JP2008253176A - Linker for obtaining highly affinitive molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently screening a protein capable of linking to a target molecule with high affinity, and a nucleic acid linker designed so as to adapt to the method. <P>SOLUTION: Disclosed is a nucleic acid linker for producing a complex formed between mRNA and a protein encoded by the mRNA. The nucleic acid linker links to a single-stranded polynucleotide portion hybridizable to the mRNA in the state of branching from the single-stranded polynucleotide portion, wherein the nucleic acid linker includes an arm portion having a protein-binding portion in its terminal end and the arm portion includes a photocleavage site or a cleavage site for single-stranded nucleic acid nicking enzyme. Also disclosed is a method for screening proteins by using the nucleic acid linker. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a linker construct for associating a gene with a protein encoded thereby, and a method for screening a protein that binds to a target molecule using the linker construct.

  When acquiring functional peptides / proteins, so-called protein engineering techniques designed by human knowledge based on the structure of proteins, and evolutionary engineering techniques for acquiring molecules with the desired functions from the creation of libraries with various random shapes. Has become the mainstream. In particular, in recent years, functional peptides can be obtained in a relatively short period of time by the advancement of genotype-phenotype association technology, which is an elemental technology of evolutionary engineering represented by the phage display method.

In addition to the phage display method, there is a cell display method for displaying on the cell surface of Escherichia coli, yeast, etc., in addition to the phage display method. These are all useful methods for obtaining functional peptides and proteins. However, since the number of cells per unit volume is limited, it is impossible to have a library size of 10 ⁸ / ml or more. , There was a drawback that screening efficiency was low. In order to overcome this problem, a method for associating a genotype with a phenotype using a cell-free translation system has been developed. Typical examples of these include “ribosome display”, which links mRNA and its translated protein via a ribosome, and “in vitro virus,” which connects mRNA and the protein encoding it via puromycin, an antibiotic. (In vitro virus) method (mRNA display method) ". These have a library size of 10 ¹² / ml or more, and problems such as cytotoxicity and membrane permeation caused by the use of cells by the phage display method can be eliminated, so that the screening efficiency can be dramatically improved. It was. In addition, a cDNA display method has been developed in which a reverse-transcribed cDNA is directly covalently linked to a protein-linking linker, and screening is performed in a variety of environments because of the dramatically stable mRNA / cDNA-protein conjugate structure compared to conventional methods. It became possible to do.

  The cDNA display method is disclosed, for example, in JP-A-2004-97213. In this method, a complex is formed in which a protein and a polynucleotide encoding the protein are bound via puromycin. This complex includes mRNA-puromycin-protein, mRNA / cDNA-puromycin-protein, cDNA-puromycin-protein, and the like. Puromycin is a protein synthesis inhibitor having a structure similar to the 3 'end of aminoacyl-tRNA, and has a property of specifically binding to the 3' end of a growing protein on a ribosome under predetermined conditions. When mRNA and puromycin are bound via an appropriate linker and the protein is synthesized from the mRNA in a cell-free translation system, the synthesized protein and the mRNA encoding the same are bound via puromycin. A complex is formed (Nemoto et al, FEBS Lett., 414, 405-408, 1997). Next, a protein having a desired function is selected from the library of the complex. After selecting using mRNA and protein complex, DNA may be synthesized by reverse transcription, or after synthesizing DNA from mRNA by reverse transcription to form DNA / mRNA hybrid and protein complex, A protein may be selected. However, since a single-stranded nucleic acid has a high possibility of non-specific interaction as an aptamer, an mRNA / cDNA-protein complex is preferred when a library containing a random sequence is used. After selecting a protein having a desired function, the protein can be identified by analyzing the DNA sequence.

  The method of mapping genotypes and phenotypes using a cell-free translation system has been established as a practical technique, leading to the cDNA display method. In ordinary in vitro screening, there is a much wider and more flexible selection than before. It became possible. If this cDNA display method enables screening targeting molecules such as cell surface receptors that were previously only possible with structurally stable phage display, GPCR (G protein-coupled receptor) and sugar chains There is a possibility that screening for drug discovery targets such as can be performed more efficiently than phage display. However, when targeting cell surface molecules, there are many membrane proteins on the cell surface, so it is possible to obtain proteins and peptides with high affinity and specificity for the target molecule. It was difficult. In phage display, for example, Morphosys' “Cys Display” has been devised to efficiently remove the phage body, which is a gene part, by adding a reducing agent from a protein or peptide strongly bound to the target molecule. This is because a phage having a protein that binds strongly to a target is less likely to be eluted by washing or the like, and as a result, a phage having a high affinity protein cannot be recovered.

Similarly, the cDNA display method has a problem that it is difficult to recover mRNA / cDNA-protein conjugates presenting high affinity proteins and peptides. In the existing cDNA display method, DTT, etc. are contained at a high concentration in the cell-free translation system used for protein synthesis, so it is difficult to incorporate an SS bond into the linker as in the Cys Display method. For this reason, especially when using membrane proteins on the cell surface as target molecules, it is difficult to form a “biotinylated-SS-target molecule” as used in in vitro screening. There is no easy way. In addition, since it is required that the recovery method (buffer, etc.) does not affect the cells themselves, conventional methods screen for mRNA / cDNA-protein conjugates that present proteins and peptides with high affinity. It is extremely difficult.
JP 2004-97213 A Nemoto et al, FEBS Lett., 414, 405-408, 1997

  An object of the present invention is to provide a method for efficiently screening a protein that binds to a target molecule with high affinity, and a nucleic acid linker designed to be compatible with this method.

  The inventor previously introduced a base that can be easily cleaved by ultraviolet light or a single-stranded nucleolytic enzyme into a specific location of the puromycin linker by chemical synthesis, and the puromycin linker-protein bound to the target molecule. We found that mRNA / cDNA can be efficiently recovered by applying ultraviolet light or single-stranded nuclease to the complex.

The present invention provides a nucleic acid linker for producing a complex of mRNA and a protein encoded by the mRNA. Nucleic acid linker is
A single-stranded polynucleotide portion capable of hybridizing with the mRNA; and an arm portion that is linked in a branched state from the single-stranded polynucleotide portion and has a protein linking portion at the end;
And the arm portion includes a photocleavable site or a single-stranded nucleolytic enzyme cleavage site.

  In a preferred embodiment of the present invention, in the nucleic acid linker of the present invention, the single-stranded polynucleotide portion has a sequence capable of hybridizing with the 3 ′ end region of the mRNA in the 3 ′ side region, and the 3 ′ end. Functions as a primer for reverse transcription of the mRNA into cDNA.

  In another preferred embodiment of the present invention, in the nucleic acid linker of the present invention, the single-stranded polynucleotide portion has a sequence capable of hybridizing to the 3 ′ end region of the mRNA in the 3 ′ side region, It has a restriction enzyme recognition site on the 5 'side.

  In another preferred embodiment of the present invention, in the nucleic acid linker of the present invention, the single-stranded polynucleotide portion has a sequence capable of hybridizing to the 3 ′ end region of the mRNA in the 3 ′ side region, It has a single-stranded RNA nucleolytic enzyme cleavage site on the 5 ′ side.

  In another preferred embodiment of the present invention, in the nucleic acid linker of the present invention, the protein linking moiety is a puromycin, 3′-N-aminoacylpuromycin aminonucleoside or 3′-N-aminoacyl adenosine aminonucleoside chemical structural skeleton. including.

  In another aspect, the present invention includes mRNA, a cDNA complementary thereto, a protein encoded by the mRNA, and the nucleic acid linker of the present invention, wherein the cDNA and the protein are Provided is an mRNA / cDNA-nucleic acid linker-protein complex linked via a nucleic acid linker. In the present specification, the expression “mRNA / cDNA” indicates that mRNA and a complementary cDNA are hybridized to form a double strand. The expression “mRNA / cDNA-nucleic acid linker-protein complex” indicates that the mRNA / cDNA hybrid and the nucleic acid linker and the nucleic acid linker and the protein are covalently bonded, respectively.

In another aspect, the present invention relates to an mRNA / cDNA-nucleic acid in which mRNA, a complementary cDNA, and a protein encoded by the mRNA are linked via the nucleic acid linker of the present invention described above. A method for producing a linker-protein complex is provided. This method
Annealing the mRNA and the nucleic acid linker;
Ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker;
By synthesizing a protein from the mRNA in a cell-free protein translation system, an mRNA-nucleic acid linker-protein complex in which the C-terminus of the protein is bound to the protein linking portion of the nucleic acid linker is generated.
Subjecting the mRNA-nucleic acid linker-protein complex to a reverse transcription reaction to produce an mRNA / cDNA-nucleic acid linker-protein complex;
Including each step.

In another aspect, the present invention provides a method for selecting a cDNA encoding a protein that binds to a target molecule. This method
An mRNA / cDNA-nucleic acid linker-protein complex in which mRNA, a complementary cDNA, and a protein encoded by the mRNA are linked via the nucleic acid linker of the present invention described above is prepared, and the mRNA is prepared. contacting the / cDNA-nucleic acid linker-protein complex with the target molecule,
Cleave the mRNA / cDNA-nucleic acid linker-protein complex bound to the target molecule by irradiating light at the site of the photocleavable group of the nucleic acid linker, or act on a single-stranded nucleic acid cleaving enzyme to bind to the target molecule Each of the mRNA / cDNA-nucleic acid linker-protein complex is cleaved at the single-stranded nucleic acid cleaving enzyme cleavage site of the nucleic acid linker and the cleaved mRNA / cDNA is recovered from the mRNA / cDNA-nucleic acid linker-protein complex. Process.

In another aspect, the present invention provides a method for identifying a protein that binds to a target molecule. This method
An mRNA / cDNA-nucleic acid linker-protein complex in which mRNA, a complementary cDNA, and a protein encoded by the mRNA are linked via the nucleic acid linker of the present invention described above is prepared, and the mRNA is prepared. contacting the / cDNA-nucleic acid linker-protein complex with the target molecule,
Cleave the mRNA / cDNA-nucleic acid linker-protein complex bound to the target molecule by irradiating light at the site of the photocleavable group of the nucleic acid linker, or act on a single-stranded nucleic acid cleaving enzyme to bind to the target molecule The mRNA / cDNA-nucleic acid linker-protein complex is cleaved at the single-stranded nucleic acid cleaving enzyme cleavage site of the nucleic acid linker, and the mRNA / cDNA cleaved from the mRNA / cDNA-nucleic acid linker-protein complex is recovered and recovered. Each step of identifying the protein encoded by the recovered cDNA by determining the base sequence of the obtained cDNA is included.

  According to the present invention, even if a cDNA display molecule is strongly bound to the cell surface, DNA can be recovered by irradiating the cell with ultraviolet rays (350 nm) for about 10 minutes. In general, it is considered that there is little effect on cells when irradiated with ultraviolet rays of a long wavelength to such a degree, and a cDNA portion encoding a high affinity protein or peptide can be rapidly recovered from the cell surface. . In addition, when a single-stranded nuclease is used, the cell is hardly affected.

Nucleic acid linker The nucleic acid linker of the present invention is a linker for linking mRNA or cDNA and a protein encoded by the mRNA or cDNA. The nucleic acid linker includes a single-stranded polynucleotide portion capable of hybridizing with mRNA and an arm portion that is bonded in a state branched from the single-stranded polynucleotide portion and has a protein-linking portion at the end. The basic structure of this nucleic acid linker is already known as a linker used in the cDNA display method.

  The structure of the nucleic acid linker of the present invention will be described with reference to FIG. The nucleic acid linker of the present invention comprises a single-stranded polynucleotide portion capable of hybridizing with mRNA, and an arm portion that is bound in a state branched from the single-stranded polynucleotide portion and has a protein linking portion at the end, The arm portion is characterized by including a photocleavable site or a single-stranded nucleic acid cleaving enzyme cleavage site. The single-stranded polynucleotide moiety may be DNA or a nucleic acid derivative such as PNA, but is preferably modified DNA imparted with nuclease resistance. Examples of the modified DNA include any modified DNA known in the art, such as DNA having an internucleoside bond such as phosphoramidite and phosphorothioate, and DNA having a sugar modification such as 2′-fluoro and 2′-O-alkyl. It may be used. The single-stranded polynucleotide part has a sequence capable of hybridizing with the 3 'terminal region of the mRNA to be screened in the 3' side region. Further, the 3 'end of the single-stranded polynucleotide portion of the nucleic acid linker of the present invention can function as a primer for reverse transcription from this mRNA to cDNA.

  The arm portion of the nucleic acid linker of the present invention functions as a spacer that holds the mRNA and protein linking portion at a desired distance. The arm part is joined in a branched state from the single-stranded polynucleotide part, and has a protein linking part at the end. The term “branched” means that one end of the arm part is joined to the single-stranded polynucleotide part at a position other than both ends of the single-stranded polynucleotide part to form a T-shaped structure. Means that

  The linkage between the single-stranded polynucleotide part and the arm part consists of a modified nucleotide (for example, an amino-modified nucleotide) present at the linking position on the single-stranded polynucleotide part and a modified nucleotide (for example, a thiol modification) present at the end of the arm part. Nucleotide) with a bifunctional reagent. The linking site on the single-stranded polynucleotide portion is preferably a position several bases upstream from the 3 ′ end of the single-stranded polynucleotide portion, which allows the single-stranded polynucleotide portion to be used in the subsequent cDNA synthesis step. The 3 ′ end of can function as a primer.

  There is a protein linking portion at the other end of the arm portion. The protein linking moiety means a structure having a property of specifically binding to the 3 'end of the extending protein on the ribosome under a predetermined condition, and a typical one is puromycin. Puromycin is a protein synthesis inhibitor having a structure similar to the 3 'end of aminoacyl-tRNA. As the protein linking moiety, any substance can be used as long as it has a function of specifically binding to the 3 ′ end of the extending protein. For example, 3′-N-aminoacylpuromycin aminonucleoside or 3 ′ Puromycin derivatives such as -N-aminoacyl adenosine aminonucleoside can be used. The arm portion may be composed of a nucleic acid or a nucleic acid derivative, or may be composed of a polymer such as polyethylene glycol. The arm may be further modified to increase the stability of puromycin or a label for detecting the complex.

  The arm portion of the nucleic acid linker of the present invention includes a photocleavable site or a single-stranded nucleic acid cleaving enzyme cleavage site, which allows mRNA (corresponding to the protein to be associated with the protein without dissociating the bond between the protein and the target substance). Or DNA) can be recovered. The photocleavable site refers to a group having the property of being cleaved when irradiated with light such as ultraviolet rays. For example, PC Linker Phosphoramidite (Glen research), a composition for photocleavage of nucleic acids containing nucleic acids (full nucleic acid) Composition for photocleavage: JP-A-2005-245223), chain breakage by photolysis (SBIP) technique, and the like. As the photocleavable site, any group that is commercially available or known in the art may be used. The term “single-stranded nucleic acid cleaving enzyme cleavage site” refers to a nucleic acid group that can be cleaved by a single-stranded nucleic acid cleaving enzyme such as deoxyribonuclease and ribonuclease, and includes nucleotides and derivatives thereof.

  In a preferred embodiment of the nucleic acid linker of the present invention, the single-stranded polynucleotide moiety has a restriction enzyme recognition site in the 5 'region. In the cDNA display method, when a mRNA-nucleic acid linker-protein complex is synthesized in a cell-free translation system and then cDNA is synthesized from the mRNA by reverse transcription, reverse transcription occurs when there is a contaminant derived from the cell-free translation system. Therefore, it is preferable to purify the mRNA-nucleic acid linker-protein complex before the reverse transcription reaction. If an affinity molecule such as biotin is bound to the single-stranded polynucleotide portion, a protein is synthesized from mRNA in a cell-free translation system, and then the mRNA-nucleic acid linker-protein complex is bound to streptavidin beads via biotin. The mRNA / cDNA-nucleic acid linker-protein complex can be easily purified from the cell-free translation system by binding and recovering, and then cleaving with a restriction enzyme. In addition, if a nucleic acid linker is designed so that a ribonuclease-cleavable site is included in the single-stranded polynucleotide portion, the mRNA / cDNA-nucleic acid linker-protein complex can be similarly converted into a cell-free translation system by allowing ribonuclease to act. Can be easily purified. In this case, it will be easily understood that the arm portion includes a deoxyribonuclease-cleavable site and the single-stranded polynucleotide portion includes a ribonuclease-cleavable site.

mRNA / cDNA-nucleic acid linker-protein complex Using the nucleic acid linker of the present invention, an mRNA / cDNA-nucleic acid linker-protein complex can be produced. First, DNA encoding the protein to be screened is prepared and transcribed with mRNA polymerase to prepare mRNA. As the DNA, any DNA or DNA aggregate to be examined for binding to the target molecule can be used. For example, a cDNA library derived from mRNA obtained from a sample tissue, a synthetic random sequence DNA library, a DNA library in which a part of the sequence is randomized, or the like can be used. The mRNA is designed to have a sequence that hybridizes with the 3 ′ region of the single-stranded polynucleotide of the nucleic acid linker of the present invention in its 3 ′ region. Next, the mRNA is annealed with the nucleic acid linker of the present invention, and the 3 ′ end region of the mRNA is hybridized with the 3 ′ side region of the single-stranded polynucleotide portion of the nucleic acid linker of the present invention. When a protein is synthesized, a complex is formed in which mRNA and a protein encoded by mRNA are linked via the nucleic acid linker of the present invention.

  If the 3 'end of mRNA and the 5' end of the nucleic acid linker are ligated before protein synthesis, the complex becomes more stable. Protein synthesis is typically performed in a cell-free protein translation system. As the cell-free protein translation system, any translation system known in the technical field, such as wheat germ-derived cell-free protein translation system and Escherichia coli-derived cell-free protein translation system, can be used.

  Next, cDNA is synthesized from the mRNA by reverse transcription to form a more stable complex. A reverse transcriptase is allowed to act on the mRNA-nucleic acid linker-protein complex obtained as described above in the presence of dNTP to synthesize cDNA complementary to mRNA. At this time, the sequence at the 3 'end of the single-stranded polynucleotide of the nucleic acid linker of the present invention acts as a primer for synthesis. The mRNA / cDNA-nucleic acid linker-protein complex thus obtained is used in the form of an mRNA / cDNA hybrid.

In another aspect of protein screening , the present invention relates to a method for selecting a cDNA encoding a protein that binds to a target molecule using the nucleic acid linker of the present invention described above. A target molecule is any molecule that is set up to explore and study proteins that bind to that molecule. For example, when searching for a protein that binds to a receptor using the method of the present invention, the receptor is called a target molecule. In the method of the present invention, as described above, mRNA / cDNA in which mRNA, cDNA complementary thereto, and protein encoded by the mRNA are linked via the nucleic acid linker of the present invention described above. Prepare a nucleic acid linker-protein complex. Next, after contacting the mRNA / cDNA-nucleic acid linker-protein complex with the target molecule, the mRNA / cDNA-nucleic acid linker-protein complex that does not bind to the target molecule is removed. The contact and removal conditions can be appropriately set according to the degree of binding affinity between the target molecule and the protein. For example, when the binding affinity is high or predicted to be high, stricter conditions (for example, higher salt concentration, higher surfactant concentration, longer time, etc.) can be set.

  Next, when the arm portion of the nucleic acid linker has a photocleavable group, the mRNA / cDNA-nucleic acid linker-protein complex bound to the target molecule is irradiated with light, and the photocleavable group site of the nucleic acid linker Disconnect with. Alternatively, when the arm portion of the nucleic acid linker has a single-stranded nucleic acid cleaving enzyme cleavage site, the single-stranded nucleic acid cleaving enzyme is allowed to act so that the mRNA / cDNA-nucleic acid linker-protein complex bound to the target molecule is nucleic acid. Cleavage at the single-stranded nucleic acid cleavage enzyme cleavage site of the linker. By collecting the cleaved cDNA from the mRNA / cDNA-nucleic acid linker-protein complex in this way, a cDNA encoding a protein that binds to the target molecule can be obtained. According to the method of the present invention, cDNA corresponding to a protein that binds to a target molecule with high affinity can be efficiently recovered without dissociating the target molecule and the protein.

  Furthermore, by determining the base sequence of the thus recovered cDNA by a conventional method, the amino acid sequence of the protein encoded by the recovered cDNA, that is, the protein that binds to the target molecule can be obtained.

  In a particularly preferred embodiment, the method of the present invention is used in molecular evolution techniques by cDNA display methods. Prepare an appropriate mRNA or DNA library, perform the above-described mRNA synthesis, protein synthesis, cDNA synthesis, selection of the protein that binds to the target molecule, and cDNA recovery, and then use the recovered cDNA to perform each step. Repeatedly. DNA encoding a protein having high affinity with the target molecule can be concentrated by repeating several to several tens of times. Such molecular evolution techniques using puromycin are well known in the art.

  Since the method of the present invention can identify a protein without dissociating the target molecule and the protein, it is particularly useful for searching for a protein having a high binding affinity for the target molecule.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

  In order to demonstrate the technology of the present invention, the following novel puromycin linker (FIG. 2) was designed and synthesized, and the cleavage efficiency by ultraviolet rays (UV) was examined. At that time, not only the puromycin linker was present in the liquid phase, but also the case of immobilization on the magnetic beads was examined in order to investigate the problem of UV shielding by the magnetic beads.

Synthesis of puromycin linker
1-1 Materials The following three types of materials (1) and (2) were purchased from Gene World Co., Ltd.
(1) Short Biotin Flagment (26mer) [sequence; 5′-CC (rG) C (TB) C (rG) CCC CGCCG CCCCC CG (T) CC T-3 ′ (SEQ ID NO: 1): where (rG ) Is Ribo G, (T) is Amino-Modifier C6 dT, (TB) is Biotin-dT, all manufactured by Glen Research Search.
(2) Puro-FS * [sequence: 5 '-(S)-(PL) -C (F)-(Spc18)-(Spc18)-(Spc18)-(Spc18) -CC- (Puro) -3' ]
However, (S): Thiol-Modifier C6 SS, (PL): PC Linker Phosphoramidite, (F): Fluorescent-dT, (Spc18): Spacer Phosphoramidite 18, (Puro): Puromycin CPG * Follow.
The crosslinker EMCS (344-05051) was purchased from Dojindo.

1-2 Synthesis and purification methods Material (2) Dissolve 10 nmol of Puro-FS * in 100 ul of 50 mM phosphate buffer (pH 7.0), add 1 ul of 100 mM TCEP (final 1 mM), and let stand at room temperature for 6 hours. 2) Thiol of Puro-FS * was reduced. TCEP was removed using NAP5 (Amersham, 17-0853-02) equilibrated with 50 mM phosphate buffer (pH 7.0) immediately before the crosslinking reaction. Add 0.2M phosphate buffer (pH7.0) to 100ul, 500pmol / ul material (1) Short Biotin Fragment 20ul, 100mM EMCS 20μl, stir well and let stand at 37 ℃ for 30 minutes, then unreacted EMCS Removed. The precipitate was dried under reduced pressure, dissolved in 10 ul of 0.2 M phosphate buffer (pH 7.0), and the reduced Puro-FS * (˜10 nmol) was added and left at 4 ° C. overnight. Add TCEP to the final sample to 4 mM and let stand at room temperature for 15 minutes, then remove unreacted Puro-FS * by ethanol precipitation and remove unreacted material (2) Short Biotin Flagment by the following conditions. HPLC purification was performed.
Column; nacalai tesque CSOMOSIL 37918-31 10x250mm C18-AR-300 (Waters)
Buffer A: 0.1M TEAA, Buffer B; 80% acetonitrile (diluted with ultrapure water)
Flow rate: 0.5ml / min (B%: 15-35% 33min)

  The HPLC fraction was analyzed on an 18% acrylamide gel (8M urea, 62 ° C.). The desired fraction was dried under reduced pressure and then dissolved in DEPC-treated water to 10 pmol / μl.

Cleavage of the linker in the liquid phase by ultraviolet light After dissolving 20 pmol of Puro-PF-S * in 20 μl of 20 mM PBS buffer in a 1.5 ml Eppendorf tube, a UV lamp (365 nm: UVP, model number UVL-56) is used. Used and irradiated at a distance of 2 cm from the tube. The irradiation time was 5 minutes, 15 minutes, and 30 minutes. 10 μl was removed from each sample and electrophoresed on 20% 8M urea polyacrylamide gel electrophoresis. The amount of cleaved linker was determined by checking the FITC band.

  The time required for cleavage of the puromycin linker by UV in the liquid phase is shown in FIG. It was found that the portion connecting the protein and the portion connecting the mRNA / cDNA portion were cleaved with an efficiency of about 90% by irradiating with ultraviolet rays for about 5 minutes.

400 μg of linker-cleaved streptavidin-coated magnetic beads “Dyna Beads (Dynal, Norway)” solid-phased on magnetic beads by ultraviolet rays was washed according to the attached protocol. Washed beads were mixed with 40 μmol of puromycin linker synthesized by the above method and 80 μl of 1 × Binding buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 M NaCl, 0.1% Triton X-100). Incubated for 10 minutes. The puromycin linker was immobilized on the magnetic beads by the above operation. Next, it was set on a magnet, the beads were collected, and the supernatant was discarded. The collected beads were divided into four and dispensed into tubes each containing 20 μl of 20 mM phosphate buffer.

  These four tubes were irradiated with UV for 0, 5, 15, and 30 minutes. The protein linking part of the linker cleaved by UV irradiation will be present in the solution. Furthermore, since this part has FITC, the amount of the cleaved linker was measured by electrophoresis of the solution after UV irradiation and confirming the FITC band. In addition, the linker remaining on the magnetic beads without being cleaved by UV was separately separated from the bead surface by RNAseT1, and the amount of the linker that was not cleaved was also measured by checking in the same manner. These samples were analyzed with a fluorescence imager Typhoon (GE) after 20% 8M urea-denaturing polyacrylamide gel electrophoresis. FIG. 4 shows the time required for cleavage of the linker immobilized on the beads via biotin by UV irradiation. From these results, it was found that when the puromycin linker according to the present invention is present on the magnetic beads, it can be cleaved with an efficiency of about 90% by irradiation with UV for 30 minutes.

FIG. 1 shows the structure of the nucleic acid linker of the present invention. FIG. 2 shows one embodiment of the nucleic acid linker of the present invention synthesized in the examples. FIG. 3 shows cleavage of the nucleic acid linker by UV irradiation. FIG. 4 shows cleavage of the bead-immobilized nucleic acid linker by UV irradiation.

Claims (9)

a nucleic acid linker for producing a complex of mRNA and a protein encoded by the mRNA, the nucleic acid linker comprising:
A single-stranded polynucleotide portion capable of hybridizing with the mRNA; and an arm portion which is bound in a branched state from the single-stranded polynucleotide portion and has a protein linking portion at the end;
A nucleic acid linker, wherein the arm part contains a photocleavable site or a single-stranded nucleic acid cleaving enzyme cleavage site. The single-stranded polynucleotide portion has a sequence capable of hybridizing with the 3 'end region of the mRNA in the 3' side region, and the 3 'end functions as a primer for reverse transcription from the mRNA to cDNA. Item 9. The nucleic acid linker according to Item 1. The single-stranded polynucleotide portion has a sequence capable of hybridizing with the 3 'end region of the mRNA in its 3' side region and a restriction enzyme recognition site in its 5 'side region. Nucleic acid linker. The single-stranded polynucleotide portion has a sequence capable of hybridizing with the 3 ′ end region of the mRNA in the 3 ′ side region, and a single-stranded RNA nucleic acid cleaving enzyme cleavage site in the 5 ′ side region. The nucleic acid linker according to claim 1. The nucleic acid linker according to claim 1, wherein the protein linking moiety comprises a chemical structural skeleton of puromycin, 3'-N-aminoacylpuromycin aminonucleoside or 3'-N-aminoacyl adenosine aminonucleoside. An mRNA, a cDNA complementary thereto, a protein encoded by the mRNA, and the nucleic acid linker according to any one of claims 1 to 5, wherein the mRNA / cDNA and the protein comprise the nucleic acid linker. MRNA / cDNA-nucleic acid linker-protein complex linked via each other. An mRNA / cDNA-nucleic acid linker-protein complex in which mRNA, cDNA complementary thereto, and a protein encoded by the mRNA are linked via the nucleic acid linker according to any one of claims 1 to 5. A method of manufacturing
Annealing the mRNA and the nucleic acid linker;
Ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker;
By synthesizing a protein from the mRNA in a cell-free protein translation system, an mRNA-nucleic acid linker-protein complex in which the C-terminus of the protein is bound to the protein linking portion of the nucleic acid linker is generated,
Subjecting the mRNA-nucleic acid linker-protein complex to a reverse transcription reaction to produce an mRNA / cDNA-nucleic acid linker-protein complex;
The method including each process of these. A method for selecting a cDNA encoding a protein that binds to a target molecule, comprising:
An mRNA / cDNA-nucleic acid linker-protein complex in which mRNA, cDNA complementary thereto, and a protein encoded by the mRNA are linked via the nucleic acid linker according to any one of claims 1 to 5. To prepare
Contacting the mRNA / cDNA-nucleic acid linker-protein complex with a target molecule;
Cleave the mRNA / cDNA-nucleic acid linker-protein complex bound to the target molecule by irradiating light at the site of the photocleavable group of the nucleic acid linker, or act on a single-stranded nucleic acid cleaving enzyme to bind to the target molecule Cleaving the resulting mRNA / cDNA-nucleic acid linker-protein complex at the single-stranded nucleic acid cleavage enzyme cleavage site of the nucleic acid linker;
recovering the cleaved mRNA / cDNA from the mRNA / cDNA-nucleic acid linker-protein complex,
The method including each process of these. A method for identifying a protein that binds to a target molecule, comprising:
An mRNA / cDNA-nucleic acid linker-protein complex in which mRNA, cDNA complementary thereto, and a protein encoded by the mRNA are linked via the nucleic acid linker according to any one of claims 1 to 5. And contacting the mRNA / cDNA-nucleic acid linker-protein complex with the target molecule,
Cleave the mRNA / cDNA-nucleic acid linker-protein complex bound to the target molecule by irradiating light at the site of the photocleavable group of the nucleic acid linker, or act on a single-stranded nucleic acid cleaving enzyme to bind to the target molecule Recovering mRNA / cDNA from the resulting mRNA / cDNA-nucleic acid linker-protein complex and determining the base sequence of the recovered cDNA to identify the protein encoded by the recovered cDNA;
The method including each process of these.