JP2007181454A - Method for detecting dna sequence encoding protein specifically selected by molecular display method using micro-array with high sensitivity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a molecule selected by a molecular display method with high sensitivity. <P>SOLUTION: The method is carried out as follows. A tiling array which immobilizes each probe designed on the basis of sequence information about an mRNA encoding a candidate protein with ≤25 bases of a gap between the probes on the average or which is designed so as to provide an overlap between the probes of ≤50% the probe length on the average is prepared. A DNA selected by a screening method using an assigned molecule in which the protein is bound to a nucleic acid encoding the protein is subjected to an RNA amplification with an RNA polymerase in the presence of a nucleotide labeled with a ligand substance without inhibiting the RNA-DNA hybridization to hybridize the amplified RNA with the tiling array. The resultant hybridization product is labeled with a conjugate of an adaptor substance binding to the ligand substance and a fluorescent substance or a substance activating chemifluorescence or chemiluminescence and the intensity of the fluorescence or light emission is measured to detect an interaction by analyzing the resultant results of measurement. Thereby, the nucleic acid encoding the protein interacting with a target molecule is detected. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting a molecule selected by a molecular display method.

  The in vitro virus (IVV) method uses a cell-free translation system to translate mRNA linked to one of the antibiotics puromycin, thereby mapping the protein to the mRNA that encodes it, that is, an in vitro virus ( This is a technique for producing in vitro virus (IVV) (Patent Document 1). So far, this technique has been used to develop a method for screening novel functional proteins and proteins with protein-protein interactions from cDNA and random sequence libraries (Patent Document 2). (Non-patent document 1; Non-patent document 2).

  In the protein interaction screening method based on the existing IVV method, the mRNA site of the selected IVV is amplified and amplified by RT-PCR after multiple cycles of panning to determine the specifically selected sequence. The strategy is to clone the DNA into E. coli and analyze and identify many sequences with a DNA sequencer. However, since a large amount of sequence analysis using a DNA sequencer is difficult in terms of cost and labor, analysis of sequences exceeding 1,000 clones is not realistic. Therefore, it can be said that in the existing method, in order to detect a sequence specifically selected after screening, the number of molecules must be at least 0.1% of the total. However, there is a concern that under such conditions that enrichment is achieved, a “false negative” problem may occur in which a low-expressed gene sequence or an interaction candidate sequence having a relatively low affinity is not detected. .

“False positive”, which is a problem in many screening technologies, can be eliminated to some extent by conducting verification experiments after screening, but false negatives are problems that cannot be solved by subsequent experiments, etc. It is. In fact, based on existing screening methods, a protein that binds to the mouse transcription factor Jun was selected from a mouse brain cDNA library and selected from the results of an experiment that determined the sequence of about 500 clones. Several examples of false negatives where no power sequence was obtained were shown.
WO98 / 016636 WO03 / 014734 Horisawa et al., (2004) Nucleic Acids Res. 32, e169 Miyamoto-Sato et al., (2005) Genome Res. 15, 710-717

  The present invention provides a sensitive detection method for molecules selected by the molecular display method.

In a specific embodiment, the present inventors combined a method of fluorescently labeling a selected DNA and immobilizing a probe designed in a specific mode on a microarray in a screening method based on an existing IVV method. Instead of cloning and DNA sequencing, detection by oligo DNA microarrays can be performed, and compared with existing analysis by cloning and DNA sequencing, highly sensitive and specifically selected sequences It has been found that a large number of sequences that are false negatives can be detected by existing methods.
Based on these findings, the present invention has been completed. The present invention provides the following.

[1] A method for detecting a protein that interacts with a target molecule or a nucleic acid encoding the same, comprising the following steps (a) to (e):
(a) A tiling array in which probes designed based on the sequence information of mRNA encoding a candidate protein for detection are immobilized, and the average gap between probes is 25 bases or less, or the average overlap between probes The process of preparing a tiling array designed to be 50% or less of the probe length.
(b) A step of preparing DNA from an associating molecule selected for interaction with a target substance by a screening method using an associating molecule in which a protein and a nucleic acid encoding the protein are bound.
(c) A step of performing RNA amplification by RNA polymerase using the DNA prepared in (b) as a template in the presence of a nucleotide labeled with a ligand substance that does not inhibit RNA-DNA hybridization.
(d) The RNA amplified in (c) is hybridized to the tiling array prepared in (a), and an adapter substance that binds to the ligand substance and a fluorescent substance or a substance that activates chemiluminescence or chemiluminescence A step of labeling with a conjugate and measuring the intensity of fluorescence or luminescence.
(e) A step of detecting a protein that interacts with a target molecule or a nucleic acid that encodes the protein by analyzing the measurement result of (d).
[2] The method according to [1], wherein the RNA polymerase initiates transcription by a promoter contained in the DNA prepared in step (b).
[3] The method according to [2], wherein the promoter is an SP6 promoter.
[4] The method according to any one of [1] to [3], wherein the ligand substance is biotin and the adapter substance is streptavidin or avidin.
[5] The screening method in step (b) comprises (b1) producing a library of mapping molecules in which a protein and a nucleic acid encoding the protein are bound from a nucleic acid library, (b2) the mapping molecule (B3) a step of selecting an association molecule bound to the target substance, and (b4) a nucleic acid library based on the nucleic acid of the association molecule selected in (b3). The method according to any one of [1] to [3], including a step of preparing, and including repeating the operations of (b1) to (b3).

[6] A protein that interacts with a target molecule, or a protein that interacts with the target molecule, which comprises detecting a protein that interacts with the target molecule or a nucleic acid that encodes the protein by the method according to any one of [1] to [5] A method for screening a nucleic acid encoding

[7] A method for detecting an interaction site of a protein that interacts with a target molecule, comprising the following steps (a) to (e):
(a) A tiling array to which probes designed based on the sequence information of mRNA encoding a protein that interacts with a target molecule are fixed, and the average gap between probes is 25 bases or less, or over-probing between probes Preparing a tiling array that is designed so that the average wrap is 50% or less of the probe length.
(b) A step of preparing DNA from an association molecule selected for interaction with a target substance by a screening method using an association molecule using an association molecule in which a protein and a nucleic acid encoding the protein are bound.
(c) A step of performing RNA amplification by RNA polymerase using the DNA prepared in (b) as a template in the presence of a nucleotide labeled with a ligand substance that does not inhibit RNA-DNA hybridization.
(d) The RNA amplified in (c) is hybridized to the tiling array prepared in (a), and an adapter substance that binds to the ligand substance and a fluorescent substance or a substance that activates chemiluminescence or chemiluminescence A step of labeling with a conjugate and measuring the intensity of fluorescence or luminescence.
(e) A step of detecting an interaction site by analyzing the measurement result of (d).

  Compared with the existing analysis by cloning and DNA sequencing, it is possible to detect a specifically selected sequence with high sensitivity and to detect a sequence that becomes false negative in the existing method.

<1> Detection Method of the Present Invention The detection method of the present invention replaces the existing molecular display method, particularly the cloning / DNA sequencing operation in the screening method based on the IVV method, as a method for solving the above false negative problem. It is characterized by detecting with a DNA microarray (FIG. 1).
The method of the present invention is a method for detecting a protein that interacts with a target molecule or a nucleic acid encoding the same, and includes the following steps (a) to (e).
(a) A tiling array in which probes designed based on the sequence information of mRNA encoding a candidate protein for detection are immobilized, and the average gap between probes is 25 bases or less, or the average overlap between probes The process of preparing a tiling array designed to be 50% or less of the probe length.
(b) A step of preparing DNA from an associating molecule selected for interaction with a target substance by a screening method using an associating molecule in which a protein and a nucleic acid encoding the protein are bound.
(c) A step of performing RNA amplification by RNA polymerase using the DNA prepared in (b) as a template in the presence of a nucleotide labeled with a ligand substance that does not inhibit RNA-DNA hybridization.
(d) The RNA amplified in (c) is hybridized to the tiling array prepared in (a), and an adapter substance that binds to the ligand substance and a fluorescent substance or a substance that activates chemiluminescence or chemiluminescence A step of labeling with a conjugate and measuring the intensity of fluorescence or luminescence.
(e) A step of detecting a protein that interacts with a target molecule or a nucleic acid that encodes the protein by analyzing the measurement result of (d).

  Hereinafter, each step will be described.

<1-a> Step (a)
Step (a) is a step of preparing a tiling array to which a probe designed based on the sequence information of mRNA encoding a detection candidate protein is immobilized.

  The tiling array is a DNA microarray (DNA chip) in which detection probes having base sequences extracted in a tile shape (usually at regular intervals) based on known sequence information are fixed on a substrate.

  The structure of the tiling array used in the present invention is based on the sequence information of mRNA encoding the detection candidate protein, and the probe has an average gap between probes of 25 bases or less, or an overlap between probes on average. Other than designing to be 50% or less of the above, a normal one may be used.

  The detection candidate protein is a protein that attempts to detect the interaction with the target substance, and may be one or plural. Candidate proteins known or expected to interact with the target substance may be selected. For example, when the target substance is DNA, a candidate protein can be determined as a candidate protein by a transcription factor or various estimation methods.

  In designing the probe, the full length of mRNA encoding the detection candidate protein may be targeted, or a part thereof may be targeted according to the purpose of detection such as determination of the interaction site.

  The length of the probe may be selected in consideration of the efficiency of signal detection after hybridization, etc., and is usually 25 to 100 bases on average, preferably 36 to 70 bases, more preferably about 50 bases. is there.

  When designing probes with a gap between probes, the average gap between probes is 25 bases or less, preferably 10 bases or less, and more preferably 0 bases. If the gap is too long, the interaction cannot be detected and false negatives are likely to occur. On the other hand, when the probes are designed to overlap, the average overlap between the probes is 50% or less, preferably 25% or less of the probe length. For example, when the probe length is 50 bases, it is 25 bases or less, preferably 12.5 bases or less. A long overlap is disadvantageous in terms of screening efficiency. There may be no gap or overlap, in which case the gap is 0 bases or the overlap is 0%.

  The probe sequence may be either the sense strand, the antisense strand, or both, and may be selected according to the DNA prepared from the mapping molecule in step (b).

<1-b> Step (b)
Step (b) is a step of preparing DNA from an association molecule selected for interaction with a target substance by a screening method using an association molecule using an association molecule in which a protein and a nucleic acid encoding the protein are bound. It is.

Screening methods that use an association molecule in which a protein and a nucleic acid encoding the protein are combined include, for example, amplification of genotype (nucleic acid), linkage of phenotype (protein) and genotype, and affinity selection. This process is repeated as necessary (FIG. 1). A typical example of such a method is a screening method based on the in vitro virus method (for example, WO
02/48347, Japanese Patent Laid-Open No. 2002-176987).

  The mapping molecule by the in vitro virus method is formed by binding a phenotype molecule containing a protein and a genotype molecule containing a nucleic acid encoding the protein. The genotype molecule is formed by binding a coding molecule having a region encoding a protein in such a form that the base sequence of the region can be translated, and a spacer part. For convenience of explanation, a part derived from a phenotype molecule, a part derived from a spacer molecule, and a part derived from a coding molecule in the mapping molecule are referred to as a decoding part, a spacer part, and a coding part, respectively. Moreover, the part derived from the spacer molecule and the part derived from the coding molecule in the genotype molecule are referred to as a spacer part and a coding part, respectively.

  The spacer molecule can bind to the peptide by, for example, a donor region capable of binding to the 3 ′ end of the nucleic acid, a PEG region mainly composed of polyethylene glycol bound to the donor region, and a peptide transfer reaction bound to the PEG region. And a peptide acceptor region containing a group. There may be no PEG area.

  The donor region that can bind to the 3 ′ end of a nucleic acid usually consists of one or more nucleotides. The number of nucleotides is usually 1-15, preferably 1-2. The nucleotide may be ribonucleotide or deoxyribonucleotide.

The sequence at the 5 ′ end of the donor region affects the ligation efficiency. In order to ligate the coding part and the spacer part, it is necessary to contain at least one residue. For an acceptor having a poly A sequence, at least one residue of dC (deoxycytidylic acid) or 2 The residue dCdC (dideoxycytidylic acid) is preferred. The base type is preferably C> U or T>G> A.

  The PEG region is mainly composed of polyethylene glycol. Here, the main component means that the total number of nucleotides contained in the PEG region is 20 bases or less, or the average molecular weight of polyethylene glycol is 400 or more. Preferably, it means that the total number of nucleotides is 10 bases or less, or the average molecular weight of polyethylene glycol is 2000 or more.

The average molecular weight of polyethylene glycol in the PEG region is usually 400 to 30,000, preferably 1,000 to 10,000, and more preferably 2,000 to 8,000. Here, when the molecular weight of polyethylene glycol is lower than about 400, when a genotype molecule containing a spacer part derived from this spacer molecule is associated and translated, a post-process of associated translation may be required ( Liu, R., Barrick, E., Szostak, JW, Roberts, RW (2000) Methods in Enzymology,
vol. 318, 268-293), and using a PEG having a molecular weight of 1000 or more, more preferably 2000 or more, high-efficiency association can be achieved only by association translation, so that post-translation processing is not necessary. In addition, when the molecular weight of polyethylene glycol increases, the stability of genotype molecules tends to increase, especially when the molecular weight is 1000 or higher, and when the molecular weight is 400 or lower, the DNA spacer may not be so characteristic and unstable. .

  The peptide acceptor region is not particularly limited as long as it can bind to the C-terminus of the peptide. For example, puromycin, 3'-N-aminoacylpuromycin aminonucleoside (3'-N-aminoacylpuromycin aminonucleoside, PANS-amino acid) For example, PANS-Gly whose amino acid part is glycine, PANS-Val of valine, PANS-Ala of alanine, and other PANS-all amino acids corresponding to all amino acids can be used. In addition, 3'-Aminoacyladenosine aminonucleoside (AANS-amino acid, 3'-Aminoacyladenosine aminonucleoside, formed by dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid as a chemical bond For example, AANS-Gly having an amino acid part of glycine, AANS-Val of valine, AANS-Ala of alanine, and other AANS-all amino acids corresponding to all amino acids can be used. Further, a nucleoside or a nucleoside and an amino acid ester-bonded one can be used. In addition, nucleoside or a substance having a chemical structure skeleton similar to nucleoside and an amino acid or a substance having a chemical structure skeleton similar to amino acid can be used as long as they can be chemically bonded.

  The peptide acceptor region is preferably composed of puromycin or a derivative thereof, or puromycin or a derivative thereof and one or two residues of deoxyribonucleotide or ribonucleotide. Here, the derivative means a derivative capable of binding to the C-terminus of the peptide in the protein translation system. The puromycin derivatives are not limited to those having a complete puromycin structure, but also include those lacking a part of the puromycin structure. Specific examples of the puromycin derivative include PANS-amino acid and AANS-amino acid.

  The peptide acceptor region may be composed of puromycin alone, but preferably has a base sequence consisting of DNA and / or RNA of 1 residue or more on the 5 ′ end side. The sequence is dC-puromycin, rC-puromycin, etc., more preferably dCdC-puromycin, rCrC-puromycin, rCdC-puromycin, dCrC-puromycin, etc., and the 3 ′ end of aminoacyl-tRNA is A mimicked CCA sequence (Philipps, GR (1969) Nature 223, 374-377) is suitable. The base type is preferably C> U or T> G> A.

  The spacer molecule preferably includes at least one function-imparting unit between the donor region and the PEG region. The function-imparting unit is preferably a functional modification of at least one residue of deoxyribonucleotide or ribonucleotide base. For example, a substance into which various separation tags such as a fluorescent substance, biotin, or His-tag are introduced as a function modifying substance can be used.

  The coding molecule includes, for example, a 5 ′ untranslated region containing a transcription promoter and a translation enhancer, an ORF region encoding a protein bound to the 3 ′ end of the 5 ′ untranslated region, and a 3 ′ end of the ORF region. A nucleic acid having a bound poly A sequence and containing an affinity tag sequence 5 ′ upstream of the poly A sequence.

  The coding molecule may be DNA or RNA. In the case of RNA, the 5 'end may or may not have a Cap structure. The coding molecule may be incorporated into any vector or plasmid.

  The 3 ′ end region may comprise an affinity tag sequence and a poly A sequence downstream thereof. As a factor affecting the ligation efficiency between the spacer molecule and the coding molecule, the polyA sequence in the 3 ′ end region is important, and the polyA sequence may be a mixture of dA and / or rA having at least 2 residues or more. The poly A continuous chain is preferably 3 or more residues, more preferably 6 or more, and even more preferably 8 residues or more.

  Elements affecting the translation efficiency of the coding molecule include a 5 ′ UTR consisting of a transcription promoter and a translation enhancer, and a combination of a 3 ′ terminal region containing a poly A sequence. The effect of the poly A sequence in the 3 ′ end region is usually exerted with 10 residues or less. T7 / T3 or SP6 can be used as the 5 ′ UTR transcription promoter, and there is no particular limitation. SP6 is preferable, and SP6 is particularly preferable when an omega sequence or a sequence containing a part of the omega sequence is used as an enhancer sequence for translation. The translation enhancer is preferably part of the omega sequence, and part of the omega sequence may be part of the TMV omega sequence (O29; Gallie DR, Walbot V. (1992) Nucleic Acids Res., Vol. 20, 4631 -4638 and WO 02/48347 (see FIG. 3) and O ′ (SEQ ID NO: 10) are preferred.

  The affinity tag sequence is not particularly limited as long as it is a sequence for using any means capable of detecting a protein such as an antigen-antibody reaction. Preferably, a Flag-tag sequence or a His-tag sequence, which is a tag for affinity separation analysis by antigen-antibody reaction.

The ORF region may be any sequence consisting of DNA and / or RNA. A gene sequence, exon sequence, intron sequence, random sequence, or any natural or artificial sequence is possible, and there is no sequence limitation. A nucleic acid library having such a sequence can be used. Further, by setting the 5′UTR of the coding molecule as SP6 + O29 and the 3 ′ terminal region as, for example, Flag + XhoI + A _n (n = 8), each length is about 60 bp in 5′UTR, The length of the 3 ′ end region is about 32 bp, and it can be incorporated as an adapter region into a PCR primer. For this reason, a coding molecule having a 5 ′ UTR and a 3 ′ terminal region can be easily prepared by PCR from any vector, plasmid, or cDNA library. In the coding molecule, translation may be done beyond the ORF region. That is, there may not be a stop codon at the end of the ORF region.

  The coding molecule was bound to the 5 'untranslated region containing the transcription promoter and translation enhancer, the ORF region encoding the protein bound to the 3' end of the 5 'untranslated region, and the 3' end of the ORF region. Or a nucleic acid containing a 3 ′ terminal region containing a poly A sequence.

  The genotype molecule is obtained by converting the above coding molecule into a form in which the base sequence of the protein coding region can be translated (for example, after transcription), if necessary, and the 3 ′ end of the coding molecule and the spacer molecule. The donor region can be produced by binding by a normal ligase reaction. The reaction conditions usually include conditions at 4 to 25 ° C. for 4 to 48 hours. When adding polyethylene glycol having the same molecular weight as the polyethylene glycol in the PEG region of the spacer molecule containing the PEG region, to the reaction system It is also possible to shorten to 0.5 to 4 hours at 15 ° C.

  The combination of spacer molecule and coding molecule has an important effect on ligation efficiency. In the 3 ′ terminal region of the coding part corresponding to the acceptor, there should be a DNA and / or RNA polyA sequence of at least 2 residues, preferably 3 residues, more preferably 6-8 residues, The UTR translation enhancer is preferably a partial sequence of an omega sequence (O29 or O '), and the spacer region donor region is at least one dC (deoxycytidylic acid) or two residues dCdC (dideoxy). Cytidylic acid) is preferred. By using RNA ligase, the problem of DNA ligase can be avoided and the efficiency can be maintained at 60 to 80%.

  (a) a 5 ′ untranslated region containing a transcription promoter and a translation enhancer, an ORF region encoding a protein bound to the 3 ′ end of the 5 ′ untranslated region, and a 3 ′ end of the ORF region The 3 'end of the coding molecule, which is RNA containing the 3' end region containing the poly A sequence, and (b) the donor region of the spacer molecule consisting of RNA constitutes the PEG region in the spacer molecule It is preferable to bind with RNA ligase in the presence of free polyethylene glycol having the same molecular weight as polyethylene glycol.

  By adding polyethylene glycol having the same molecular weight as the PEG region of the spacer part including the PEG region during the ligation reaction, the ligation efficiency is improved to 80 to 90% or more regardless of the molecular weight of the polyethylene glycol of the spacer part. The separation step can also be omitted.

  The mapping molecule in this embodiment is linked to a phenotype molecule that is a protein encoded by the ORF region in the genotype molecule by translating the genotype molecule in a cell-free translation system. (A in FIG. 4). The cell-free translation system is preferably that of wheat germ or rabbit reticulocytes. The conditions for translation may be those normally employed. For example, the conditions are 15 to 240 minutes at 25 to 37 ° C. In addition, the nucleic acid portion of the mapping molecule of this embodiment can be a hybrid of RNA and DNA by reverse transcription after translation.

  The screening method based on the in vitro virus method usually comprises (b1) a step of producing a library of mapping molecules in which a protein and a nucleic acid encoding the protein are bound from a nucleic acid library; A step of mixing a library of attached molecules and a target substance, and (b3) a step of selecting a corresponding molecule bound to the target substance. The method further includes a step of preparing a nucleic acid library based on the nucleic acid of the corresponding molecule selected in (b4) and (b3), and the nucleic acid library prepared in (b4) is used as the nucleic acid library in (b1). And may include repeating the operations (b1) to (b3).

  Target substances include peptides (which may be chemically synthesized, isolated from nature, or partially digested proteins), proteins, nucleic acids (DNA or RNA), saccharides, various low molecular compounds, metals All compounds and substances such as metal compounds are applicable.

The target substance used in the present invention is bound to a solid phase such as a resin or a bead depending on the embodiment, and the timing of binding may be before or after mixing with the corresponding molecule, and is selected according to the binding method. . Examples of the method for binding to a solid phase include a method of binding via a modifying substance and a method of binding using a moiety other than that.

  The modifier used for binding via a modifier is usually a molecule that specifically binds to a specific polypeptide (hereinafter sometimes referred to as a “ligand”) and is attached to the solid surface. Binds a specific polypeptide that binds to the ligand (hereinafter sometimes referred to as "adapter protein"). Adapter proteins also include binding proteins, receptor proteins that constitute receptors, antibodies, and the like. Examples of adapter protein / ligand combinations include biotin-binding proteins such as avidin and streptavidin / biotin, maltose-binding protein / maltose, G protein / guanine nucleotide, polyhistidine peptide / nickel or cobalt or other metal ions, glutathione-S -Transferase / glutathione, DNA binding protein / DNA, antibody / antigen molecule (epitope), calmodulin / calmodulin binding peptide, ATP binding protein / ATP, or various receptor proteins such as estradiol receptor protein / estradiol / ligands thereof It is done.

  Among these, adapter protein / ligand combinations include biotin-binding proteins such as avidin and streptavidin, maltose-binding proteins / maltose, polyhistidine peptides / metal ions such as nickel or cobalt, glutathione-S-transferase / glutathione, The combination of streptavidin / biotin is most preferable. These binding proteins are known per se, and the DNA encoding the protein has already been cloned.

  The adapter protein can be bound to the solid phase surface by a method known per se. Specifically, for example, tannic acid, formalin, glutaraldehyde, pyruvaldehyde, bis-diazotized benzidone, toluene- A method utilizing a 2,4-diisocyanate, an amino group, a carboxyl group that can be converted into an active ester, or a hydroxyl group or amino group that can be converted into a phosphoramidide can be used.

  When binding to the solid phase by a moiety other than the modifying substance, known methods commonly used for binding proteins, nucleic acids, sugar chains, low molecular weight compounds to the solid phase, specifically, for example, tannic acid, formalin, Use a method that uses glutaraldehyde, pyruvic aldehyde, bis-diazotized benzidone, toluene-2,4-diisocyanate, amino group, carboxyl group that can be converted to active ester, hydroxyl group or amino group that can be converted to phosphoramidide, etc. be able to.

  The solid support is preferably agarose beads or agarose beads in which a magnetic material is embedded.

  The library of the mapping molecule and the target substance may be mixed under the condition that the target protein of the mapping molecule interacts with the target substance. This condition is appropriately selected according to the interaction to be detected and the type of target substance. When the target substance is a protein, RNA encoding the protein may be prepared and synthesized together during the synthesis of the corresponding molecule (cotranslation). Such a method is described in WO03 / 048363.

The process of selecting the mapping molecule bound to the target substance is a process of separating the mapping molecule bound to the target substance and the mapping molecule that does not bind to the target substance. Usually, the target substance is fixed to the solid phase. By separating or immobilizing, separation can be performed by washing the solid phase on which the target molecule after mixing with the corresponding molecule is immobilized. Washing conditions are appropriately selected according to the interaction to be detected and the type of target substance. Here, immobilization on the solid phase means that the conjugate of the mapping molecule and the target substance can be separated from the non-bonded molecule. For example, when the target substance is a membrane protein, the cell membrane of the cell Membrane proteins expressed in the above or proteins embedded in artificial membranes are also included in the target substance immobilized on the solid phase.

  The step of preparing a nucleic acid library based on the nucleic acid of the selected mapping molecule usually includes preparing DNA by reverse transcription. The prepared DNA is used for sequence analysis, or used as a nucleic acid library for the synthesis of mapping molecules.

  A specific example of the screening method will be described with reference to FIG. In [1], cDNA libraries encoding various proteins are amplified by PCR, reverse transcribed, and linked to a PEG spacer containing puromycin to prepare an IVV template RNA library. In [2], the IVV template RNA library is cotranslated together with the bait template RNA encoding the bait protein, and a complex of the IVV library and the bait protein is obtained. In [3], the complex of IVV library and bait protein is subjected to affinity screening. In [4], the RNA portion of the selected IVV is reverse transcribed and amplified by PCR to obtain an RT-PCR product. The RT-PCR product is transcribed at [5] and subjected to the next selection round, or subjected to array analysis at [6].

<1-c> Step (c)
Step (c) is a step of performing RNA amplification with RNA polymerase in the presence of a nucleotide labeled with a ligand substance that does not inhibit RNA-DNA hybridization, using the DNA of the library prepared in step (b) as a template. It is.

  This step can be performed according to an RNA amplification method using RNA polymerase using DNA as a template (for example, Everwine method (Proc Natl Acad Sci USA 87, 1663-1667)). Alternatively, a commercially available RNA amplification kit (for example, MEGAscript (registered trademark) kit from Ambion) can be used.

  The RNA polymerase is preferably one that initiates transcription by a promoter contained in the DNA prepared in step (b). This eliminates the need to introduce a promoter corresponding to RNA polymerase into DNA.

  The promoter is, for example, SP6 promoter.

  The fact that RNA-DNA hybridization is not inhibited means that in step (d), the nucleotide incorporation efficiency during RNA amplification is affected, or the hybridization efficiency in step (d) is affected. It means that there is substantially no variation or decrease in the intensity of fluorescence or luminescence measured. Examples of the ligand substance include biotin and digoxigenin.

  The nucleotide labeled with the ligand substance may be one or more of ATP, GTP, CTP, and UTP. For example, two kinds of CTP and UTP may be used. Nucleotides modified with the above ligand substances are available as commercial products. The amount of nucleotides labeled with the ligand substance is preferably 10 to 15 mol% of the total unlabeled nucleotides.

<1-d> Step (d)
In the step (d), the RNA amplified in the step (c) is hybridized to the tiling array prepared in the step (a), and an adapter substance and a fluorescent substance or a chemiluminescent or chemiluminescent substance that binds to the ligand substance are combined. This is a step of labeling with a conjugate of a substance to be activated and measuring the intensity of fluorescence or luminescence.

  Examples of the adapter substance include streptavidin or avidin against biotin, anti-digoxigenin antibody against digoxigenin, and the like. Examples of the substance that activates chemiluminescence or chemiluminescence include a chemiluminescence substrate or an enzyme that decomposes the chemiluminescence substrate to generate fluorescence or luminescence.

  Hybridization, labeling, and fluorescence or luminescence intensity measurements can be made by methods commonly applied to tiling arrays (eg, Stolc V, Samanta MP, Tongprasit W, Marshall WF. Genome-wide transcriptional Analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. See Proc Natl Acad Sci US A. 2005 Mar 8; 102 (10): 3703-7. Epub 2005 Feb 28.).

<1-e> Step (e)
Step (e) is a step of detecting a protein that interacts with the target molecule or a nucleic acid that encodes the protein by analyzing the measurement result of step (d).

  The analysis of the measurement results can be performed according to a normal method (for example, Kim TH, Barrera LO, Zheng M, Qu C, Singer MA, Richmond TA, Wu Y, Green RD, Ren B. A high-resolution map of Nature. 2005 Aug 11; 436 (7052): 876-80. Epub 2005 Jun 29.).

  For example, using a Find Peaks program implemented in SignalMap software (NimbleGen), parameters such as a detection window width and a signal threshold value can be appropriately optimized. Examples of the optimization method include a method of optimizing parameters using a protein having a known interaction as a detection candidate protein.

  Based on the probe detected by the analysis of the measurement result, mRNA is specified, and thereby the protein and the nucleic acid encoding it are detected.

<2> Application of the detection method of the present invention By the detection method of the present invention, a protein that interacts with a target molecule or a nucleic acid that encodes the same is detected by detecting a protein that interacts with the target molecule or a nucleic acid that encodes it. Screening with sensitivity becomes possible. Therefore, the detection method of the present invention provides a screening method for a protein that interacts with a target molecule or a nucleic acid that encodes the protein, which comprises detecting a protein that interacts with the target molecule or a nucleic acid that encodes the protein.

  In this application, the target molecule is selected according to the purpose of screening. For example, in the case of transcription factor screening, DNA can be used. Further, drug candidate small molecules can be used in screening for drug discovery purposes.

  In the detection method of the present invention, if the sequence information of mRNA encoding the protein that interacts with the target substance is used instead of the sequence information of mRNA encoding the detection candidate protein, the detection result and the detected probe mRNA in the mRNA are detected. The interaction site can be detected based on the position information. Therefore, there is provided a method for detecting an interaction site of a protein that interacts with a target molecule, using the main part of the detection method of the present invention.

  In this case, it is preferable that the gap between the probes is made smaller or the overlap is made larger than in the case of screening.

  Hereinafter, examples of the present invention will be specifically described. However, the following examples should be regarded as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is limited by the following examples. It is not something.

(1) Preparation of In vitro virus (IVV) template RNA library First, a cDNA library was prepared using poly (A) + RNA (BD Biosciences) derived from mouse brain. A cDNA library was prepared by random priming using an oligonucleotide (3'random + adp) (SEQ ID NO: 1) having a random sequence of 9 bases (unless otherwise noted, all oligo DNAs are based on the date concept) Synthesis). Superscript double strand cDNA synthesis kit (Invitrogen) was used for the preparation of the cDNA library, and the operation was performed according to the protocol of the kit. Specifically, first, single-stranded cDNA was prepared using SuperScript double strand cDNA synthesis kit and 3′random + adp. In order to add a common sequence necessary for PCR, an adapter that has been pre-annealed with two complementary oligonucleotides (5'adp_FW (SEQ ID NO: 2) and 5'adp_RV (5'-GGAATTCG-3 ')) is used. Ligation high kit (Toyobo) was used for connection. After ethanol precipitation, the single-stranded cDNA library to which the adapter was added was PCR amplified using 5′F3 (SEQ ID NO: 3) and 3 ′ lib_PCR (SEQ ID NO: 4) primers. The PCR product was purified with a QIAquick PCR purification kit (QIAGEN), and short molecules of 200 bp or less containing almost no mRNA-derived sequence were eliminated with a CHROMA SPIN-1000 gel filtration column (BD Biosciences). The cDNA library was then in vitro transcribed using RiboMax large RNA production system-SP6 (Promega), PEG Puro spacer using T4 RNA ligase (Takara) (Nemoto et al., (1997) FEBS Lett. 414, 405 -408) and purification with RNeasy mini kit (QIAGEN) to obtain an IVV template RNA library.

(2) Preparation of bait protein template RNA Primer (5'Jun-BamHI (SEQ ID NO: 6)) to which BamHI and XhoI sites were added so as to include the 167th to 319th amino acid sequences (SEQ ID NO: 5) of mouse Jun protein ; 3'Jun-XhoI (SEQ ID NO: 7)) was used for PCR amplification from the cDNA. The PCR product was purified with the QIAquick PCR purification kit and treated with BamHI and XhoI enzymes (Toyobo), and the pCMV-CBPzz vector (Horisawa et al., (2004) Nucleic Acids Res. 32, e169) in the BamHI / XhoI site. Inserted into. Thereafter, the subcloned plasmid was amplified by PCR using 5'SP6 (O ') T7 (SEQ ID NO: 8) and 3'FosCBPzz (SEQ ID NO: 9) primers, and simultaneously with in vitro transcription. SP6 promoter necessary for E. coli and O ′ sequence (SEQ ID NO: 10) necessary for efficient cell-free translation were added. The PCR product was purified by QIAquick PCR purification kit. The purified PCR product was transcribed in vitro with RiboMax large-scale RNA production system-SP6 and purified with RNeasy mini kit.

(3) Affinity selection and amplification of in vitro virus (IVV) molecules after selection First, 10 pmol of template RNA library was put into 50 μl of wheat germ-derived cell-free translation system (Promega) to form IVV. At that time, 10 pmol of Jun's template RNA (SEQ ID NO: 11) serving as a bait was simultaneously introduced and expressed [bait (+)]. As a control, an experiment [bait (-)] without bait template RNA was also performed. Translation was performed at 26 ° C. for 1 hour. After translation, add 50 μl of IPP150 buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40) to the total volume, and rabbit IgG agarose beads (Sigma) pre-washed twice with 500 μl of IPP150 buffer in advance. Inverted at 4 ° C for 2 hours. The supernatant is then discarded, twice with 800 μl of IPP150 buffer, and once with 800 μl of TEV cleavage buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA, 1 mM DTT). Washed. 100 μl of TEV cleavage buffer containing 100 U of TEV protease (Invitrogen) was added to rabbit IgG agarose beads and treated at 16 ° C. for 2 hours to release the bait and IVV molecules bound thereto to the supernatant and collect them.

  5 μl of the collected supernatant was added as a template to a 50 μl RT-PCR reaction system. The RT-PCR reaction was performed with the OneStep RT-PCR kit (QIAGEN) using 5′F3 (SEQ ID NO: 3) and 3 ′ Flag-1AL (SEQ ID NO: 12) primers. The RT-PCR program examined conditions between 24-32 PCR cycles and determined conditions that did not reach a plateau for each round. The PCR product was again used as an IVV library template RNA by the above method and repeatedly screened. Screening was performed for 5 rounds for both bait (+) and bait (-).

(4) Oligo DNA microarray design The oligo DNA microarray used is a photolithographic technology using the NimbleGen digital mirror through the “anything array” service of GeneFrontier (Nuwaysir et al., (2002) Genome Res. 12 , 1749-1755). The base sequences to be analyzed in this example are mouse transcription factors and mRNA sequences of genes considered to be transcription factor candidates in terms of primary sequence structure. This is a 1,602 mRNA sequence centered on a list prepared by Gunji et al. (Gunji W., (2004) Biochem Biophys Res Commun. 325, 265-275). The probe length was set to 50 mer, which is expected to obtain high sensitivity from past results (Nuwaysir et al., (2002) Genome Res. 12, 1749-1755), and designed to have no gap on the mRNA sequence group. . As a result, the total number of probes designed for both sense and antisense strands was 167,186. In order to confirm reproducibility, two similar probe sets were constructed on the same array.

(5) Introduction of labeling substance of DNA library after affinity selection DNA of bait (+) and bait (-) obtained by RT-PCR after 5 rounds of affinity selection obtained in step (3) A suitable method for fluorescent labeling of samples was verified. Labeling methods include random prime methods commonly used for labeling DNA samples, such as the ChIP on chip method, and the Eberwine method (Van Gelder RN, (1990) Proc Natl) used for amplification and labeling of mRNA samples in expression analysis. Acad Sci USA 87, 1663-1667) was used to compare the results.

In the random prime method, Random 9mer Buffer (125 mM Tris-HCl, 12.5 mM MgCl ₂ , 0.00175% β-mercaptoethanol) was prepared by diluting 9 mer random oligo DNA (Trilink Biotechnologies) labeled with Cy5 or Cy3 to 2 OD. ) Was produced. 1 μg of this solution
40 μl was added to the DNA sample, and further made 68 μl with distilled water. It was heated at 98 ° C. for 5 minutes and quenched on ice. After rapid cooling, 10 μl of 30 × dNTP mix (1 × TE, 6 mM dATP, 6 mM dGTP, 6 mM dTTP, 6 mM dCTP) and 40 U of Klenow enzyme (NEB) were added to make 100 μl, and incubated at 37 ° C. for 2 hours. After the reaction, 10 μl of 0.5 M EDTA was added to stop the reaction, and after purification by isopropanol precipitation, the generated DNA was suspended in 10 μl of distilled water and used for hybridization.

  The modified Eberwine method is a method of synthesizing cRNA incorporating biotinylated CTP and UTP using the T7 promoter added during reverse transcription of mRNA in the usual Eberwine method, whereas it is always attached to the IVV template molecule. This is a newly devised method to generate biotin-labeled sRNA using the existing SP6 promoter (in the case of DNA-RNA hybridization, since direct fluorescence is labeled with RNA, the efficiency is reduced, so indirect via biotin. A fluorescent labeling method). Specifically, biotin labeling was performed on 1 μg of sample DNA by performing an in vitro transcription reaction at 37 ° C. for 5 hours using MEGAscript kit (Ambion), Bio-11 CTP and Bio-16 UTP (both Enzo). SRNA probes were generated. Detailed procedures were in accordance with the kit usage. After the reaction, sRNA was purified using RNeasy mini kit (QIAGEN), diluted with distilled water to a concentration of 1.0 μg / μl, and used for hybridization.

(6) Hybridization, signal detection and data processing Samples directly fluorescently labeled by the random prime method should be hybridized to the microarray according to the experimental conditions of Kim et al. (Kim TH (2005) Nature 436, 876-880). went. Cy3-labeled bait (+) sample and Cy5-labeled control bait (-) sample were mixed together and competitively hybridized. On the other hand, the sample labeled with biotin by the modified Eberwine method is coupled with Streptoavidin-Cy3 (Amersham) after hybridization based on the method of Nuwaysir et al. (Nuwaysir EF (2002) Genome Res. 12, 1749-1755). And fluorescently labeled. Here, the control sample hybridized to another array. Measure fluorescence intensity of each spot using the Axon 4000B fluorescence scanner (Axon Instruments) according to the method of Kirmizis et al. (Kirmizis A. (2004) Genes Dev. 18, 1592-1605). did. After measurement, Kim et al. (Kim TH (2005) Nature 436,
876-880), and the data was processed. Specifically, the logarithm with a base of 2 for each of the bait (+) and bait (-) data was taken, and the ratio was calculated (log2 [bait (+)] / log2 [bait (-)]). Subsequent analysis was performed using this data as "ratio".

(7) Analysis and Evaluation of Detection Results Significant signals were detected from the ratio data obtained by the experiment using the Find Peaks program implemented in SignalMap software (NimbleGen). The Find Peaks signal detection algorithm surveys the nucleic acid sequence of a limited region with an arbitrary window width (expressed in base number), and the value of the probe set in the limited region is equal to or greater than the threshold value. A region where there are four or more having a is detected as a peak. In this example, as a parameter condition of a specific detection algorithm, the maximum on the mRNA sequence is within a window of 300 bases (6 probes of 50 bases) for continuous probes on an arbitrary mRNA sequence. A region where four or more probes having a value of 20% or more of the numerical value of the probe showing the above signal are detected as peaks. In the peak detection algorithm, it is practically impossible to change the minimum number of probes necessary to detect a peak to 4 or less in order to prevent an increase in noise. It is necessary to fix to about 50 bases in order to obtain a good signal. Therefore, the parameters that can be changed as parameters are the detection window width and the signal threshold, but the 20 interactions obtained from the results of screening by the usual IVV method using the bZIP domain of the transcription factor Jun as the bait When the protein candidate group (Horisawa et al., (2005) J Biochem. 137, 121-124) was examined as an index, the conditions under which the number of false negatives and false positives were minimized were the above values.

As a result, in the system that performed labeling, hybridization, and detection by the modified Eberwine method, all 20 interacting protein candidate groups detected by the above existing method were specifically detected as signals. In the system in which labeling was performed by the random prime method and detection was performed by competitive hybridization using two-color fluorescently labeled samples, there were sequences that were not detected. An example (gene name: 9130229H14Rik) is shown in FIG. Reasons for this difference in the results are as follows: (1) In the random prime method, a single fluorescent molecule is labeled at the end of the random primer, so that a large number of fluorescent molecules are labeled per fragment. (2) In the modified Eberwine method, the direct labeling with biotin-streptavidin is not performed with the fluorescent molecule that inhibits efficient RNA-DNA hybridization, but the sensitivity is lower than the modified method. The signal was sensitized and the sensitivity was improved. (3) In the random prime method, the bias of the random primer sequence may bias the abundance ratio of the amplified sequence, whereas in the modified Eberwine method Since RNA amplification by transcription is relatively linear and difficult to cause quantitative bias, it was possible to detect the selected sequence accurately. Erareru. Therefore, it is considered effective to use the modified Eberwine method in order to detect the enrichment of DNA fragments that have undergone specific selection by the IVV method with a tiling array. Therefore, the following analysis used data by the modified Eberwine method. In this example, as a protocol for the modified Eberwine method, biotin-conjugated UTP and CTP were incorporated into RNA using an Ambion MEGAscript kit, and indirectly labeled with fluorescence. Most of the resulting DNA fragments have a short chain length, and the efficiency of labeling is unlikely to be greatly affected by the inherent performance of the kit for cell-free transcription, so other cell-free transcription kits using RNA polymerase It is expected that almost the same result will be obtained even if is used. Similarly, regarding biotin-binding NTP to be incorporated into RNA, it is considered that the same results can be obtained when using other than UTP and CTP (ATP, GTP) adopted this time. In addition to using RNA as a fluorescently labeled sample, it is possible to obtain results equivalent to the modified Eberwine method by incorporating fluorescently labeled dNTP or biotinylated dNTP when replicating DNA and increasing signal sensitivity However, DNA amplification such as PCR has low linearity compared to RNA amplification by transcription, and therefore there is no denying the possibility that a sequence abundance will be biased. In addition, since the Eberwine modified method can be labeled in one step from a cDNA library concentrated by the IVV method, there is little room for bias and it is considered that it is advantageous in terms of ease of operation.

  Focusing on the individual peaks obtained from the ratio data by the Find Peaks program, a signal with a range that is close to the minimum necessary for binding to the bait protein is obtained for proteins with known binding sites to the bait protein. It has been found. An example (gene name: Maf) is shown in FIG. The minimum region necessary for protein interaction detected by the IVV method is often a region with a short motif level. In fact, most of the sequences obtained as a result of past research are also motifs of about 200 to 350 bp. Most of the fragments were limited to the sequence. Considering this, the signal of the specifically enriched sequence was obtained efficiently this time when designing a tiling array, a custom probe array with no gaps between probes was created. It is thought that it was a factor of success to have designed. Because the probe design of commercially available tiling arrays generally used in the ChIP on chip method or the like usually has an average gap of about 50 to 300 bp between the probes, the resolution is low and DNA in a short region is used. This is because it is considered difficult to detect fragments. Therefore, due to the nature of the IVV method, specifically selected DNA fragments are relatively short and in a limited region, and the algorithm conditions for peak detection are fixed to some extent for the reasons given above. In addition, considering the fact that a probe chain length of about 50 bases or more is necessary for efficient signal detection, the DNA fragment selected by the IVV method is effectively detected using a DNA microarray. It was found that it is essential to design an oligo DNA probe having a chain length of about 50 bases with a small gap between the probes. In this example, the interval between probes was set to O base, but considering the above conditions, gaps and overlaps between probes that do not greatly exceed the chain length of the probe can be allowed by design. Conceivable.

  Among the ratio data of all sequences, the one with the maximum peak signal intensity of 3.0 or more was selected with good reproducibility. Previous reports (Horisawa et al., (2004) Nucleic Acids Res. 32, In addition to the 20 sequences detected in e169), 23 sequences could be detected as signals (Table 1). The amount of these 23 sequences in the library before and after screening was quantified using LightCycler (Roche), SYBR Premix PCR kit (Takara), and sequence-specific primer set. It was found to be specifically concentrated (Table 1). The sequence of the sequence-specific primer is shown in the sequence listing by adding “_F” (forward primer) and “_R” (reverse primer) to the target gene name (SEQ ID NOs: 13 to 58). Of the 16 sequences, 5 were genes known as Jun interactors and the rest were novel interaction candidates. With the cloning method (WO 03/014734), which is a sequence detection method in the existing IVV screening, analysis exceeding 1,000 clones is practically difficult, and the detection limit at that time is theoretically 0.1% in terms of the number of molecules. . The actual results (Horisawa et al., (2004) Nucleic Acids Res. 32, e169) also agreed with this. In the candidate group detected by the present example, the lowest concentration after screening is 0.0001%. This indicates that the sensitivity is about 100 to 1,000 times higher than that of the existing IVV screening.

References in Table 1 are as follows.
Horisawa et al. (2004): Nucleic Acids Res. 32, e169
Kataoka et al. (1994): Mol. Cell Biol. 14, 700-712
Chatton et al. (1994): Oncogene 9, 375-385
Hai and Curran (1991): Proc Natl Acad Sci USA. 88, 3720-3724
Kerppola and Curran (1993): Mol. Cell Biol. 13, 5479-5489
Kouzarides and Ziff (1989): Nature 340, 568-571

Tiling array analysis at various probe intervals was performed. More specifically, Example 1
In the design of the oligo DNA macroarray, the same operation as in Example 1 was performed except that the probe interval was changed, and the influence of the probe interval was examined. Specifically, it was as follows.

The cDNA library sample to be analyzed by the DNA microarray was the one prepared in (1) to (3) of Example 1. The genes shown in Table 2, such as the gene having high signal intensity in Example 1, were selected, and the probe overlap was 25, 22, 20, 17, 15, 12 by the method of Example 4 (4), respectively. , 10, 7, 5, 0, -20 base (minus indicates the interval between probes). In Example 1 (5), a modified Eberwin method was used as a method for introducing a labeling substance into a sample. Hybridization, signal detection, data processing, and analysis were carried out in the same manner as (6) to (7) in Example 1.
The results are shown in Table 2.

When the overlapping region of the probe is increased (0 to 50% of the probe chain length), the number of detected genes is greatly increased, although there is some variation compared to the case where there is no overlap. While it did not fluctuate, it was found that the number of genes detected was significantly reduced when the probes were spaced apart. This is thought to be due to the length of the DNA fragment selected by the IVV method and the characteristics of the tiling array technology that requires a signal to be continuously obtained in a certain region. This is an effective and essential condition for the use of tiling arrays in the IVV method.

  In order to compare the detection sensitivity of the conventional IVV method and the method of the present invention, it is measured using real-time PCR how much the DNA encoding the detected candidate protein is concentrated in the cDNA library. did. Specifically, it was as follows.

  As the cDNA library, the DNA sample after 5 rounds of screening prepared in (1) of Example 1 was used. Real-time PCR was performed by the method shown in Non-Patent Document 1 except that the primers used in Example 1 (SEQ ID NOs: 12 to 58) were used as primers.

  The results are shown in FIG. The left column (A) in FIG. 5 is a candidate for which detection by the conventional IVV method was reported and was also detected in the method of the present invention (Example 1), and the right column (B) Candidates detected only by the method (Example 1). Each numerical value indicates the number of molecules of each candidate in the cDNA library (1.0E + 10 molecules) after screening. In the conventional IVV method, since each sequence is analyzed through cloning, detection is difficult unless it is finally concentrated to 0.001% or more in the library (sequence analysis of about 1,000 clones). On the other hand, the method of the present invention can detect abundance candidates of about 0.000001% (sequence analysis of about 1,000,000 clones). This can be said to be about 100 to 1,000 times more sensitive than the conventional method.

  This improvement in sensitivity is considered to be very effective for candidates that cannot ultimately increase the enrichment in the library, such as proteins with low expression levels and proteins with relatively weak affinity with baits. It is done. Actually, when Atf3, which is a known Jun interacting protein detected in this example, was confirmed by real-time PCR for the abundance before screening, the initial abundance was determined as the source of the cDNA library. It was found that only a few copies were expressed in one cell out of 1,000 cells in brain tissue. From this, it is considered that the method of the present invention is very effective for an example such as somatic stem cells in which the quantitative limitation of sample acquisition makes the interaction analysis difficult.

Schematic diagram of conventional screening strategy and screening strategy according to the present invention. The example which compared the result of the fluorescence labeling by the random prime method and the modified Eberwine method about the same gene. An example of a gene in which a site necessary for known binding is selectively detected. Explanatory drawing of an example of IVV screening method. A is an explanatory diagram of the principle of IVV formation on a ribosome in a cell-free translation system. B is an explanatory diagram of repeated selection of protein interactions using IVV. The example which compared the density | concentration in the final cDNA library with the candidate protein detected by the conventional method and the method of this invention.

Claims (7)

A method for detecting a protein that interacts with a target molecule or a nucleic acid that encodes the protein, comprising the following steps (a) to (e):
(a) A tiling array in which probes designed based on the sequence information of mRNA encoding a candidate protein for detection are immobilized, and the average gap between probes is 25 bases or less, or the average overlap between probes The process of preparing a tiling array designed to be 50% or less of the probe length.
(b) A step of preparing DNA from an associating molecule selected for interaction with a target substance by a screening method using an associating molecule in which a protein and a nucleic acid encoding the protein are bound.
(c) A step of performing RNA amplification by RNA polymerase using the DNA prepared in (b) as a template in the presence of a nucleotide labeled with a ligand substance that does not inhibit RNA-DNA hybridization.
(d) The RNA amplified in (c) is hybridized to the tiling array prepared in (a), and an adapter substance that binds to the ligand substance and a fluorescent substance or a substance that activates chemiluminescence or chemiluminescence A step of labeling with a conjugate and measuring the intensity of fluorescence or luminescence.
(e) A step of detecting a protein that interacts with a target molecule or a nucleic acid that encodes the protein by analyzing the measurement result of (d). The method according to claim 1, wherein the RNA polymerase initiates transcription by a promoter contained in the DNA prepared in step (b). The method according to claim 2, wherein the promoter is an SP6 promoter. The method according to any one of claims 1 to 3, wherein the ligand substance is biotin and the adapter substance is streptavidin or avidin. The screening method of step (b) comprises: (b1) producing a library of mapping molecules in which a protein and a nucleic acid encoding the protein are bound from a nucleic acid library; (b2) the library of mapping molecules (B3) a step of selecting the mapping molecule bound to the target substance, (b4) a step of preparing a nucleic acid library based on the nucleic acid of the mapping molecule selected in (b3) The method according to any one of claims 1 to 3, further comprising repeating the operations (b1) to (b3). Detection of a protein that interacts with a target molecule or a nucleic acid that encodes the same, comprising detecting a protein that interacts with the target molecule or a nucleic acid that encodes the protein by the method according to any one of claims 1 to 5. Screening method. A method for detecting an interaction site of a protein that interacts with a target molecule, comprising the following steps (a) to (e):
(a) A tiling array to which probes designed based on the sequence information of mRNA encoding a protein that interacts with a target molecule are fixed, and the average gap between probes is 25 bases or less, or over-probing between probes Preparing a tiling array that is designed so that the average wrap is 50% or less of the probe length.
(b) A step of preparing DNA from an association molecule selected for interaction with a target substance by a screening method using an association molecule using an association molecule in which a protein and a nucleic acid encoding the protein are bound.
(c) A step of performing RNA amplification by RNA polymerase using the DNA prepared in (b) as a template in the presence of a nucleotide labeled with a ligand substance that does not inhibit RNA-DNA hybridization.
(d) The RNA amplified in (c) is hybridized to the tiling array prepared in (a), and an adapter substance that binds to the ligand substance and a fluorescent substance or a substance that activates chemiluminescence or chemiluminescence A step of labeling with a conjugate and measuring the intensity of fluorescence or luminescence.
(e) A step of detecting an interaction site by analyzing the measurement result of (d).

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