification be performed. Furthermore, if a new microarray MMV developed in recent years is applied, the functions of 10 ³ to 10 ⁶ different peptides and proteins can be assayed in parallel. For this purpose, a stop codon must be introduced into the mRNA, and the ribosome needs to turn over (see FIGS. 2A and 2B).

  For this reason, it was necessary not to attach a linker to the 3 'end of mRNA but to link it to the 5' end. On the other hand, proteins need to be linked not on the C-terminal side but on the N-terminal side. That is, head-to-head connection is required. There was a strong social demand for a display method that can recycle ribosomes in a completely different manner from that using a puromycin linker.

The present invention has been completed under the above circumstances.
That is, an embodiment of the present invention includes a linker production step for producing a linker comprising a target domain binding site and a fluorescent label; and a DNA construct comprising at least a target domain, a variable region, and a base sequence of a stop codon. A construct preparation step; a corresponding mRNA is prepared from the DNA construct by transcription; a transcription step; an mRNA-linker ligation step in which the 5 ′ end of the mRNA is treated with an enzyme to bind to the 3 ′ end of the linker; A translation step in which one ribosome binds to the mRNA contained in the mRNA-linker conjugate to generate the target domain by translation; the translated target domain binds to the target domain binding site of the linker Form an mRNA-linker-protein conjugate Together with the ribosome the stop codon recognized by the mRNA- linker - ribosome free process liberated from protein conjugate automatically; comprises a mRNA display methods which can turnover ribosome.

Here, the desired domain used in the DNA construct production step is any one selected from the group consisting of O ⁶ -methylguanine-DNA-methyltransferase (MGMT) and a functional mutant protein of MGMT. Is preferred. Among these, O ⁶ -methylguanine-DNA-methyltransferase is more preferable.

  The DNA construct includes a spacer having a desired length, an α-stand peptide having an α-helical structure having a desired length, and a purification tag on the downstream side of the base sequence, and further includes a variable peptide sequence or variable It is preferable that any one of protein is included. The spacer is 3 to 5 amino acids long, the α stand peptide is preferably 17 to 21 amino acids long, and the purification tag is composed of a His tag, a FLAG tag, and a biotin-like peptide. It is preferably any one selected from the group.

  The enzyme used for the enzyme treatment is a triphosphate removing enzyme that removes triphosphate from the 5 ′ end of mRNA and a kinase that adds phosphate to the 5 ′ end of mRNA from which triphosphate has been removed. It is more preferable that the triphosphate removing enzyme is an atactic phosphatase and the kinase is a polynucleotide kinase. More preferably, said polynucleotide kinase is T4 polynucleotide kinase.

  Yet another aspect of the present invention comprises a linker generating step of generating a linker comprising a target domain binding site and a fluorescent label; at least either the variable peptide sequence or the variable protein sequence, and the target domain preceding the stop codon A target DNA construct production step for preparing a target DNA construct comprising the nucleotide sequence of: a target mRNA construct; a corresponding mRNA is prepared by transcription from the target DNA construct; and a transcription step; An mRNA-linker ligation step for binding to the 3 ′ end of a linker; a translation step in which one ribosome binds to the mRNA contained in the mRNA-linker conjugate to produce the target domain by translation; Target domain binding of the linker to the target domain A ribosome releasing step of binding to a site to form an mRNA-linker-protein conjugate, wherein the ribosome recognizes the stop codon and is automatically released from the mRNA-linker-protein conjugate; A reverse transcription step in which the mRNA in the protein conjugate is reversely transcribed to synthesize cDNA; and an mRNA display method capable of ribosome turnover.

  It is preferable to further include a solid phase binding step for binding the mRNA-linker-protein conjugate to a solid phase. Moreover, it is preferable to further comprise a purification step for purifying the (mRNA + cDNA) -linker-protein conjugate obtained in the reverse transcription step.

The desired domain used in the construct production step is preferably any one selected from the group consisting of O ⁶ -methylguanine-DNA-methyltransferase (MGMT) and a functional mutant protein of MGMT. Of these, O ⁶ -methylguanine-DNA-methyltransferase is more preferable.

  The construct includes a spacer having a desired length on the downstream side of the base sequence, an α stand peptide having an α helix structure having a desired length, and a purification tag, and includes either a variable peptide sequence or a protein sequence. It is preferable to further include such a sequence. Furthermore, the spacer is preferably 3 to 5 amino acids long, and the α-stand peptide is preferably 17 to 21 amino acids long.

  The purification tag is preferably any one selected from the group consisting of a His tag, a FLAG tag, and a biotin-like peptide. The enzyme used in the enzyme treatment step is a triphosphate removing enzyme that removes triphosphate from the 5 ′ end of mRNA, a kinase that adds phosphate to the 5 ′ end of mRNA from which triphosphate has been removed, And a combination of an enzyme such as T4 RNA ligase that directly links the linker and the 5 ′ triphosphate end of the mRNA. The triphosphate removing enzyme is preferably an atactic phosphatase, and the kinase is preferably a polynucleotide kinase.

  The linker and the 5 'triphosphate mRNA can be directly linked with T4 RNA ligase without using the two enzymes, the triphosphate removal enzyme and the kinase, used in the enzyme treatment step.

According to the present invention, by creating a linker that links the 5 ′ end of mRNA and the amino-terminal region of the nascent protein, it is a strong method against the use of ribosome turnover in a completely different manner from that using puromycin. An mRNA display method is provided that can meet potential needs.
The protein to be associated with the desired sequence (mRNA) translated by including the sequence of the protein to be associated with the mRNA in the linker bound to the 5 ′ end of the mRNA with the desired sequence. Can be associated with each other and information can be associated with functions.

  Furthermore, since a region where the primer sticks to the 3 'end of mRNA is included, cDNA can be obtained by reverse transcription and can also be applied as a cDNA display method.

  According to the present invention, there are provided a head-to-head-bound mRNA display method and a cDNA display method capable of ribosome turnover. In these methods, the ribosome used for synthesis can be used for turnover, so that the amount of reagent used is reduced by almost an order of magnitude compared to the conventional method (Tail-to-Tail). At the same time, in vitro translation of the protein can be performed with this construct.

FIG. 1 is a diagram showing various display methods for in vitro selection and library sizes that can be handled by each method. FIG. 2 is a diagram schematically showing an example (A) of the conventional display method and the display method (B) of the present invention. FIG. 3 is a diagram showing a linker used in the present invention. FIG. 3A is a schematic diagram showing a linker, and FIG. 3B shows a specific structure of the linker.

FIG. 4 is a schematic diagram showing a preparation example of a DNA construct containing an MGMT coding sequence. 4A shows a schematic diagram of the construct 1, and FIG. 4B shows a schematic diagram of the construct 2. FIG. 5 shows the success or failure of demethylation and PCR amplification. FIG. 6 is a schematic diagram showing the ribosome turnover and the protein region synthesized by the ribosome when the linker of the present invention is used. (A) shows ribosome turnover, and (B) shows the overall structure of the translated protein.

FIG. 7 is a schematic diagram showing a method for performing head-to-head coupling. FIG. 8 is a schematic diagram showing the structure of myoglobin based on the design of a peptide having an α helix structure.

FIG. 9 is a schematic diagram (1) showing a mode in which a spacer having an α helix structure (hereinafter referred to as “α stand spacer”) is bound to a desired peptide aptamer. FIG. 10 is a schematic diagram (2) showing a mode in which an α-stand spacer is bound to a desired peptide aptamer.

FIG. 11 is a gel electrophoresis image showing the result of amplification of the MGMT sequence by PCR.

FIG. 12 is a gel electrophoresis image showing the result of amplifying a T7 promoter-MGMT DNA fragment in which the sequence of the T7 promoter region was connected by upstream PCR upstream of MGMT. FIG. 13 is a gel electrophoresis image showing the results of PCR using a primer that adds a portion downstream of MGMT and a His tag sequence upstream of the random region. FIG. 14 is a gel electrophoresis image of a full construct DNA obtained by connecting a T7 promoter-MGMT sequence and a His tag-random region sequence using overlap PCR.

FIG. 15 is a gel electrophoresis image showing the result of purifying the full construct DNA obtained in FIG.

FIG. 16 is a gel electrophoresis image showing the result of amplifying the full construct DNA obtained in FIG. 15 by PCR and confirming the chain length. FIG. 17 is a gel electrophoresis image of mRNA obtained by transferring the full construct DNA obtained in FIG.

FIG. 18 is a gel electrophoresis image when a linker is bound to the 5 'end of mRNA. (A) is Syber Green 2, and (B) is FITC, which are gel electrophoresis images showing detection results when labeled. FIG. 19 is a gel electrophoresis image showing the results of ligation with a linker after dephosphorylation of mRNA. (A) The detection result by FITC, and (B) the detection result by SYBER Green 2. FIG. 20 is a gel electrophoresis image showing that a bond was formed at the 5 ′ end of the mRNA and the 3 ′ end of the linker, not between the linkers. (A) is a gel electrophoresis image after the mRNA is excised from the gel electrophoresis shown in FIG. 19, (B) is a gel electrophoresis image of the mRNA after dephosphorylation, and (C-1) is a BG linker-mRNA. FITC detection result of gel electrophoresis image of ligation product, (C-2) shows the detection result of BGBER-mRNA ligation product gel electrophoresis image by SYBER Green 2.

FIG. 21 is a gel electric diagram showing the result of binding of 5 ′ end of triphosphate of mRNA and —OH of 3 ′ end of linker when another T4 RNA ligase (manufactured by Takara Bio Inc.) is used. It is an electrophoretic image. (A) shows the detection result by FITC, and (B) shows the detection result by SYBER Green 2. FIG. 22 is a gel electrophoresis image showing a change in the amount of each conjugate produced when the in vitro translation time is changed. FIG. 23 is a gel electrophoresis image showing the result of treating an in vitro translation product with proteinase K (hereinafter sometimes referred to as “PNK”, manufactured by Invitrogen).

FIG. 24 is a gel electrophoresis image showing the results of treating in vitro translation products with RNase A. FIG. 25 is a gel electrophoresis image showing the results of His tag purification. FIG. 26 is a diagram schematically showing the MGMT-spacer (4-amino acid-α stand) -His tag structure of the construct based on the structure prediction by SWISS-MODEL. (A) schematically shows the structure of construct 1, and (B) schematically shows the structure of construct 2. FIG. 27 is a gel electrophoresis image showing the result of His tag purification after spacer introduction.

Hereinafter, the present invention will be described in more detail with reference to FIGS.
As shown in FIG. 2 (B), the present invention is an mRNA display method capable of utilizing ribosome turnover, (a1) a linker production step for producing a linker containing a target domain binding site and a fluorescent label; a2) a DNA construct production step for producing a DNA construct comprising at least a variable region, a stop codon, and a base sequence of a target domain; (a3) a transcription step for preparing a corresponding mRNA by transcription from the DNA construct; (A4) an mRNA-linker ligation step in which the 5 ′ end of the mRNA is treated with an enzyme to bind to the 3 ′ end of the linker; (a5) one ribosome in the mRNA contained in the mRNA-linker conjugate A translation step wherein the target domain is generated by translation with binding; (a6) And a; - target domains, said binding to the target domain binding site of the linker, the ribosome recognition to mRNA- linker the stop codon automatically liberated ribosomes free process from protein conjugate.

Next, the target domain contained in the linker (a1) is not particularly limited. For example, O ⁶ -methylguanine-DNA-methyltransferase (hereinafter sometimes referred to as “MGMT”), and those. It is preferable that it is any one selected from the group consisting of the functional mutant proteins of the above because the target peptide to which these domains bind can be accurately captured and covalently bound.

  As used herein, “functional variant protein” refers to a protein that maintains the function it had before the mutation, even though a certain protein, for example, MGMT, has been mutated.

For example, the case where MGMT is used will be described as an example. MGMT is essentially an enzyme that repairs base pair mismatches and has methyl transfer activity. For example, when MGMT acts on temozolomide (alkylating agent), which is known as a therapeutic agent for malignant glioblastoma among brain tumors, the effect of this drug is reduced. When it acts on O ⁶ -methylguanine, which is one of the substrates, it is demethylated into guanine. MGMT having such an action can be used for mRNA display for searching for peptides or proteins (see FIG. 6A).

On the other hand, O ⁶ -benzylguanine (hereinafter sometimes referred to as “O ⁶ BG”) is an inhibitor of MGMT and strongly covalently binds to MGMT. For this reason, it can be used for connection with MGMT.

  Examples of the fluorescent label contained in the linker (a1) include FITC, Cy3, TAMRA, BODIPY and the like, and the use of FITC is preferable from the viewpoint of filter penetration of the detector. Examples of the variable region include various enzyme-inhibiting peptides, receptor agonists, and the like.

When constructing a construct containing the MGMT sequence, a linker utilizing the fact that MGMT and its substrate analog O ⁶ BG form a covalent bond can be produced. For example, a linker that is modified with O ⁶ BG is attached to the 5 ′ end of mRNA, and the sequence of the protein to be associated with the MGMT sequence is inserted into the mRNA, thereby forming a covalent bond between the translated MGMT and O ⁶ BG. And the information which these arrangement | sequences have can be matched with the function of the peptide represented by these arrangement | sequences.

  Furthermore, by inserting a region where the primer sticks to the 3 ′ end of the mRNA (primer hybridization region) (see FIGS. 4A and 4B), reverse transcription is performed after completion of the mRNA display structure, and cDNA is obtained. You will be able to get The display in this state is called a cDNA display. Since mRNA and protein are bound to the leading 5 'end of mRNA and the leading N-terminal side of the protein, they are hereinafter referred to as Head-to-Head Linking (H2H) type mRNA display.

  Specific examples of the base sequence of the linker include those shown in SEQ ID NO: 1 in the sequence listing. In addition, the linker has a main chain and a side chain, and has a peptide binding site to which a peptide containing a target domain can be bound at the tip of the side chain, and a FITC for detection is bound (FIG. 3).

Next, in the above (a2) DNA construct production step, for example, a desired DNA fragment containing the target domain is connected using an overlap PCR method, and a construct having the following sequence can be produced. As the target domain, for example, an enzyme sequence such as O ⁶ -methylguanine-DNA-methyltransferase (MGMT) or a derivative of MGMT can be used.

  A case where a construct having the base sequence described in SEQ ID NO: 2 in the sequence listing (hereinafter referred to as “construct 1”) is prepared will be described as an example. Construct 1 is 839 bp, and as schematically shown in FIG. 4 (A), 6 spacers, T7 promoter, linker hybridization region, RBS (ribosome binding site), MGMT coding sequence, His-Tag, variable region sequence A stop codon and a primer hybridization region, and cNewYTag is used as the primer hybridization region.

  The construct of the present invention is prepared using the overlap PCR method. In the case of a construct containing the base sequence of MGMT as described above, overlap PCR is performed using primers such as MGMT primers 1 and 2, T7 primer, biotin-cNew Ytag primer, reverse transcription primer and the like. Such primers can have, for example, the base sequences shown in SEQ ID NOs: 2 to 7 below.

(1) Amplification of target domain by PCR For example, a desired amount of Taq polymerase, Taq reaction buffer, dNTP mixture and two kinds of primers (forward primer and reverse primer), template, reaction product of Scheme 1, and distilled water Prepare containing PCR mix. As such a PCR mix, for example, a mixed solution having a composition as described later is preferably used from the viewpoint of reaction efficiency. In this specification, the demethylation reaction which is the original function of MGMT is not used, but the property of being covalently bonded to a substrate analog is merely used. For this reason, in the reaction of Scheme 1, it is confirmed whether or not MGMT is correctly expressed when translation is performed with the prepared construct.

  The PCR mix is then mixed at about 95 ° C for about 1.5-2.5 minutes, then about 95 ° C for about 15-25 seconds, then about 66 ° C for about 25-35 seconds, about 72 ° C for about 25-35 The second incubation cycle can be performed 25-35 times, followed by about 3-7 minutes at about 72 ° C. and then cooled to about 4 ° C. to stop the reaction and run in a thermal cycler.

(2) Addition of T7 region to target domain by overlap PCR Desired amount of prime star, prime star reaction buffer, dNTP mixture and two types of primers (forward primer and reverse primer), template, PCR product of target domain and An overlap PCR solution containing distilled water is prepared. As such overlap PCR solution, for example, it is preferable from the viewpoint of reaction efficiency to prepare according to the instructions attached to Prime Star (manufactured by Takara Bio Inc.).

  Then, the amplification solution was incubated at 95 ° C. for 1.5 to 2.5 minutes, 95 ° C. for 10 to 30 seconds, 65 ° C. for 3 to 7 seconds, and 72 ° C. for 15 to 45 seconds, 25 to 35 times, Cool at 72 ° C. for 4-6 minutes and then to 4 ° C. to obtain the target domain-T7 region amplification product. The amplification product thus obtained is taken in a desired amount, for example, 1 μL, and a desired amount of distilled water and 2 × STR (manufactured by Promega), for example, 7 μL of distilled water and 8 μL of 2 × STR are mixed, and about 2 at 95 ° C. Heat for ~ 4 minutes and subject to gel electrophoresis.

  For example, a sample is applied to a mini gel (3 to 5% acrylamide gel containing 6 to 9 M urea), and 0.25 to 1 × TBE buffer is used as a running buffer, under conditions of 150 to 250 V and 60 ° C. for 60 to 120 minutes. Electrophoresis can be performed. After electrophoresis, it can be stained with a staining reagent and detected with an imager. Examples of such a reagent include SYBR Green II Nucleic Acid Gel Stains (hereinafter abbreviated as “SYBR Green 2”, manufactured by Takara Bio Inc.), ethidium bromide, and the like.

(3) Amplification of random region Desired amount of DNA polymerase, DNA polymerase reaction buffer, magnesium sulfate and two kinds of primers (target domain-tag primer (for example, SEQ ID NO: 8 in the sequence listing)) and biotin tag primer (for example, arranged A solution for amplifying a random region is prepared, which comprises a sequence listing (SEQ ID NO: 6)), a random template (eg, SEQ ID NO: 14), a target domain PCR product and distilled water. As such a solution, it is preferable from the viewpoint of reaction efficiency to use a random region amplification buffer as shown in Example 2 (1) (1-3) described later.

  The random region amplification solution is then cycled 25-35 times, for example, at 95 ° C for 1.5-2.5 minutes, 95 ° C for 10-30 seconds, 65 ° C for 15-45 seconds, and then at 75 ° C. The amplification product in the random region can be obtained by cooling to 0.5 to 2 minutes, 4 to 6 minutes at 72 ° C, and then cooled to 4 ° C. A desired amount of PCR product, eg, 1 μL and an equal amount of 2 × STR, are mixed, heated at 95 ° C. for 2-4 minutes, and subjected to gel electrophoresis.

  Next, for example, the sample is applied to a microgel (6 to 10% acrylamide gel containing 6 to 9 M urea), and electrophoresis is performed at 100 V and 55 ° C. for 5 to 15 minutes using 1 × TBE buffer as a running buffer. . After the gel electrophoresis is completed, the single-stranded DNA is stained using the same staining reagent as described above, and detected by an imager.

  Subsequently, in order to measure the concentration of the generated DNA, for example, 1/10 volume of Quick-Precip (registered trademark) Plus Solution (manufactured by EdgeBio; 10 mM Tris-HCl (pH 7.5 to 8.5), 1 mM EDTA, (Containing 5M NaCl), 2.5 volumes of 99% ethanol, and the PCR product are mixed and centrifuged at the desired g, for example, 13,000 rpm (rotor diameter: 60 mm) for 10-20 minutes. The supernatant is discarded, leaving the white precipitate obtained, mixed with 70% ethanol, and centrifuged again at the same g for 5-15 minutes. The supernatant is discarded and ethanol is completely volatilized using an evaporator to obtain a DNA pellet. The obtained DNA pellet is dissolved in distilled water and the DNA concentration is measured. For example, nanodrop (manufactured by Thermo Fisher Scientific) or the like can be used for measuring the DNA concentration.

(4) Purification using beads in a random region A desired amount, for example, 50 μL of streptavidin beads (manufactured by Takara Bio Inc.) is placed in a low adsorption tube (manufactured by Biomedical Science Co., Ltd.) and attached to a magnet. Discard the supernatant. The desired amount, eg, 100 μL, of 1 × binding buffer is then added and mixed. Attach this tube to a magnet and discard the supernatant. This operation is further repeated 2-3 times. The desired amount, eg, 20 μL (about 37 μg) of the random region PCR product is then mixed with the desired amount, eg, 30 μL of distilled water, the desired amount, eg, 50 μL of 2 × binding buffer.

  Place in a rotator (Tytec) and rotate at 4 ° C overnight. Thereafter, the operation of attaching to the magnet and discarding the supernatant is repeated three times. This is followed by the addition of the desired amount, eg, 50 μL, of dissociation buffer (93-98% formaldehyde containing 10 mM EDTA), incubated in a rotator for 15 minutes at 65-75 ° C., attached to a magnet and supernatant Recover. Again, add the desired amount, eg 50 μL of dissociation buffer, incubate at 65-75 ° C. for 10-20 minutes in a rotator, attach to magnet and collect supernatant. DNA is obtained from the collected supernatant by the same operation as described above, and its concentration is measured as described above.

(5) Production of full construct Including desired amount of DNA polymerase, DNA polymerase reaction buffer, magnesium sulfate and dNTP mixture, target domain-T7 addition product and tagged random region obtained as described above, and distilled water Prepare a solution for making a full construct. As such a solution, for example, it is preferable from the viewpoint of reaction efficiency to use a buffer for construct preparation having a composition shown in Example 2 (1) (1-5) described later.

  This solution is then placed in a thermal cycler and incubated, for example, at 95 ° C for 1.5-2.5 minutes and at 75 ° C for 5-7 minutes. Then, for example, add the desired amount of T7 primer and the same amount of Biotin-cNew Ytag primer and place in thermal cycler, cycle 5-15 seconds at 95 ° C, 15-45 seconds at 65 ° C, 1 minute at 75 ° C Is repeated 25 to 35 times, incubated at 75 ° C. for 3 to 7 seconds, and then cooled to 4 ° C. to obtain an amplification product.

  Thereafter, the amplification product obtained contains 1/10 volume of Quick-Precip Plus Solution (containing 10 mM Tris-HCl (pH 7.5 to 8.5), 0.5 to 2 mM EDTA and 4 to 6 M NaCl, manufactured by QIAGEN). Add 2.5 volumes of 99% ethanol, mix and repeat the operation of centrifugation at 10,000-15,000 rpm (7,000-14,000 xg) for 10-20 minutes, using pellets dissolved in distilled water as above. The DNA concentration is measured by operation. After that, for example, a sample is applied to a mini gel (2 to 6% acrylamide gel containing 6 to 9 M urea), 0.25 to 0.75 × TBE buffer is used as a running buffer, and the conditions are about 200 V and about 60 ° C. for 60 to 120 minutes. Electrophoresis is performed, and after completion of gel electrophoresis, single-stranded DNA is stained with the same staining reagent as described above and detected by an imager.

  The band at the desired position, eg, 839 bp, is then cut from the gel using a scalpel. Place the gel cut into a tube of the desired size, for example, a 1.5 mL tube, and crush it with a spatula etc., and then add the desired amount, for example, 600 to 1,000 μL of dilution buffer (5 to 15 mM magnesium acetate, 0.25 to 0.75 M ammonium acetate. , 0.5-2 mM EDTA, and 0.05-0.2% SDS, pH 7.5-8.5) is added to the tube and incubated overnight at 65-75 ° C.

  After that, as described above, add 1/10 volume of Quick-Precip Plus Solution and 2.5 volume of 99% ethanol, mix, for example, centrifuge at 10,000-15,000 rpm for 10-20 minutes, Discard the supernatant, add 70% ethanol, mix, and centrifuge again. The supernatant is discarded leaving a white precipitate, ethanol is evaporated to completely volatilize, the DNA pellet is dissolved in distilled water, and the DNA concentration is measured as described above. In addition, the DNA after excision and purification is subjected to electrophoresis using a minigel under the conditions as described above to confirm whether or not it is a single band. Next, direct sequence analysis is performed to check whether the sequence is correct.

(6) Transcription to mRNA A transcription solution containing the DNA full construct obtained as described above, an rNTP mixture, a T7 transbuffer, a T7 enzyme mix, and an RNase inhibitor in desired amounts is prepared. As such a transfer solution, for example, a transfer buffer having a composition shown in Example 2 (1) (1-6) described later is preferably used from the viewpoint of transfer efficiency.

  Next, this transfer solution is put into a thermal cycler and incubated at 37 ° C. for 1.5 to 2.5 hours, for example. Subsequently, RNA is purified. For example, adding 0.5-2 μL of RQ1 RNase-Free DNase (0.5-2 U / μL), incubating at 37 ° C. for 10-20 minutes, and purifying RNA using RNeasy Mini Kit (QIAGEN) it can.

  Prepare RNA purification solution containing desired amount of RNase free water, RLT buffer (provided in QIGEN RNeasy Mini Kit) and 99% ethanol. As such an RNA purification solution, for example, an RNA purification buffer having a composition shown in Example 2 (2) described later is preferably used from the viewpoint of purification efficiency.

  This RNA purification solution is put into an RNeasy purification column, and centrifuged at a desired g for a desired time, for example, 10,000-15,000 rpm (7,000-14,000 × g) for 10-20 seconds. The column is loaded with the desired amount of RPE buffer (provided in the kit above), eg, 500 μL, and centrifuged at 12,000 rpm for 10-20 seconds. Next, a desired amount of 80% ethanol, for example, 400 to 600 μL is added thereto, and centrifuged at 10,000 to 15,000 rpm for 1 to 4 minutes to perform ethanol precipitation. Subsequently, RNase free water is added in a desired amount, for example, 10 to 20 μL, and left at room temperature for 2 to 4 minutes, and then centrifuged at a desired g for a desired time, for example, 10,000 to 15,000 rpm for 0.5 to 2 minutes. . Thereafter, the concentration of RNA is measured by nanodrop. If the sample obtained as described above is not used immediately, store it at -80 ° C.

(7) Ligation of linker (7-1) Ligation in one step Ligation in one step is performed by, for example, linking mRNA and a linker in an incubation solution having a desired composition. As such an incubation solution, for example, it is preferable from the viewpoint of reaction efficiency to use a buffer for one-step binding shown in Example 2 (3) (3-1) described later. The reaction conditions are, for example, incubation at 88 to 92 ° C. for 1 to 3 minutes, lowering the temperature to 0.05 to 0.15 ° C./second to 65 to 75 ° C., incubation at about 65 to 75 ° C. for 1 to 3 minutes, and subsequently 0.05 to The temperature can be lowered to 20-30 ° C. at 0.15 ° C./second.

  A ligation solution is added thereto to form a mixed solution, and the above mixed solution is incubated at 20-30 ° C. for 45 minutes to 1.5 hours to obtain a ligation product. From this ligation product, take the desired amount, for example 0.5-2 μL, mix with the same amount of buffer, for example 2 × STR and heat at 92-98 ° C. for 2-4 minutes. Thereafter, in the same manner as described above, the sample is applied to the microgel and gel electrophoresis is performed. As electrophoresis conditions, for example, 1 × TBE buffer is used as a running buffer, and can be set at about 100 V and 50 to 60 ° C. for 10 to 15 minutes.

  After completion of electrophoresis, FITC is detected by an imager and the success or failure of ligation is confirmed. Next, staining is performed using a staining solution, and the presence or absence of RNA degradation products is confirmed. As such a staining solution, for example, it is preferable to use one that can stain single-stranded DNA such as SYBR Green 2. When the ligation product obtained as described above is not used immediately, it is preferably stored at −80 ° C.

(7-2) Ligation in three stages Ligation in three stages involves removal of phosphate groups in a phosphate group removal buffer. As such a phosphate group removing solution, for example, a phosphate group removing buffer having a composition shown in Example 2 (3) (3-2), which will be described later, is preferably used from the viewpoint of reaction efficiency. As reaction conditions, it is possible to incubate at 36 to 38 ° C. for 10 to 20 minutes, then at 68 to 72 ° C. for 4 to 8 minutes, and then cooled to 3 to 5 ° C. Next, a phosphate group addition solution is added thereto, and the mixture is mixed and incubated at a desired temperature for a desired time. As such a phosphate group addition buffer, a ligation buffer shown in Example 2 (3) (3-2) described later is preferably used from the viewpoint of reaction efficiency.

  The reaction conditions were incubation at 36-38 ° C. for 25-35 minutes, followed by addition of a desired amount, for example, 0.5-15 μM of 5-15 μM linker, and incubation at 85-95 ° C. for 1-3 minutes, 0.05 The temperature can be lowered to 65 to 75 ° C. at −0.15 ° C./second, and the temperature can be lowered to 20 to 30 ° C. at the same rate of temperature reduction.

  Here, a desired amount of RNA ligase is added and incubated for a desired time to obtain a ligation product. For example, 0.05 to 0.15 μL of 5 to 100 unit / μL T4 RNA ligase (New England Biolabs) can be added and incubated at 20 to 30 ° C. for about 45 minutes to 1.5 hours to obtain a ligation product. The obtained ligation product is taken in a desired amount, for example, 0.5 to 2 μL, and mixed with the same amount of buffer, for example, 2 × STR Loading Buffer (hereinafter abbreviated as “2 × STR”, Promega) 90-98 ° C. Heat for 2 to 4 minutes.

  Thereafter, a sample is applied to a microgel under the same conditions as described above, and gel electrophoresis is performed. FITC is detected by an imager to confirm whether or not ligation is performed. Next, staining is performed using a staining solution in the same manner as described above, and the presence or absence of RNA degradation products is confirmed. Such a staining solution is preferably one that can stain single-stranded DNA such as SYBR Green 2 from the viewpoint of detection sensitivity. If the ligation product is not used immediately, it is preferably stored at -80 ° C.

(8) Translation in vitro First, a translation solution 1 containing a ligation product, an RNase inhibitor and the like is prepared and incubated at 36 to 38 ° C. for 10 minutes to 4 hours. As such a translation solution 1, for example, a translation buffer having a composition shown in Example 3 (1) described later is preferably used from the viewpoint of reaction efficiency. The desired amount of loading solution is then added here and incubated for the desired time. For example, the translation product can be obtained by incubating at 92 to 96 ° C. for 1 to 5 minutes. As such a loading solution, it is preferable to use a loading buffer having the composition shown in Example 3 (1) described later from the viewpoint of separation accuracy.

  The resulting translation product is confirmed by SDS gel electrophoresis. For gel electrophoresis, it is preferable to use, for example, an SDS-PAGE running buffer having the composition shown in Example 3 (1) described later, from the viewpoint of separation efficiency. Next, a stacking gel and a running gel are prepared, and gel electrophoresis is performed. As such a gel, it is preferable from the viewpoint of separation efficiency to use a stacking gel and a running gel having the composition shown in Example 3 (1) described later. A gel can be prepared according to a conventional method, for example, gel electrophoresis can be performed at about 15 to 30 mA for 1 to 4 hours.

(9) Tag purification Next, the obtained translation product is purified using a tag. First, PBS having a desired composition and an elution buffer as described below are prepared, and purified by reacting in a low adsorption tube using tagged magnetic beads. For example, when using a His tag, it is purified as follows. First, 1 × PBS containing 130-140 mM NaCl, 2.5-3.0 mM KCl, 5-15 mM disodium hydrogen phosphate, 1.5-2.0 mM potassium dihydrogen phosphate (adjusted to pH 7.2-7.4 with 6M NaOH), And 2 × HisMag elution buffer (adjusted to pH 7.2-7.6 with 6M NaOH) containing 5-15 mM sodium phosphate and 450-550 mM imidazole.

  For example, a desired amount of His Mag Sepharose Ni (manufactured by GE Healthcare Science; for example, 30-50 μL) is taken into a desired size, for example, a 1.5 mL low adsorption tube, where a desired amount, for example, 150 The operation of suspending in 250 μL of 1 × PBS, attracting beads with a magnet, discarding the supernatant, and suspending in a desired amount of 1 × PBS is repeated a desired number of times.

  Next, the in-vitro translation product to be purified is added thereto, and a desired buffer such as 1 × PBS is added so that the total volume becomes, for example, 100 μL. Place the tube at 8-12 ° C overnight and discard the supernatant. Thereafter, an operation of adding a desired amount of 1 × PBS, suspending, attracting beads with a magnet, and discarding the supernatant is repeated a desired number of times. Next, 10-30 μL of elution buffer is added and suspended to recover the supernatant. Suspend the same amount of elution buffer, collect the translation product again by recovering the supernatant, and use SDS-PAGE to confirm the translation product. As described above, a full construct containing a linker containing a desired target domain binding site and a desired domain can be obtained.

  If tag purification of the constructed construct is not successful due to the interaction between the tag and the enzyme protein, etc., some spacer is included between the random region and the desired tag. As a result, the interaction between the tag and the enzyme protein main body can be prevented, and the purification can proceed efficiently.

(10) New display method using covalent bond between target domain and target domain binding site The method of binding linker and mRNA by head-to-head bond using the DNA construct obtained above is as follows. Explained.

First, using tritactic phosphatase (for example, manufactured by Takara Bio Inc.), triphosphate is removed from the 5 ′ end of mRNA, and then a polynucleotide kinase (for example, manufactured by Takara Bio Inc.) is acted on. Then, a phosphate group is attached to the 5 ′ end of the mRNA and linked to the linker prepared as described above (FIG. 7).
Alternatively, these phosphatase treatments and kinase treatments can be omitted and T4 RNA ligase treatment can be performed to directly link the linker and the mRNA.

  When translation is performed under the following conditions, ribosomes first bind to mRNA. The ribosome synthesizes a protein containing the target domain, and the synthesized protein binds to the target domain binding site. The ribosome reads the stop codon contained in the mRNA, finishes protein synthesis, and leaves the mRNA. Ribosomes away from the mRNA are turned over and used for the next protein synthesis (ribosome turnover, see FIG. 6A).

  For example, a case where a 809 bp mRNA (hereinafter, also referred to as “construct 1” or “mRNA 1”, respectively) shown in SEQ ID NO: 9 in the sequence listing, which is a PCR product obtained by the above-described PCR, is obtained. Will be described.

  Here, N represents any base selected from the group consisting of G, A, U, and C, and K represents G or U. The mRNA1 represents a protein composed of 230 amino acids (SEQ ID NO: 10 in the sequence listing).

  A desired amount of product is taken from the PCR product obtained as described above, mixed with the same amount of desired buffer to prepare a measurement sample, and heated at a desired temperature for a desired time. Thereafter, the binding between the linker, the construct 1, and the PCR product (mRNA1) is confirmed by fluorescence observation. For example, 0.5 to 1.5 μL of the above PCR product is taken and mixed with the same amount of 2 × STR to prepare a measurement sample, heated at 93 to 96 ° C. for 1 to 5 minutes, and then subjected to fluorescence measurement. It can be confirmed whether or not the linker is bound to the mRNA1.

  Subsequently, by preparing a spacer sequence in advance in the synthesized protein, it is possible to facilitate the subsequent purification and purification of the protein using the His tag. As such a spacer, one having an α helix structure (hereinafter referred to as “α stand spacer”) can be used.

  The desired protein, for example, myoglobin A chain (A1 to A16, see SEQ ID NO: 11 in the Sequence Listing and FIG. 8) can be prepared by substituting Gln for the hydrophobic amino acid and using this sequence (patent application) During).

  Using the amino acid sequence of this peptide, N-terminal free type α-stand peptide (SEQ ID NO: 12 in the sequence listing) and C-terminal free type α stand peptide (sequence listing) having the following sequences by in vitro selection described below. SEQ ID NO: 13) can be obtained.

  The in vitro selection method is performed as follows. Here, the linkage between the mRNA and the linker can be performed directly or indirectly, chemically or physically, using a known technique. For example, if a sequence complementary to the end of the linker is provided at the 3 'end of mRNA, both can be linked by hybridization.

  In the present invention, “ligation” or “ligation reaction” refers to a reaction promoted by an enzyme. Specifically, a ligase such as T4 RNA ligase or TS2126 thermostable phage-derived DNA ligase can be suitably used. It is more preferable to use T4 RNA ligase (manufactured by Takara Bio Inc.) for reasons of ligation efficiency.

  For annealing before the ligation reaction, heat at 60 to 100 ° C for 2 to 60 minutes, heat with a heat block, aluminum block, water bath or other heating equipment, and then leave it at room temperature for about 2 to 60 minutes. It is preferable to carry out by lowering the temperature gently and further cooling to −5 to 10 ° C. For example, warm on an aluminum block for about 5 minutes at about 90 ° C, then warm on an aluminum block for about 5 minutes at about 70 ° C, and finally leave it on ice for about 10 minutes at room temperature. Is preferred.

The composition of the ligation reaction solution was 0.1 to 2.5 U / μL of T4 polynucleotide kinase (manufactured by Takara Bio Inc.); 0.4 to 5 U / μL of T4 RNA ligase (manufactured by Takara Bio Inc.); 10 to 250 mM Tris -Hydrochloric acid (pH of about 7.0 to about 8.0); 2.0-50 mM magnesium chloride; 2.0-50 mM dithiothreitol (DTT); 0.2-5.0 mM ATP. In terms of reaction efficiency, about 0.5 U / μL T4 polynucleotide kinase; about 2.0 U / μL T4 RNA ligase; about 50 mM Tris-HCl (about pH 7.5); about 10 mM magnesium chloride; about 10 mM DTT; Preferably it contains about 1.0 mM ATP. If necessary, an RNA-degrading enzyme inhibitor such as SUPERase / In ^™ RNase inhibitor (manufactured by Ambion) may be added to this reaction solution.

  The volume of the reaction system for performing the ligation reaction is preferably 4.0 to 100 μL. The reaction efficiency is preferably 20 to 40 μL, and the reaction efficiency is highest when it is about 20 μL. The amount ratio of RNA to linker in this reaction system is preferably such that the amount of mRNA relative to the linker of 5.0 to 100 pmol is 5.0 to 100 pmol.

  The annealing reaction can be performed in a ligation reaction solution excluding T4 polynucleotide kinase and T4 RNA ligase. Then, the ligation reaction can be started by introducing a necessary amount of these enzymes into the reaction solution after the annealing. This ligation reaction is preferably performed at 10 to 40 ° C. for about 1 minute to several hours. From the viewpoint of working efficiency and reaction efficiency, it is preferably 4 to 12 hours at 20 to 30 ° C, and most preferably about 8 hours at about 25 ° C.

Next, protein synthesis is performed by mixing ligation A (conjugate of mRNA and linker) with a cell-free translation system. Here, the cell-free translation system for synthesizing the protein can be selected from the group consisting of animal-derived cells, plant-derived cells, fungi and bacteria. Specifically, Escherichia coli, rabbit reticulocyte, wheat germ extract and the like can be used (Lamfrom H., Grunberg-Manago M., Biochem. Biophys. Res. Commun., 27, 1 (1967)).
Since the type of codon used for translation differs depending on the biological species, it is preferable to select a cell-free translation system in accordance with the target gene and the origin of the gene.

  In the embodiment of the present invention, it is preferable to use a reticulocyte lysate obtained from rabbit blood as the cell-free translation system. The lysate is obtained by degrading cell-derived mRNA with micro-coccal nuclease, adding glycol ether diamine tetraacetic acid (EGTA) to chelate calcium, and inactivating the nuclease (hereinafter referred to as “micro-coccal nuclease treatment”). It is more preferable to say “finished”).

The composition of the translation reaction solution was 4 to 85 μL of rabbit reticulocyte lysate (micrococcal nuclease-treated) and a reaction buffer containing 0.24 to 10 pmol of the above conjugate (final concentration: 16 to 400 mM potassium acetate; 0.1 to 2.5 It is preferred to use 10-100 μL of mM magnesium acetate; 0.2-50 mM creatine phosphate; each containing 0-0.25 mM amino acid. From the viewpoint of reaction efficiency, a reaction buffer (final concentration: 80 mM potassium acetate; 0.5 mM magnesium acetate; 10 mM creatine phosphate) containing 17 μL of rabbit reticulocyte lysate (same as above) and 1.2 to 2.0 pmol of the above conjugate. It is more preferred to use 25 μL of each 0.05 mM amino acid (methionine and leucine are each 0.025 mM); If necessary, to the reaction solution, may be added to RNA degradation enzyme inhibitors such as SUPERase · In ^TM RNase inhibitor (Ambion, Inc.).

  The translation reaction is preferably performed at about 20 to about 40 ° C. for about 10 to about 90 minutes, and particularly preferably at about 30 ° C. for about 20 minutes from the viewpoint of production efficiency and work efficiency.

  The conjugate can be easily detected by binding a labeling compound to a linker in advance. Examples of such labeling compounds include fluorescent compounds, antigenic epitopes, and radioisotopes. However, radioisotopes are not suitable for this experiment because they require a special facility (radioactive material handling facility) for subsequent experimental operations.

  When it is difficult to use a radioisotope, it is preferable to use a fluorescent compound. The fluorescent compound has a free functional group (for example, a carboxyl group that can be converted into an active ester, a hydroxyl group that can be converted into a phosphoramidite, or an amino group), and can be linked to a linker as a labeled base. It is preferable to use the fluorescent dye. Examples of such fluorescent dyes include fluorescein isothiocyanate (FITC), phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate. Various GFPs can also be used.

  By the procedure as described above, a full construct containing a linker, a target domain and an α-stand can be prepared, and a protein containing the target domain can be synthesized. By placing the base sequence of the α stand spacer between the base sequence of the protein containing the target domain and the tag sequence used for tag purification, the tag can be successfully placed on the surface of the protein containing the target domain. it can. Thereby, the purification efficiency can be dramatically increased.

  Furthermore, in the present invention, since a linker is bound to the 5 ′ end of the mRNA transcribed with the target domain, it is possible to design an mRNA with a stop codon and to turn over the ribosome. ing.

(Example 1) Preparation of DNA construct containing O ⁶ -methylguanine-DNA-methyltransferase (MGMT) (1) Material and method
NaCl, KCl, disodium hydrogen phosphate · 12 water, potassium dihydrogen phosphate, 6M NaOH, EDTA (sodium ethylenediaminetetraacetate), and formamide were purchased from Wako Pure Chemical Industries, Ltd. MgCl ₂ was purchased from Nacalai Tesque. HEPES was purchased from Dojindo Laboratories Co., Ltd., and dithiothreitol and Triton X-100 were purchased from SIGMA.

(2) Confirmation of MGMT activity and amplification of DNA encoding MGMT (2-1) Preparation of solution First, in order to evaluate the activity of MGMT by fluorescence intensity, 10 × D-PBS (1.37 M NaCl, 27 mM KCl, 100 mM) Disodium hydrogen phosphate, 12 hydrate, 18 mM potassium dihydrogen phosphate (adjusted to pH 7.4 with 6 M NaOH), 1 × reaction buffer (10 mM MgCl ₂ , 1 mM dithiothreitol, 100 mM NaCl, 18 mM HEPES (pH 7.5) ), 50 mM KCl, 0.025% Triton X-100, 1 μg / mL BSA), and stop solution (90% formamide and 20 mM EDTA (pH 8.0)).

(2-2) Preparation of enzyme solution etc. Next, substrate FQ (2.5 pmol / μL: 2 μL of 10 μM MGMT F (FITC-MGMT23), 2 μL of 10 μM MGMT Q (MGMT-BHQ1), 3.2 μL of water and 0.8 μL of 2 types including 10 × D-PBS (−); total 8 μL) or substrate F100 (2.5 pmol / μL: 1 μL 10 μM MGMT F100, 2.6 μL water and 0.4 μL 10 × D-PBS (−); total 4 μL) A substrate solution was prepared.

The above two kinds of substrate solutions were each incubated at 95 ° C. for 1 minute, and then cooled to 37 ° C. at 1 ° C./second. After incubating at 37 ° C. for 5 minutes, the temperature was further decreased to 4 ° C. at 1 ° C./second, and hybridization was performed at 4 ° C. These substrate solutions were stored as MGMT substrate stock solutions at −20 ° C. until use. In addition, since the fluorescence intensity of the substrate decreased with time, it was used on the adjusted day. Also, O ⁶ is a substrate analog of MGMT - -benzylguanine A stock solution of (. Hereinafter, "O ⁶ -BG" or, simply may be referred to as "BG") was prepared using the above 1x reaction buffer.

(2-3) Production of BG linker The linker used in the mRNA display method and cDNA display method of the present invention was commissioned to Tsukuba Oligo Service Co., Ltd.

Example 2 Preparation of Two DNA Constructs Two constructs (hereinafter referred to as “construct 1” and “construct 2”) were prepared as follows. The schematic overall structure of these constructs is shown in FIGS. The DNA sequence (839 bp), mRNA sequence (809 bp) and protein sequence (230 aa) of the construct 1 are shown in SEQ ID NOs: 2 to 4 in the sequence listing. Similarly, the DNA sequence (896 bp), mRNA sequence (866 bp) and protein sequence (249aa) of construct 2 are shown in SEQ ID NOs: 5 to 7 in the sequence listing.

(1) Preparation of construct 1 (1-1) Amplification of MGMT region In order to amplify the MGMT region, MGMT amplification solution (0.5 μL of Prime Star (Takara Bio Inc.), 10 μL of 5 × Prime Star reaction buffer) 4 μL of 2.5 mM dNTP mixture, 1 μL of 10 μM MGMT Primer 1, 1 μL of 10 μM MGMT Primer 2, 0.5 μL of SF-Construct-MGMT and 33 μL of DDW (double distilled water) were prepared (total 50 μL). Next, PCR amplification was performed by repeating 30 cycles of 95 ° C for 2 minutes, 95 ° C for 20 seconds, 65 ° C for 5 seconds, 72 ° C for 30 seconds, heated at 72 ° C for 5 minutes, and then cooled to 4 ° C. Thus, the reaction was stopped, and PCR product 1 was obtained. Thereafter, 1 μL was taken from the obtained PCR product 1, mixed with 7 μL of distilled water and 8 μL of 2 × STR, and heated at 95 ° C. for 3 minutes to obtain a sample for gel electrophoresis.

  Acrylamide, N, N'-methylene-bis (acrylamide) (BIS), ammonium persulfate (APS), N, N, N ', N'-tetramethyl-ethylenediamine (TEMED), urea, 2-mercaptoethanol, lauryl sulfate Sodium (SDS) was purchased from Wako Pure Chemical Industries, Ltd. and used for the preparation of polyacrylamide gel. Boric acid and tris (hydroxymethyl) aminomethane were also purchased from Wako Pure Chemical Industries, Ltd. and used for the preparation of 5 × TBE buffer.

  Samples were applied to a minigel (4% acrylamide gel containing 8 M urea), and gel electrophoresis was performed at 200 V, 60 ° C. for 90 minutes using 0.5 × TBE buffer as a running buffer. After gel electrophoresis, single-stranded DNA was stained with SYBR Green II Nucleic Acid Gel Stains (abbreviated as SYBR Green 2 (manufactured by Takara Bio Inc.)) and detected with an imager. Show.

(1-2) Addition of T7 region to amplified MGMT region In order to add the T7 region to the MGMT region obtained in (1-1) above by overlap PCR, a T7 region addition solution (0.5 μL) was added. Prime Star, 10 μL 5 × Prime Star Reaction Buffer, 4 μL 2.5 mM dNTP Mixture, 1 μL 10 μM T7 Primer, 0.3 μL 10 μM T7-Linker Hybrid-MGMT, 1 μL 10 μM MGMT Primer 2, 1 μL MGMT PCR Product and 33 μL A total of 50 μL containing DDW was prepared.

  The above T7 region addition solution was placed in a thermal cycler, treated at 95 ° C for 2 minutes, and then cycled [95 ° C for 20 seconds, 65 ° C for 5 seconds, 72 ° C for 30 seconds] 30 times, then 72 ° C For 5 minutes and then cooled to 4 ° C. 1 μL of the PCR product, 7 μL of distilled water, and 8 μL of 2 × STR were mixed and heated at 95 ° C. for 3 minutes. This was applied as a sample to a mini gel (4% acrylamide gel, containing 8M urea), and gel electrophoresis was performed at 200 V, 60 ° C. for 90 minutes using 0.5 × TBE buffer as a running buffer. After completion of gel electrophoresis, single-stranded DNA was stained with SYBR Green 2 and detected with an imager.

  A PCR product 2 containing an MGMT sequence was obtained from the target band at 621 bp (see FIG. 11). Next, the sequence of the T7 promoter region is mixed upstream of MGMT, and two kinds of DNAs (for T7 promoter and MGMT) whose 3 ′ ends overlap each other are mixed in equal amounts, and overlap PCR is performed under normal PCR conditions. A 733 bp T7-MGMT sequence was obtained in a row (see FIG. 12).

(1-3) Amplification of random region Subsequently, in order to amplify the random region, a random region amplification buffer (4 μL of Vent _R (exo-) DNA polymerase (manufactured by BioLabs), 20 μL of 10 × Vent _R (exo-) DNA Polymerase reaction buffer, 4 μL of 100 mM MgSO ₄ , 16 μL of 2.5 mM dNTP mixture, 2 μL of 100 μM MGMT-His tag Xa primer, 2 μL of 100 μM biotin-cNew Ytag primer, 1 μL of 10 μM random template and 151 μL of DDW in total 200 μL ) Was prepared. This buffer was placed in a thermal cycler, treated at 95 ° C for 2 minutes, and then cycled [95 ° C for 20 seconds, 65 ° C for 30 seconds, 75 ° C for 1 minute] 30 times, and heated at 72 ° C for 5 minutes. Later, it was cooled to 4 ° C. to obtain PCR product 3.

  1 μL was taken from the PCR product 3 obtained as described above, mixed with 1 μL of 2 × STR, and heated at 95 ° C. for 3 minutes. Next, this solution was applied as a sample to a microgel (8% acrylamide gel containing 8M urea), and gel electrophoresis was performed at 100 V and 55 ° C. for 10 minutes using 1 × TBE buffer as a running buffer. After gel electrophoresis, single-stranded DNA was stained with SYBR Green 2 and detected with an imager.

Next, 1/10 volume of Quick-Precip ^™ Plus Solution (containing 10 mM Tris-hydrochloric acid (pH 8.0), 1 mM EDTA and 5 M NaCl, manufactured by EdgeBio) and 2.5% volume of 99% of the PCR product 3 above. Ethanol was added and mixed, followed by centrifugation at 13,000 rpm (12,000 × g) for 15 minutes. Next, the supernatant was discarded so as not to drop the white precipitate, 70% ethanol was added and mixed, and the mixture was again centrifuged at 13,000 rpm for 10 minutes. The supernatant was discarded so as not to drop the white precipitate, ethanol was completely blown off with an evaporator, and then the DNA pellet was dissolved in distilled water, and the DNA concentration was measured with nanodrop (manufactured by Thermo Fischer Scientific).

(1-4) Bead purification of PCR amplified random region
50 μL of streptavidin beads (Dynabeads MyOne Streptavidin C1, Invtrogen) was placed in a low adsorption tube, this tube was attached to a magnet, and the supernatant was discarded. Here, 100 μL of 1 × binding buffer was added and mixed, attached to a magnet, and the supernatant was discarded. The operation of adding 100 μL of 1 × binding buffer, mixing, attaching to a magnet, and discarding the supernatant was repeated two more times.

  Here, 20 μL (about 37 μg) of PCR product 3 (random region) obtained in (1-2) above, 30 μL of distilled water, and 50 μL of 2 × binding buffer were added and mixed. This solution was placed in a rotator and rotated at 4 ° C. and left overnight. The tube was attached to a magnet and the supernatant was discarded. The operation of adding 100 μL of 1 × binding buffer, mixing, attaching to a magnet, and discarding the supernatant was repeated three times.

  50 μL of dissociation buffer (95% formamide containing 10 mM EDTA) was added, placed in a rotator, reacted at 70 ° C. for 15 minutes, attached to a magnet, and the supernatant was collected. The operation of adding 50 μL of dissociation buffer and placing it in a rotator, reacting at 70 ° C. for 15 minutes, attaching to a magnet and collecting the supernatant was performed again.

  The recovered supernatant was purified by the ethanol precipitation method using Quick-Precip Plus Solution in the same manner as the purification of PCR product 3 in (1-3) above. The DNA pellet obtained by centrifugation was dissolved in distilled water, and the DNA concentration was measured by nanodrop. A PCR amplification product containing a His tag-random region sequence was obtained from the band of interest at the position of 128 bp (see FIG. 13).

(1-5) Production of full construct In order to produce construct 1 as a full construct using each PCR amplification product for construct 1 obtained as described above, a buffer for producing full construct (4 μL of Vent _R (exo-) DNA polymerase (manufactured by BioLabs), 20 μL of 10 × Vent _R (exo-) DNA polymerase reaction buffer, 4 μL of 100 mM MgSO ₄ , 16 μL of 2.5 mM dNTP mixture, 9 μL of T7 to MGMT region (15 pmol), 1.3 μL His tag to random region (15 pmol) and 141.7 μL DDW in total 196 μL) were prepared.

  The full construct preparation buffer was placed in a thermal cycler and incubated at 95 ° C. for 2 minutes and then at 75 ° C. for 6 minutes. To this was added 2 μL of 100 μM T7 primer and 2 μL of 100 μM Biotin-cNew Ytag primer. Place this mixture in a thermal cycler and repeat the cycle of 95 ° C for 10 seconds, 65 ° C for 30 seconds, 75 ° C for 1 minute 30 times, then incubate at 75 ° C for 5 minutes, cool to 4 ° C, and construct 1 Obtained as a full construct.

PCR product 4 (construct 1) obtained as described above was added to 1/10 volume of Quick-Precip ^™ Plus Solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA and 5 M NaCl) and 2.5 volumes. 99% ethanol was added and mixed, and centrifuged at 13,000 rpm (12,000 × g) for 15 minutes. Next, the supernatant was discarded so as not to drop the white precipitate, 70% ethanol was added and mixed, and the mixture was again centrifuged at 13,000 rpm for 10 minutes. The supernatant was discarded so as not to drop the white precipitate, ethanol was completely blown off with an evaporator, then the DNA pellet was dissolved in distilled water, and the DNA concentration was measured with nanodrop.

  1 μL of PCR product 4, 7 μL of distilled water and 8 μL of 2 × STR were mixed and heated at 95 ° C. for 3 minutes. This was applied as a sample to a minigel (4% acrylamide gel containing 8 M urea), and gel electrophoresis was performed at 200 V, 60 ° C. for 90 minutes using 0.5 × TBE buffer as a running buffer. After completion of gel electrophoresis, single-stranded DNA was stained with SYBR Green 2 and detected with an imager. Of the two bands visible in the lane in which the full construct of FIG. 14 was migrated, the band visible on the lower side is the remainder of the T7 promoter-MGMT sequence (733 bp), and the band visible on the upper side is the full construct containing the random region (839 bp). ). When the chain length was confirmed, a band was confirmed around the target 839 bp.

  The gel was cut with a scalpel at the 839 bp band position, placed in a 1.5 mL tube and ground with a spatula. To this, 800 μL of dilution buffer (10 mM magnesium acetate, 0.5 M ammonium acetate, 1 mM EDTA (pH 8.0), 0.1% SDS) was added and incubated at 70 ° C. overnight.

  The PCR product 4 was purified in the same manner as described above, and the DNA concentration was measured by nanodrop. The DNA cut out from the gel and purified was further subjected to gel electrophoresis with a minigel to confirm whether it was a single band. The results are shown in FIG. 15 and FIG. Subsequently, direct sequence analysis was performed to confirm whether the sequence was correct.

(1-6) Transcription to mRNA (for concentration measurement)
To transcribe the resulting construct 1 into mRNA, a transcription buffer (2 μL of DNA full construct (3.6 pmol), 3 μL of 2.5 mM rNTP mixture, 2 μL of 5 × T7 transbuffer, 2 μL of T7 enzyme mixture and 1 μL of RNase inhibitor A total of 10 μL) was prepared. The transfer buffer was placed in a thermal cycler and incubated at 37 ° C. for 2 hours. Next, 1 μL of RQ1 RNase-Free DNase (1 U / μL, manufactured by Promega) was added thereto and incubated at 37 ° C. for 15 minutes to obtain a sample for RNA purification. RNeasy Mini Kit (manufactured by QIAGEN) was used for RNA purification. First, an RNA purification buffer (90 μL of RNase free water, 350 μL of RLT buffer and 250 μL of 99% ethanol) was added to the RNA purification sample to obtain a mixed solution.

  The above mixture was placed in an RNeasy purification column and centrifuged at 12,000 rpm for 15 seconds. The collection tube was discarded, the column was placed in a new collection tube, 500 μL of RPE buffer was placed in the column, and centrifuged at 12,000 rpm for 15 seconds. The collection tube was discarded, the column was placed in a new collection tube, 500 μL of 80% ethanol was added, and the mixture was centrifuged at 12,000 rpm for 2 minutes. The collection tube was discarded, the column was placed in a new collection tube, the column lid was opened, and the mixture was further centrifuged at 12,000 rpm for 5 minutes.

  The collection tube was discarded, the column was placed in a new collection tube, 15 μL of RNase free water was added, and the mixture was allowed to stand at room temperature for 3 minutes. Centrifugation was performed at 12,000 rpm for 1 minute. 15 μL of RNase free water was added again and left at room temperature for 1 minute. Then, it centrifuged for 1 minute at 12,000 rpm, and the density | concentration of RNA was measured by nanodrop.

(2) Transcription to mRNA (for size measurement)
In order to transcribe the DNA construct 1 obtained in the above (1-5) into mRNA, a buffer solution for transcription (2 μL of DNA full construct (3.6 pmol), 3 μL of 2.5 mM rNTP mixture, 2 μL of 5 × T7 transfer buffer, 2 μL 1 μL of RNase inhibitor (RNasin Plus PNase Inhibitor, manufactured by Promega) (total 10 μL) was prepared. This transfer buffer was placed in a thermal cycler and incubated at 37 ° C. for 2 hours. Subsequently, 1 μL of RQ1 RNase-Free DNase (1 U / μL) was added thereto and incubated at 37 ° C. for 15 minutes.

  RNA was purified using RNeasy Mini Kit. First, a buffer for RNA purification (containing 90 μL of RNase free water, 350 μL of RLT buffer and 250 μL of 99% ethanol, total 700 μL) was placed in RNeasy Mini Kit, and centrifuged at 12,000 rpm for 15 seconds. The collection tube was discarded, the column was placed in a new collection tube, 500 μL of RPE buffer was placed in this column, and the mixture was centrifuged again at 12,000 rpm for 15 seconds. After centrifugation, the collection tube was discarded, the column was placed in a new collection tube, 500 μL of 80% ethanol was added to the column, and the mixture was centrifuged at 12,000 rpm for 2 minutes. After the centrifugation, the collection tube was discarded, the column was put into a new collection tube, the column lid was opened, and the mixture was further centrifuged at 12,000 rpm for 5 minutes.

  After centrifuging, the collection tube was discarded and a column was placed in a new collection tube. 15 μL of RNase free water was added to the collection tube, left standing at room temperature for 3 minutes, and then centrifuged at 12,000 rpm for 1 minute. After centrifuging, the collection tube was discarded and a column was placed in a new collection tube. 15 μL of RNase free water was added thereto, left at room temperature for 1 minute, and centrifuged at 12,000 rpm for 1 minute. Thereafter, the concentration of RNA was measured.

  After confirming the approximate size of the mRNA obtained as described above by gel electrophoresis (see Fig. 17), it was used as the 809nts (base number) mRNA in the following experiment, and its validity was confirmed in the subsequent experiment. did. The exact measurement at this stage is very difficult because the amount of mRNA is small and unstable.

(3) Linker ligation (3-1) Binding in one step Next, the FITC-labeled BG linker sequence obtained in (2) above was added to the 5 ′ end of the obtained 809 bp mRNA by a hybridization method. Complementary binding was followed by enzymatic binding with T4 RNA ligase. Linker bound to mRNA was detected by FITC fluorescence after gel electrophoresis. Details will be described below according to the procedure.

  For linker ligation, using the mRNA and BG linker at the concentrations shown in Table 1 below, four types of one-step binding buffers (x μL mRNA (xpmol), yμL 10 μM BG-PEG-linker (20 × pmol), And 21.5- (x + y) μL of RNase free water (21.5 μL in total). This buffer was placed in a thermal cycler, incubated at 90 ° C. for 2 minutes, cooled to 70 ° C. at 0.1 ° C./second, incubated at 70 ° C. for 2 minutes, and cooled to 25 ° C. at 0.1 ° C./second. Next, a ligation buffer (containing 2 μL of RNase inhibitor (40 units / μL), 2.5 μL of 10 × T4 RNA ligase buffer and 1 μL of T4 RNA ligase (10-50 units / μL), total 5.5 μL) is prepared here. In addition, a mixture was obtained.

  The mixture was incubated at 25 ° C. for 1 hour to ligate the linker. 1 μL was taken from the resulting ligation product, mixed with 1 μL of 2 × STR, and heated at 95 ° C. for 3 minutes. Using this solution as a sample, it was applied to a microgel similar to the above (8% acrylamide gel containing 8 M urea), and gel electrophoresis was performed at 100 V and 55 ° C. for 10 to 15 minutes using 1 × TBE buffer as a running buffer. It was.

  After the gel electrophoresis, FITC was detected by an imager to confirm whether or not ligation was performed. Next, single-stranded DNA was stained with SYBR Green 2 to confirm whether or not there was a degradation product of RNA. The results are shown in FIG.

  As shown in FIG. 18, no band was seen in the detection by FITC, and no ligation product was confirmed, indicating that no ligation was performed. This is because T4 RNA ligase is an enzyme that links the 5 'phosphate end oligonucleotide and the 3'-OH oligonucleotide, so that the 5' end of the triphosphate of mRNA is not bound to the BG-linker. It was thought to be due to.

(3-2) Three-step binding Phosphate removal buffer (8 μL of mRNA (8 pmol), 1 μL of 10 × antarctic phosphate buffer and 1 μL of an antitactic phosphatase (New England Biolabs) A total of 10 μL) was prepared. This buffer was placed in a thermal cycler and incubated at 37 ° C. for 15 minutes. Subsequently, it incubated at 70 degreeC for 5 minute (s), cooled to 4 degreeC, and removed the phosphate group from 5 'terminal of mRNA. Ligation buffer (10.5 μL of RNase free water, 2.5 μL of 10 × T4 RNA ligase buffer and 1 μL of T4 polynucleotide kinase (10 units / μL), 24 μL in total) was prepared and added to this solution to obtain a mixture.

  This mixture was incubated at 37 ° C. for 30 minutes, then 1 μL of 10 μM BG-linker was added, placed in a thermal cycler and incubated at 90 ° C. for 2 minutes. The temperature was decreased to 70 ° C. at 0.1 ° C./second, incubated at 70 ° C. for 2 minutes, and then decreased to 25 ° C. at 0.1 ° C./second.

  To this sample, 1 μL of T4 RNA ligase (10-50 units / μL) was added and incubated at 25 ° C. for 1 hour to obtain a ligation product. 1 μL was taken from the resulting ligation product, mixed with 1 μL of 2 × STR, and heated at 95 ° C. for 3 minutes. This sample was applied to a microgel in the same manner as in (2) above, and gel electrophoresis was performed at 100 V and 55 ° C. for 10 to 15 minutes using 1 × TBE buffer as a running buffer. The results are shown in FIG.

  As shown in FIG. 19, a FITC-labeled band was observed around about 200 bases. When staining with SYBER Green2, a band was detected at the same position, and a band was further detected at a position corresponding to 809 bases. When the 809-base mRNA and the 50-base mRNA fragment are combined, an 859-base band should be seen, but the band seen this time seems to move much slower than that. This was thought to be due to the bulky structure with a branched structure.

  In addition, mRNA obtained by transcription contained mRNA fragments having a size of about 150 bases or 180 bases. When a linker binds to the mRNA of these fragments, it matches the size of the band seen in FITC, so the bands detected by staining are those that have these linkers bound to these mRNA fragments, or It was thought that it might have been combined.

  In order to confirm that the bond between the 5 ′ end of mRNA and the 3 ′ end of the linker was formed instead of the bond between the linkers, the mRNA band was cut out from the above gel electrophoresis result and the same as above. Purification was performed under the conditions, and mRNA was converted to a single band, and ligation was performed under the same conditions as described above. The results are shown in FIG.

As shown in Fig. 20, the linker-mRNA ligation product should actually be about 50 bases larger than the mRNA, but what was seen at 400 to 500 nts was thought to be a partial degradation of the mRNA. . Conversely, the size of the linker (50nts) binding is too large to explain. This confirmed that the linker and mRNA were bound.
On the other hand, it became clear that mRNA was gradually shortened in the course of the reaction, indicating that it was necessary to prevent the degradation of mRNA in order to use it in subsequent reactions. The cause of the shortened mRNA is that the mRNA is an unstable substance, RNase is easily decomposed or hydrolyzed by heating, and zinc phosphate is contained in the dephosphorylation enzyme reaction solution. It was considered that hydrolysis during heating might have been accelerated.

(3-3) Transcription and binding with a linker—Part 2
Using another lot of T4 RNA ligase, another attempt was made to bind the triphosphate at the 5 ′ end of the mRNA to —OH at the 3 ′ end of the linker. This time, BG-linker was added to the mRNA in an excess amount and hybridized (see Table 2). When T4 RNA ligase was added at 100 units / μL and reacted, the BG-linker was bound to the mRNA. I was able to confirm. The results are shown in FIG.

  Usually, T4 RNA ligase links a 5 'phosphate-terminal oligonucleotide and a 3'-OH oligonucleotide, but the other lot of enzymes used here is more active than previously used. It was considered that the 5 'end of the triphosphate of mRNA and 3'-OH of the linker were bound in a star-active manner. Furthermore, since the linker and mRNA could be combined in a single step reaction, degradation of mRNA was also suppressed. Therefore, it was confirmed that this method is suitable for linking a linker and mRNA.

(Example 3) In vitro translation using PUREflex (1) In vitro translation The ligation product of BG linker mRNA produced in Example 1 above was translated using PUREflex (manufactured by Nihon Millipore Corporation). First, it contains xμL ligation product (xpmol), 25μL solution 1, 2.5μL solution 2, 2.5μL solution 3, 5μL RNase inhibitor (40 units / μL) and (15-x) μL RNase free water. A translation buffer (total 50 μL) was prepared and incubated at 37 ° C. for 1 to 4 hours to obtain a translation product.

  Next, a gel electrophoresis solution containing 6 μL of translation product, 4 μL of 0.5 M EDTA and 10 μL of 2 × loading buffer was prepared and incubated at 95 ° C. for 3 minutes. As described above, a sample for gel electrophoresis was obtained.

For gel electrophoresis, 1.5M Tris-hydrochloric acid (pH 8.8), 0.5M Tris-hydrochloric acid (pH 6.8), 2 × SDS-PAGE running buffer (25 mM Tris (hydroxymethyl) aminomethane, 192 mM glycine and 0.1% SDS), 2 × SDS loading buffer (containing 0.1 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, dyes BPB and XC), PBS-T buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ · 12H ₂ O, 1.8 mM K ₂ HPO ₄ (adjusted to pH 7.4 with 6M NaOH) and 0.1% Tween 20), and 40% acrylamide stock (acrylamide / bisacrylamide = 19/1 38% (w / v) acrylamide and 2% (w / v) N, N′-methylene-bisacrylamide) were prepared.

  Contains 0.5 mL of 40% acrylamide stock, 1.25 mL of 0.5 M Tris-HCl (pH 6.8), 250 μL of 2% SDS (add 16 μL of 20% APS and 5 μL of TEMED as needed), distilled water Was added to 5 mL to prepare a stacking gel. Also contains 1.5 mL of 40% acrylamide stock, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 500 μL of 2% SDS (add 33 μL of 20% APS and 10 μL of TEMED as needed), The running gel was prepared by making up to 10 mL with distilled water.

  The sample for gel electrophoresis obtained as described above was applied to each well of the gel, and gel electrophoresis was performed at 20 mA for 2 hours. After the gel electrophoresis was completed, FITC was detected with an imager to confirm whether or not ligation was performed. Subsequently, single-stranded DNA was stained with SYBR Green 2, and it was confirmed whether or not there was an RNA degradation product.

  After translation, a band was observed at a position of a size larger than the ligation product of the BG linker / mRNA, and it was considered that the translated MGMT was bound to the linker BG. Furthermore, a band of a protein / BG linker conjugate formed by binding the mRNA and unreacted remaining BG linker to the translated protein was also confirmed. This indicated that the synthesis of MGMT was successful and that the linkers BG and MGMT were bound.

  When the translation time was set to 0.5 hours, 1 hour, and 3.5 hours, and the change in the translation product in vitro was examined, the amount of the protein / BG linker conjugate increased as the in vitro translation time increased. This was thought to be due to the turnover of ribosomes and the production of excess amounts of MGMT protein (see FIG. 22).

  Further, when 0.1 μg of proteinase K was allowed to act on 500 pg of the obtained translation product, the band considered to be MGMT protein-BG linker / mRNA conjugate and the band of MGMT protein / BG linker conjugate were thinned. From this, it was confirmed that any band was a conjugate with protein. Further, MGMT and BG linker were reacted under the reaction conditions for translating the linker-mRNA conjugate, and the position of the band of the protein / BG linker conjugate was confirmed (see FIG. 23).

  When 0.5 pmol of the obtained translation product was treated with 6 μg of RNase A at 37 ° C. for 30 minutes, both the band that appeared to be protein-BG linker / mRNA conjugate and the band of BG linker / mRNA conjugate disappeared. For this reason, it was confirmed that both of these two bands are RNA conjugates. Furthermore, it was also confirmed that only the protein (MGMT) -BG linker conjugate band remained (see FIG. 24). This indicates that the BG linker is bound to both mRNA and protein (MGMT) as intended.

(2) His Tag Purification of Translation Product Next, for purification of the His tag, 1 × PBS containing 137 mM NaCl, 2.7 mM KCl, 10 mM disodium phosphate · 12 hydrate and potassium dihydrogen phosphate (pH 7.4 with 6M NaOH) And a HisMag elution buffer containing 20 mM sodium phosphate and 500 mM imidazole (adjusted to pH 7.4 with 6 M NaOH).

  40 μL of His Mag Sepharose Ni was taken and placed in a 1.5 mL low adsorption tube. Here, 200 μL of PBS was suspended and attached to a magnet, and the supernatant was discarded. The operation of adding 200 μL of PBS to suspend, attaching to a magnet and discarding the supernatant was repeated twice. Next, the in vitro translation product to be purified was added, PBS was added so that the total volume was 100 μL, placed in a rotator, and placed at 10 ° C. overnight. The same operation as above was repeated twice, in which PBS was added and suspended, and the supernatant was discarded. 20 μL of elution buffer was added and suspended therein, and the supernatant was collected by attaching to a magnet. The operation of recovering the supernatant was performed again, and the recovered product was confirmed by SDS-PAGE. As a result, even when the His tag purification eluate was subjected to gel electrophoresis, no band was observed, and it was revealed that His tag purification was not possible. The results are shown in FIG.

(Example 4) Improvement of display of His tag by improvement of construct (1) Prediction of three-dimensional structure of construct 1 As described above, the construct 1 obtained in Example 1 is a spacer having a length of 4 amino acids behind MGMT. It is designed so that the His tag sequence comes across. For this reason, the His tag should be bound to the Ni-NTA beads and the translation product should be recovered. However, since the reverse result was obtained, based on SWISS-MODEL structure prediction, we examined how the His tag part was folded by computer simulation.

  In the above construct 1, it was presumed that the His tag interacted with the β sheet of MGMT, and it was considered that the purification of the His tag did not succeed. This is due to steric hindrance or the fact that the His tag is entangled in the protein during protein folding and does not serve as a purification tag, and as a result does not bind to Ni-NTA beads. It was thought to be due to

(2) Prediction of the structure of construct 2 Therefore, the SWISS-MODEL structure prediction method uses a structure in which a spacer of 4 amino acids and an α-stand peptide of 19 amino acids are sandwiched between MGMT and His tag sequence. Based on this, a computer simulation was performed (see FIGS. 26A and 26B). As a result, it has been clarified that in this construct 2, the His tag portion appears on the protein surface and has a structure suitable for purification of the His tag. For this reason, DNA construct 2 was prepared as follows, and His tag purification was attempted.

(3) Construction 2 and practical evaluation In the same manner as construction 1, each region constituting the construct 2 is amplified, and finally, these are ligated, and the 896 bp construct 2 (construct) using the overlap PCR method. For the sequence of the His Tag display portion (57 nts) inserted in 2, see SEQ ID NO: 15 in the Sequence Listing).

  That is, the MGMT region was amplified by PCR in the same manner as when construct 1 was prepared. Next, the T7 region was added to the MGMT region using the overlap PCR method in the same manner as described above. Thereafter, the random region was purified with beads, the MGMT region added with the T7 region, and the random region added with the His-tag containing the spacer were amplified by PCR in the same manner as described above, and construct 2 (full construct) was obtained. Obtained.

  The construct 2 was transcribed into mRNA by the same method as described above to obtain 866mer mRNA (SEQ ID NO: 16 in the Sequence Listing). Here, N represents any base selected from the group consisting of G, A, U, and C, and K represents G or U.

  When this mRNA was treated in the same manner as in the above construct 1, first, from the in vitro translation lane, a triplet conjugate of “protein / BG linker / mRNA”, an over-translated protein and an excess BG linker And a “protein / BG linker” conjugate formed by binding to each other. A band (triplicate) indicating that the His-tag purified product was picked up by the His-tag was traced from before the purification and was slightly detected at the top of the gel. On the other hand, in the His-tag purified eluate (third and fourth lanes from the left), the “protein-BG linker” conjugate produced by overtranslation clearly appears in this photograph. On the other hand, the BG linker / mRNA conjugate containing no His tag disappeared, which clearly means the presence of a protein sequence purified by the His tag.

  This showed that Construct 2 was working. The protein obtained here was estimated to be a protein composed of 249 amino acids (SEQ ID NO: 17 in the sequence listing) (see FIG. 27). Here, X represents any amino acid.

(4) Results From the above, it was confirmed that a BG linker was bound to the 5 ′ end side of mRNA, and that the translated protein was bound to the BG linker, and Head-to-Head Linking (H-to-H Bond) could be formed. This technique allows in vitro translation without using large amounts of ribosomes. When using H-to-H bonds for actual in vitro selection, it was expected that this method could be improved by adding an unreacted linker step before translation.

  The present invention is useful in the fields of evolutionary engineering, peptide drug screening, medical fields, and diagnostic fields.

SEQ ID NO: 1: Base sequence of H2H linker SEQ ID NO: 2: DNA sequence of MGMT SEQ ID NO: 3: Primer for MGMT DNA amplification SEQ ID NO: 4: Primer for MGMT DNA amplification SEQ ID NO: 5: T7 primer SEQ ID NO: 6: Biotin-cNew Ytag primer

SEQ ID NO: 7: Reverse transcription primer SEQ ID NO: 8: His tag-XaR primer SEQ ID NO: 9: MGMT mRNA amplified by PCR
SEQ ID NO: 10: amino acid sequence of protein translated from SEQ ID NO: 9: SEQ ID NO: 11: amino acid sequence of artificial myoglobin protein

SEQ ID NO: 12: N-terminal free stand peptide SEQ ID NO: 13: C-terminal free stand peptide SEQ ID NO: 14: Random primer SEQ ID NO: 15: His-tag presentation sequence inserted in construct 2 SEQ ID NO: 16: mRNA of construct 2
SEQ ID NO: 17: amino acid sequence of the protein translated from SEQ ID NO: 16

Claims (6)

Creating a linker comprising a target domain binding site and a linker comprising a fluorescent label;
Producing a DNA construct comprising at least a target domain, a variable region, and a DNA sequence comprising a base sequence of a stop codon;
Preparing a corresponding mRNA from the DNA construct by transcription; a transcription step;
An mRNA-linker ligation step in which the 5 ′ end of the mRNA is treated with an enzyme to bind to the 3 ′ end of the linker;
A translation step in which one ribosome binds to the mRNA contained in the mRNA-linker conjugate to produce the target domain by translation;
The translated target domain binds to the target domain binding site of the linker to form an mRNA-linker-protein conjugate, and the ribosome recognizes the stop codon and the mRNA-linker-protein conjugate. A ribosome release step that is automatically released from
An mRNA display method capable of ribosome turnover. The desired domain used in the DNA construct production step is any one selected from the group consisting of O ⁶ -methylguanine-DNA-methyltransferase (MGMT) and a functional mutant protein of MGMT. An mRNA display method capable of ribosome turnover according to claim 1.   The DNA construct includes, on the downstream side of the base sequence, a spacer having a desired length, an α stand peptide having an α helix structure having a desired length, and a purification tag, and further comprising a variable peptide sequence or a variable protein. The mRNA display method capable of ribosome turnover according to claim 2, comprising any one of them.   The mRNA display capable of ribosome turnover according to claim 3, wherein the spacer is 3 to 5 amino acids long and the α-stand peptide is 17 to 21 amino acids long. Law.   5. The mRNA display capable of ribosome turnover according to claim 3 or 4, wherein the purification tag is any one selected from the group consisting of a His tag, a FLAG tag, and a biotin-like peptide. Law.   a 5 ′ triphosphate and a linker of mRNA, a triphosphate removing enzyme that removes triphosphate from the 5 ′ end of mRNA, a kinase that adds phosphate to the 5 ′ end of mRNA from which triphosphate has been removed, and The linker according to any one of claims 1 to 5, wherein the linker and the 5 'triphosphate end of mRNA are directly linked by T4 RNA ligase or directly linked by T4 RNA ligase. An mRNA display method capable of ribosome turnover.

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