WO2006011589A1

WO2006011589A1 - Method of preparing artificial gene population and artificial protein population by random polymerization of motif sequences of multiple types

Info

Publication number: WO2006011589A1
Application number: PCT/JP2005/013913
Authority: WO
Inventors: Kiyotaka Shiba; Hirohide Saito
Original assignee: Japan Science And Technology Agency
Priority date: 2004-07-30
Filing date: 2005-07-29
Publication date: 2006-02-02
Also published as: JP4568723B2; JPWO2006011589A1

Abstract

In order to create an artificial functional gene or an artificial functional protein, it is intended to provide an artificial gene population obtained via the random polymerization of motif sequences of multiple types, an artificial protein population encoded by the above-described artificial gene population and a method of preparing the same. An artificial gene population obtained via the random polymerization of motif sequences of multiple types is prepared by constructing at least three types of DNA sequences each having a single-stranded DNA containing a sequence encoding an arbitrary motif sequence, transferring a complementary base sequence thereinto so as to allow the formation of a complementary base pair in part of the terminal sequences of the single-stranded DNA sequences with each other, and then treating a liquid reaction mixture, which is prepared by mixing the thus constructed single-stranded DNAs of multiple types at an arbitrary ratio, with DNA polymerase so that random polymerization proceeds among the DNAs encoding the motif sequences. By translating the artificial gene population and converting the same into artificial proteins, an artificial protein population encoded by the motif sequences of multiple types can be prepared.

Description

Specification

Production of artificial genes and artificial protein populations by random polymerization of multiple motif sequences

Technical field

[0001] The present invention aims to create a functional artificial gene or a functional artificial protein, an artificial gene population in which a large number of motif sequences are randomly polymerized, an artificial protein population encoded by the artificial gene population, and production thereof Regarding the law.

Background art

[0002] In recent years, research on elucidating the functions of human genome genes and proteins has progressed mainly in the field of drug discovery, etc. As post-genomic research, analysis of new gene functions and elucidation of protein functions have been conducted on a large scale. As the result, the functions of many genes and proteins are being revealed. By elucidating the functions of these genes and proteins, as their knowledge has accumulated, expectations and motivation to design and create artificial genes and proteins with reasonably new functions have increased. This research into “designing” creation of artificial genes and proteins with reasonably new functions was born as protein engineering in the 1980s (Science, 219, 666-671, 1983). However, after several artificial proteins were created, it was still possible to design new proteins! But we have reached a certain level! /.

[0003] In the 1990s, evolutionary molecular engineering (in vitro molecular evolution) has become a major trend in the field of artificial molecule creation. In this evolutionary molecular engineering, a strategy to obtain a molecule with the desired function and activity by "selection" from a random sequence group prepared in advance rather than rationally designing artificial molecules is adopted. (Science, 67,938-947, 1997). In other words, evolutionary molecular engineering consists of (1) selecting a small molecule with the desired activity from a combinatorial molecular population (DNA or protein) with various sequence diversity, and (2) selecting the gene The same reaction is repeated after amplification by gene amplification reaction (PCR), and (3) an artificial molecule having the desired reaction activity is evolved. Currently, this method uses gene-protein fusion technologies such as ribosome display and mRNA display. Attempts have been made to create functional artificial proteins. However, the conventional technology has the following problems. (I) An effective method for constructing a DNA population that has sequence diversity and is suitable for the experiment of interest has been established !, NA! /, GO ribosome (mRNA) displays can be selected from artificial proteins that are target molecules It is limited to substances that bind to the enzyme and is not suitable for selection of molecules that function enzymatically or function in vivo.

[0004] When creating an artificial protein with an extraordinary function that does not exist in nature by evolutionary molecular engineering, how to skillfully prepare an artificial genetic diversity population (library) that serves as a screening population Is the key to success. For this reason, it is essential to develop artificial gene and artificial protein population creation methods for the purpose of creating functional molecules effectively. Methods for preparing artificial gene populations include error prone PCR (PCR Methods Appl, 2: 28-33, 1992), DNA shuffling (Nature, 370: 389-391, 1994), or natural gene sequences. Currently, general-purpose synthesis methods that are randomized in part or in whole are widely used.

[0005] The error prone PCR method is a method of preparing a mutant population by randomly introducing base substitutions into a parent gene by performing PCR amplification in the presence of manganese ions with the parent gene as a saddle type. However, when the efficiency of introducing a base substitution mutation is increased by the error prone PCR method, there is a problem that not only amino acid substitution but also the probability that a codon encoding an amino acid is replaced with a stop codon is increased. Moreover, in this error prone PCR method, unintended deletion mutations are likely to occur under conditions that increase the mutation rate (Trends Biochem. Sci. 26: 100-106, 2001). There is a problem that the coded stop codon appears. For this reason, error prone PCR is usually performed under conditions that introduce a low mutagenesis rate, ie, substitution of 2 to 3 bases per gene and a mutation rate of 1 residue as an amino acid. For this reason, it is considered difficult to create artificial molecules that greatly change functions and substrate affinity.

[0006] DNA shuffling is a method of preparing a population of mutants by homologous recombination in vitro using a gene DNA encoding a protein and one or more similar DNAs having sequence homology. Can do. However, since the DNA shirt-fling method is based on homologous recombination, recombination between relatively similar sequences is possible. It is limited to. That is, it is difficult to shuffle between DNAs with low sequence similarity. Furthermore, in order to obtain the target mutant population, a series of complicated operations such as DNA cleavage and ligation are required.

[0007] A method using a gene population in which a part or all of a natural gene sequence is randomized by chemical synthesis is effective for the production of functional nucleic acids such as artificial RNA enzymes (ribozymes). However, functional artificial proteins have not been created. The reasons for this are as follows: (1) Genes with long ORFs (Open Reading Frames) are difficult because stop codons that stop protein translation appear frequently in populations with random mutations. 2) Since the sequence space composed of 20 types of proteins is vast compared to the sequence space of nucleic acids composed of 4 types of bases, it is extremely difficult to select functional proteins from random sequence populations. It is difficult.

In recent years, attention has been focused on the role of short sequences (motif sequences) that play an important role in the function and structure of nucleic acids and proteins. As a technique for creating an artificial gene population using a motif sequence, a technique for producing a polymer microgene polymer invented by the present inventors (Patent No. 3415995; Proc. Natl. Acad. Sci. USA, 94: 3805) —3810, 1997). By using this method, it is possible to prepare an artificial gene in which the stop codons of all translation reading frames are excluded. This method is a method of preparing a large translational reading frame by polymerizing short DNA sequences (microgenes) from which the stop codon has been eliminated in tandem. Furthermore, a microgene serving as a repetitive unit is a motif sequence corresponding to the intended function or structure in three reading frames using the multifunctional base sequence design method invented by the present inventor (JP 2001-352990 A). Since it is designed to code, an artificial gene population with high latent ability can be prepared.

[0009] However, the challenges that must be overcome are as follows: (1) The number of motifs that can be introduced into an artificial gene population is limited to three corresponding to the number of reading frames. (2) Artificial diversity To create a gene population, it depends on random frame shifts that occur during the microgene polymerization reaction. (3) It is difficult to adjust the appearance frequency of motif sequences according to the target experiment. Is mentioned. In addition, the above-mentioned method for producing a microgene polymer (Japanese Patent No. 3415995) is intended to produce a microgene repetitive polymer. It has not yet been possible to create a gene population that randomly polymerizes several motif sequences.

[0010] From the above background, an artificial protein with an extraordinary function that does not exist in nature has evolved. Effective artificial genetic diversity population (library) is the key to success in molecular engineering. A random DNA recombination technology that encodes multiple types of motif sequences at least in part, and a recombination technology that does not require sequence similarity throughout the DNA used and a long translation reading frame. The development of combinatorial artificial gene populations and artificial protein population creation technologies that have (Open Reading Frame; ORF) and can freely control the appearance frequency of motif sequences by adapting to the target experiment is required.

Patent Document 1: Japanese Patent No. 3415995.

Patent Document 2: JP 2001-352990 A.

Non-patent literature l: Science, 219, 666-671, 1983.

Non-Patent Document 2: Science, 67,938-947,1997.

Non-Patent Document 3: PCR Methods Appl, 2: 28-33, 1992.

Non-Patent Document 4: Nature, 370: 389-391, 1994.

Non-Patent Document 5: Trends Biochem. Sci. 26: 100-106, 2001.

Non-Patent Document 6: Proc. Natl. Acad. Sci. USA, 94: 3805-3810, 1997.

Disclosure of the invention

Problems to be solved by the invention

The subject of the present invention is a functional artificial gene or! For the purpose of effectively creating functional artificial proteins, Tsuji provides an artificial gene group in which multiple motif sequences are randomly polymerized, an artificial protein group encoded by the human gene group, and a method for producing the same. In addition, a single strand that encodes a motif sequence at least in part, can randomly polymerize DNA that is not subject to high overall sequence similarity, and avoids the occurrence of stop codons. It is an object of the present invention to provide a method for producing a combinatorial artificial gene and an artificial protein population that enables random polymerization of DNA sequences having a long translation reading frame (ORF) by randomly polymerizing DNA. Means for solving the problem

[0013] As a result of diligent studies to solve the above problems, the present inventors constructed at least three kinds of single-stranded DNAs containing sequences encoding arbitrary motif sequences, and the single-stranded DNA sequences. By introducing a base sequence capable of forming a complementary base pair with each other in a part of the terminal sequence of the DNA, mixing the various single-stranded DNAs in an arbitrary ratio, and allowing DNA polymerase to act on them. Depending on the frequency of base pair formation depending on the combination of base pairs and the concentration of each type of single-stranded DNA, it is possible to proceed with random polymerization reactions between DNAs encoding multiple types of motif sequences. Random recombination between DNA that encodes motif sequences in at least part of it, and long-range translational reading frames (ORFs), subject to high sequence similarity throughout Random polymerization is possible As a result, the present invention has been completed.

That is, the present invention constructs at least three kinds of DNA sequences of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and a part of the terminal sequence of the single-stranded DNA sequence is By introducing complementary base sequences so that they can form complementary base pairs with each other, a DNA polymerase is allowed to act on a reaction mixture in which various single-stranded DNAs constructed by the method are mixed at an arbitrary ratio. It consists of proceeding a random polymerization reaction between DNAs encoding, and creating an artificial gene population in which multiple motif sequences are randomly polymerized. As the motif sequence in the present invention, it is desirable to construct a single-stranded DNA in which at least 3 types, preferably 4 or more types of motif sequences exist, respectively.

[0015] In the present invention, a part of the complementary base pair of the terminal sequence of the single-stranded DNA sequence can usually be introduced at the 3 'end, and the number of the complementary base pairs is 6 bases. It is preferable that this is the case. At both ends of the complementary base pair, one or more bases that are not paired with the partner base are preferably present in the range of 1 to 3, so that the efficiency of the polymerization reaction by DNA polymerase can be increased. In the present invention, single-stranded DNA containing a sequence encoding an arbitrary motif sequence is based on a functional motif sequence, a structural motif sequence, a human peptide sequence obtained by evolutionary molecular engineering, or protein engineering knowledge. It can be a microgene constructed on the basis of the designed sequence. Within the single-stranded DNA sequence to be synthesized, stop codons can be excluded in advance for each translation reading frame. Furthermore, in the present invention, by adding a restriction enzyme recognition sequence to a single-stranded DNA that encodes multiple types of motif sequences, the frequency of occurrence of motif sequences used in random polymerization reactions can be directly monitored by restriction enzyme reactions. As described above, an artificial gene population can be produced.

[0016] The artificial gene population produced by the method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to the present invention further translates the artificial gene population and converts it into an artificial protein. Thus, an artificial protein population in which a large number of motif sequences are randomly inserted can be produced. In the production of the artificial protein population, in the present invention, an artificial gene population is produced by randomly polymerizing single-stranded DNAs encoding multiple types of motif sequences without inserting base mutations or deletions. By translating the gene population, it is possible to produce an artificial protein population in which a large number of motif sequences are randomly inserted without depending on the frame shift. Further, in the present invention, a single gene DNA encoding a multiple types of motif sequences is randomly polymerized while inserting base mutations and deletions to produce a human gene population, and the artificial gene population is translated into a frame. Depending on the shift, an artificial protein population can be created in which many types of motif sequences are randomly inserted. Thus prepared artificial gene populations in which a large number of the motif sequences of the present invention are randomly polymerized or artificial protein populations into which various motif sequences are randomly inserted are functional groups of these populations. Functional artificial genes or functional artificial proteins can be created by screening genes or functional proteins.

[0017] Specifically, the present invention provides [1] (1) constructing at least three or more DNA sequences of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, and (2) A complementary base sequence is introduced so that part of the terminal sequence of the single-stranded DNA sequence can form a complementary base pair with each other, and (3) various single-stranded DNAs constructed by the method are arbitrarily selected. Artificial gene population in which a large number of motif sequences are randomly polymerized by allowing a random polymerization reaction between the DNAs encoding the motif sequences by allowing DNA polymerase to act on the reaction mixture mixed at a ratio And [2] encode any motif sequence By constructing at least 4 types of DNA sequences of single-stranded DNA containing sequences and reacting DNA polymerase in the reaction mixture in which the single-stranded DNA is mixed in an arbitrary ratio, the motif sequence is converted. A method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized, and [3] a complementary base sequence, wherein random polymerization reactions between encoded DNAs proceed. 3, a method for producing an artificial gene population in which a large number of motif sequences according to [1] or [2] are randomly polymerized, and [4] the number of complementary base pairs is 6 The method for producing an artificial gene group in which a large number of motif sequences according to any one of the above [1] to [3] are randomly polymerized, characterized in that it is a base or more, and [5] The presence of one or more bases that cannot be paired with the other base [1] to [4], characterized by a method for producing an artificial gene population in which the multiple motif sequences described in any one of the above are randomly polymerized, and [6] The method for producing an artificial gene cluster in which a large number of motif sequences according to [5] are randomly polymerized, wherein non-pairing bases are present in any number of bases of 1 to 3, [7] Control the frequency of the occurrence of motif sequences used in random polymerization reactions by adjusting the combination of complementary base pairs of various single-stranded DNAs and the concentration of each Z or multiple single-stranded DNA. A method for producing an artificial gene group in which a large number of motif sequences described in any one of the above [1] to [6] are randomly polymerized, and [8] a variety of random polymerization reaction systems. The concentration of the double-stranded DNA varies within the range of 0.2 μΜ to 20 μΜ. Preparation methods mosquito ゝ Ranaru many species motif sequence force S randomly polymerized artificial gene cluster according to claim 7,.

The present invention also [9] mixes various constructed single-stranded DNAs into a reaction solution and acts from the 3 ′ end to the end with the complementary base sequence of the introduced single-stranded DNA as a saddle. The above-mentioned [1] to [8], characterized in that a random polymerization reaction proceeds between DNAs encoding motif sequences by the action of a thermostable DNA polymerase including exonuclease activity. (10) A single-strand D を containing a sequence encoding an arbitrary motif sequence is a functional motif sequence, structural motif sequence, evolution Based on artificial peptide sequences obtained from molecular engineering, or sequences designed based on protein engineering knowledge A method for preparing an artificial gene population in which a large number of motif sequences are randomly polymerized according to any one of the above [1] to [8], which is a constructed microgene, and [11] a mouth-mouth gene The multi-motif sequence described in [10] above, wherein the stop codon is excluded in each translation reading frame in the single-stranded DNA sequence to be synthesized. A method for creating a gene population and [12] single-strand DNA strength containing sequences encoding arbitrary motif sequences Random polymerization reaction by adding restriction enzyme recognition sequences to single-strand DNA encoding multiple motif sequences The multiple motif sequences described in any one of [1] to [11] above are randomly polymerized so that the frequency of the motif sequences used in can be directly monitored by restriction enzyme reaction Method for manufacturing an artificial gene population, consisting of [13] above [1] to [12] Artificial genes Group The fabricated by work made method of any, many species motif sequence was polymerized randomly.

Furthermore, the present invention provides [14] a beta incorporating a gene produced by a method for producing an artificial gene population in which the multiple motif sequences according to any one of [1] to [12] are randomly polymerized, A method of creating artificial protein populations that randomly insert various motif sequences characterized by introduction and expression into cells, and [15] single-stranded DNA encoding multiple motif sequences The above-mentioned [14], wherein an artificial gene population is produced by random polymerization without insertion, and a polymorphic molecular population is obtained without depending on frameshift by translating the artificial gene population. Artificial protein populations by randomly inserting various motif sequences, and [16] single strand DNA encoding multiple motif sequences are randomly polymerized with base mutations and deletions inserted, and artificial gene populations Make Then, the artificial gene population is produced by randomly inserting various motif sequences according to [14], wherein the diversity population is obtained by translating the artificial gene population depending on the frame shift. And [17] artificial protein populations prepared by randomly inserting various motif sequences, produced by the method described in any one of [14] to [16], and [18] a large number of [13] A functional gene or protein is screened for an artificial gene population in which species motif sequences are randomly polymerized or a human protein population into which various motif sequences described in [17] above are randomly inserted. Brief description of the drawings that can be used to create functional artificial genes or functional artificial proteins

FIG. 1 is a diagram showing an outline of production of an artificial protein population using the motif sequence of the present invention.

FIG. 2 is a diagram showing a design of a single-stranded DNA containing a restriction enzyme recognition sequence (top) and an electrophoretogram of a random polymerization reaction in Examples of the present invention. ^ KY-1354 ^ end force KY— 1355— KY— Designed to form a complementary base pair with the ^ end of 1357. As a result of electrophoresis, the polymerization reaction did not proceed with KY-1354 alone (lane 1), but the polymerization reaction proceeded by mixing DNA in various combinations (lanes 2-6). M represents a single DN A.

FIG. 3 is a diagram showing the results of confirming multiple types of random DNA polymerization by restriction enzyme reaction in Examples of the present invention.

FIG. 4 is a diagram showing the sequence of a DNA polymerization product having a restriction enzyme recognition motif in an example of the present invention.

FIG. 5 is a diagram showing design (A) and design (B) of single-stranded DNA having a BH1-B H4 motif present in an apoptosis-regulating protein in an example of the present invention. Design A was designed to form a complementary base pair with the 3 ends of KY-1372, 3 ends of KY-1372, KY-1375, KY-1375, and KY-1377. Design B was designed to form a complementary base pair with the ends of KY-1372 and KY-1379 and the ends of KY-1374 and KY-1375.

FIG. 6 is an electrophoretogram of a single-stranded DNA polymerization reaction containing a BH1-BH4 motif in an example of the present invention. o It was confirmed that the four types of single-stranded DNA synthesized in Design A and Design B proceeded with the polymerization reaction.

FIG. 7 is a diagram showing the results of confirmation of single-stranded DNA random polymerization containing a BH1-BH4 motif by restriction enzyme reaction in an example of the present invention. Electrophoresis after the restriction enzyme reaction confirmed that each motif polymerized randomly.

[FIG. 8] a, b and c are artificial gene populations obtained by design A in the examples of the present invention. It is a figure which shows the arrangement | sequence of the artificial protein population which is and its translation product.

FIG. 9 is a diagram showing the results of confirming the expression of an artificial protein in mammalian cells in an example of the present invention. The gene for artificial protein A6 obtained by design A was introduced into breast cancer cell line MCF-7, and its expression was confirmed by immunostaining with Alexa-His antibody. These artificial proteins were found to be localized and expressed in mitochondria.

FIG. 10 is a diagram showing the sequences of the artificial gene population obtained by design B and the artificial protein population that is the translation product in the example of the present invention.

[Fig. 11] In the examples of the present invention, the results of the design of single-stranded DNA having BH1-BH4 motif (C, D) and electrophoretic diagram of single-stranded DNA polymerization reaction present in the apoptosis-control protein are shown. FIG. In design (C), four types of single-stranded DNA (KY-137 2, KY-1389, KY-1391) were mixed and polymerized in equal amounts, whereas in design (D), KY-1372: KY -1389: KY- 1390: KY-1391 = 2: 2: 0. 4: 0. 3 Encoding 重合 Η4 and ΒΗ3 motifs 重合 — 1372 and ΚΥ— 1389 Increase the ratio of polymerization reaction Added to the system.

FIG. 12 is a diagram showing the results of confirming single-stranded DNA random polymerization containing ΒΗ1-ΒΗ4 motifs by restriction enzyme reaction in Examples of the present invention. In designs C and D, partial cleavage by three restriction enzymes was confirmed.

[FIG. 13] a and b are diagrams showing the sequences of an artificial gene population obtained by design C and an artificial protein population which is a translation product thereof, in an example of the present invention.

FIG. 14 is a diagram showing the sequences of an artificial gene population obtained by design D and an artificial protein population that is a translation product thereof, in Example of the present invention.

15] In an embodiment of the present invention, an artificial protein population (C) and an artificial protein population (D

FIG. 4 is a diagram showing the results of comparing the appearance frequencies of BH1-BH3 motifs in FIG. In the protein population (D) prepared by increasing the concentration of single-stranded DNA (KY-1389) encoding the BH3 motif, the frequency of appearance of the BH3 motif increased.

FIG. 16 is a diagram showing various localization patterns of artificial proteins in human cancer cells in Examples of the present invention.

FIG. 17 shows a gene encoding artificial protein D29 obtained in the examples of the present invention. FIG. 6 is a diagram quantifying the number of apoptotic cells induced in breast cancer cell line MCF-7 by introduction of. The effect was equivalent to the natural apoptosis-inducing protein Bax, and about 30% of the cells were apoptosis-positive cells. In cells transfected with the control artificial protein A10 gene, we found that such apoptosis-positive cells were not confirmed.

FIG. 18 is a graph showing that apoptosis is effectively induced in cells expressing D29 in the examples of the present invention. Double staining by confocal microscopy (green; expressed protein, red; apoptosis-positive cells) confirmed the correlation between cells expressing D29 and apoptosis-positive cells. It was found that apoptosis was not induced in cells expressing the control artificial protein A10.

FIG. 19 is a diagram quantifying the inhibitory activity of the growth inhibition induced by the cervical cancer cell line HeLa by introducing the gene encoding the obtained artificial protein A10 or D16 in the examples of the present invention. Natural type apoptosis-inducing protein BIM, or anticancer drug etoposide (VP-16) or staurosporine (STS) reduces the number of HeLa cells (quantified by WST-1; see control pcDNA), but introduces A10 or D16 gene As a result, the proliferation activity was partially recovered. This inhibitory effect on growth inhibition was also observed in cells transfected with a plasmid encoding the gene for Bel-xL, a natural apoptosis-inhibiting protein.

FIG. 20 is a view showing that apoptosis induced by the obtained artificial protein A10 or D16 force STS can be suppressed in Examples of the present invention. 24 hours after the gene transfer, the medium was replaced with a medium containing 125 nM STS, and the cells were further cultured for 23 hours. As a result of staining the apoptotic cells with the TUNEL method (red), apoptosis was effectively induced in the cells transfected with the control pcDNA or D29, whereas in the cells transfected with the apoptosis-inhibiting Bel—xL gene. The number of TUNEL positive cells decreased. Apoptosis was partially suppressed in cells transfected with A10 and D16 genes.

FIG. 21 is a diagram showing that cells expressing the obtained artificial protein D16 become apoptosis-negative in the examples of the present invention. As in Fig. 20, after fixing cells 23 hours after STS administration, the artificial protein was greened with Myc-secondary antibody and FITC secondary antibody. Apoptotic cells were double-stained with TMR-red label (red). In cells expressing D16 and Bcl— x L, apoptosis was suppressed. Cells that expressed D29 were confirmed to be positive for apoptosis.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] The present invention aims to effectively create a functional artificial gene or functional artificial protein, an artificial gene population in which a large number of motif sequences are randomly polymerized, and an artificial protein population encoded by the artificial gene population And providing a method for producing the same. The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to the present invention consists of (1) constructing at least three types of DNA sequences of single-stranded DNA containing sequences encoding arbitrary motif sequences. And (2) introducing a complementary base sequence so that part of the terminal sequence of the single-stranded DNA sequence can form a complementary base pair with each other, and (3) various single strands constructed by the method. Artificial DNA in which a large number of motif sequences are randomly polymerized by allowing a random polymerization reaction between DNAs encoding motif sequences by allowing DNA polymerase to act on a reaction mixture in which DNA is mixed at an arbitrary ratio. Creating a gene population is the key.

[0022] The method for producing an artificial gene population of the present invention will be specifically described with reference to an example. As shown in Fig. 1, first, multiple types (multiple types) of single-stranded DNA containing the desired motif sequence are synthesized. In the present invention, synthesis is carried out so that a part of the three rules of a plurality of types of single-stranded DNA can form complementary base pairs. The number of complementary base pairs is preferably 6 bases or more. However, it is synthesized so that there are one or more bases (preferably 1 to 3 bases) that are not paired with the partner base at both ends of the complementary base pair. By this operation, the efficiency of the polymerization reaction can be increased. In addition, multiple types of single-stranded DNA that encodes any sequence that does not limit the type, sequence, length, and combination of single-stranded DNA to be added can be used in this reaction. Furthermore, there are no restrictions on the combination of single-stranded DNAs that form complementary base pairs.

[0023] For example, when the reaction is performed using four types of single-stranded DNA, the remaining three types of single-stranded DNA 3 'sequences are complementary to the 3' sequence of one type of single-stranded DNA. It may form, or two types of single-stranded DNA with homologous ^ -terminal sequences, while the other two types of single-stranded DNA You can design your DNA sequences so that they are evenly complementary. In addition, the concentration of single-stranded DNA added to the reaction system can vary from 0.2 / z / to 20 / ζ M, and the concentration of single-stranded DNA to be used for the polymerization reaction can be increased. To the reaction system. That is, by adjusting the combination of complementary base pairs and the concentration of single-stranded DNA, the frequency of motif sequences used in random polymerization reactions can be controlled. In designing multiple types of single-stranded DNA, it is desirable to make the length and Tm of each sequence close to each other.

[0024] The complementary regions of these multiple types of single-stranded DNA function as a trapezoid for a PCR reaction using a thermostable polymerase. In the presence of multiple types of single-stranded DNA, it acts in the direction from the ^ end to the ⁵ 'end (when a PCR reaction is performed using a thermostable DNA polymerase containing 3, → 5 exonuclease activity, A double-stranded DNA polymer population in which multiple types of single-stranded DNA containing a motif sequence are randomly extended and ligated is synthesized under the conditions of PCR using DNA polymerase (eg, Vent polymerase), for example 94 One cycle of 10 seconds at 55 ° C and 60 seconds at 55 75 ° C is performed for 7 minutes at 69 ° C until DNA polymerization can be confirmed (30 to 65 cycles). Furthermore, it is preferable to carry out the reaction at 94 ° C for 10 minutes and at 69 ° C for 10 minutes, and the elongation reaction temperature is set close to Tm—5 ° C of single-stranded DNA. I prefer that.

[0025] In this way, complementary regions of multiple types of single-stranded DNA form base pairs in a random combination, and as a result of repeating extension and polymerization reactions, the DNA force encoding the motif sequence S is shuffled randomly. A double-stranded artificial DNA population is produced. In the method of the present invention, the preparation method of the polymer microgene polymer invented by the present inventors (Patent No. 3415995; Proc. Natl. Acad. Sci. USA, 94: 3805-3810, 1997) In contrast, a sequence diversity population can be created without depending on base substitution, insertion, or deletion that occurs between DNA strands during the polymerization reaction.

[0026] The method for producing the artificial gene population of the present invention has been specifically described above with examples. However, in the method for producing the artificial gene population of the present invention, a variety of single-stranded D as described above can be used. Control the frequency of motif sequences used in random polymerization reactions by adjusting the combination of NA's complementary base pairs and the concentration of each Z or multiple single-stranded DNA Is possible. In the method for producing an artificial gene population of the present invention, a plurality of motif sequences are arbitrarily selected and randomly polymerized to produce an artificial gene population and an artificial protein population. Is constructed based on sequences such as functional motif sequences, structural motif sequences, artificial peptide sequences obtained from evolutionary molecular engineering, or sequences designed based on protein engineering knowledge. It is possible to create an artificial gene population by predicting a certain degree of function with a reasonable rationality.

[0027] An artificial protein population produced by the method for producing an artificial gene population of the present invention in which various motif sequences are randomly inserted is introduced into a host cell and expressed by introducing a vector incorporating the gene into the host cell. It is possible to produce artificial protein populations in which various motif sequences are randomly inserted. Vectors used for the production of the artificial protein population, host cells and their gene expression methods include vectors, host cells and gene expression methods known in the art. An artificial gene population in which a large number of motif sequences produced by the method of the present invention are randomly polymerized, or an artificial protein population into which various motif sequences are randomly inserted, is used for the artificial gene population or artificial protein population. By screening functional genes or functional proteins, functional artificial genes or functional artificial proteins can be created.

[0028] Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

Prior to the description of the examples, FIG. 1 shows a strategy for producing an artificial protein population using the motif sequence of the present invention. This method consists of (1) design of multiple types of single-stranded DNA encoding motif sequences, (2) random polymerization reaction between multiple types of single-stranded DNA, and (3) efficiency of random polymerization reaction by restriction enzyme cleavage reaction. Study, (4) Transformation of random polymer into E. coli and sequencing, (5) Expression of artificial protein and screening of functional artificial protein in E. coli or mammalian cells. When it is desired to obtain an artificial gene cluster (when the expression of an artificial protein is not required), the reaction is completed in steps (1) to (4). In this example, an artificial gene and an artificial protein population are generally prepared by random polymerization of four types of motif sequences. The number of motifs that can be used is not limited to four, (more than that is theoretically possible). Specific experimental examples are given below.

Example 1

[0029] [Production of artificial gene cluster by random polymerization of 4 types of single-stranded DNA containing restriction enzyme recognition motif]

Design of single-stranded DNA containing restriction enzyme recognition sequence)

Four types of single-stranded DNA containing restriction enzyme recognition sequences were synthesized as follows as single-stranded DNA containing a functional sequence. KY—1354 (SEQ ID NO: 1, 5, GGGGAATTCGGC GGG A-3,), ΚΥ— 1355 (SEQ ID NO: 5′-GGGAAGCTTACCCGCCA-3 ′), ΚΥ— 1356 (SEQ ID NO: 3, 5, -0001 £ 1 ^^^^ ji 0ji-3,), 1 ^ —1357 (SEQ ID NO: 5, -GG GAGAICIACCCGCCA-3,). Here, the fourth to ninth sequences (underlined) of KY-1354, KY-1355, KY-1356, KY-1357 are the restriction enzyme recognition sequences GAAT TC (EcoRI), AAGCTT (HindIII), TCTAGA, respectively. (Xbal) and AGATCT (Bglll). In addition, the 10th to 15th 6 bases of KY-1354 (5, -GGCGGG-3,) are the 6th base of 11th to 16th of KY-1355, KY-1356, KY-1357 (5 CCCGCC -Designed to form complementary base pairs with 3 '). In addition, adenine (A) was introduced at the ^ terminal and 10th position of KY-1355, KY-1356, and KY-1357 so that a complementary base pair of 6 bases or more could not be formed with KY-1354. Furthermore, each of the four types of single-stranded DNA has a length (16-17 bases), Tm (: ~ 58 ° C), and GC content (65-69%) close to each other. Strand DNA was designed.

[0030] (Random polymerization reaction of single-stranded DNA containing restriction enzyme recognition sequence)

First, the above single-stranded DNA random polymerization reaction solution 50 / zL was prepared. This reaction solution contains 10 X ThermoPol Reaction Buffer (NEW ENGLAND BioLabs'lx ThermoPol Reaction Buffer: 20 mM 2-amino-2-hydroxymethyl-1,3, monopropanediol hydrochloride (hereinafter tris-hydrochloric acid) pH 8.8, 10 mM Vent DNA polymerases with potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton X—100) 5; z L, 350 Μ (1 ', 3' → 5 'etasonuclease activity ( 2 units / μL, NEW EN GLAND BioLabs) 2.6 L and 4 types of single-stranded DNA (KY— 1354— KY— 135 7) was mixed in the following 6 ratios. (1) KY— 1354: 20 pmol only (1 DNA), (2) KY— 1354: 20 pmol + KY— 1355: 20 pmol (2 DNA), (3) KY— 1354: 20 pmol + KY— 1356: 20 pmol (2 types of DNA), (4) KY— 1354: 20 pmol + KY— 13 57: 20 pmol (2 types of DNA), (5) KY— 1354: 20 pmol + KY— 1355: 10 pmol + KY— 1356: 10 pmol (3 types of DNA), (6) KY— 1354: 20 pmol + KY— 1355: 6.7 pmol + KY— 1356: 6.7 pmol + KY— 1357: 6.7 pmol (4 types of DNA).

[0031] These reaction solutions were placed in a 200 / z L thin wall PCR tube (Toyobo, Osaka) and subjected to temperature cycling with a thermal cycler PCR-2400 (Perkin Elmer I, Norwalk) between the multiple single-stranded DNAs. The polymerization reaction was carried out. As a pretreatment for the polymerization reaction, a reaction was performed at 94 ° C for 10 minutes and at 69 ° C for 10 minutes. The polymerization reaction temperature was set to 55 ° C in consideration of the Tm value of KY-1354-1357 (58 ° C) or less. The polymerization reaction cycle was 94 ° C. for 10 seconds and 55 ° C. for 60 seconds, and 40 cycles of the reaction proceeded. After completion of the polymerization reaction, an elongation stop reaction was carried out at 69 ° C for 7 min. The DNA polymerization product was electrophoresed at 100V for 15 minutes using 1.0% TAE agarose gel (Agarose ME Iwai Chemical Tokyo) and Mupid electrophoresis apparatus (Cosmo Bio, Tokyo) to confirm the polymerization reaction (Fig. 2). . By this reaction, it was confirmed that DNA of several kilo base pairs or more was polymerized by this method.

[0032] (Confirmation of multiple types of random DNA polymerization by restriction enzyme reaction)

In order to confirm the random polymerization of single-stranded DNA containing a restriction enzyme recognition sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. 50 μL of 5 kinds of reaction solutions containing different restriction enzymes were prepared as follows. 5 μL of the above DNA polymerization reaction solution, 10 × restriction enzyme buffer B (Hindlll, EcoRI), M (BglII), or H (Xbal) 5 μL, (Roche Diagnostics, Basel), restriction enzyme (Roche Diagnostic, Basel) has (1) no enzyme, (2) HindllKlOunits / μL) 2 μL, and (3) BglII (lOunits / μL) for each of the five reaction solutions. 2 μL, (4) Xbal (40 units / μL) was added at 2 μL, and (5) EcoRI (10 units / μL) was added at 2 / z L. The cleavage reaction was allowed to proceed at 37 ° C for 90 minutes. In order to confirm the DNA cleavage reaction, 5 L was extracted from the 50 L reaction solution and subjected to electrophoresis (100 V, 15 minutes) with 1.0% TAE agarose gel. (Figure 3). According to Figure 3, the DNA polymer in 1-2 is cleaved with the restriction enzyme sequence contained in any single-stranded DNA used in the polymerization reaction. It was also confirmed that the DNA polymer composed of multiple types of single-stranded DNA chains was cleaved by multiple types of restriction enzymes. This proved that random polymerization and elongation reactions occurred between single-stranded DNAs containing restriction enzyme recognition motifs.

[0033] (Transformation into E. coli and sequencing of DNA polymerization product)

Random polymerization reaction solution between the above DNA sequences is commercially available Zero Blunt TOPO PCR

Cloning and sequencing were performed using Cloning Kit (Invitrogen, CA). The ligation reaction is performed on a 6 μL reaction scale, and this reaction solution contains 1 μL of DNA polymerization product, 1 L of Salt Solution, 1 L of TOPO vector, and 3 μL of ultrapure water. After the reaction solution was reacted at room temperature for 30 minutes, 2 μL of the reaction solution was mixed with TopOlOcell (Invitrogen) and transformed. Select 18 clones containing the insert, purify the plasmid with QIAGEN mini kit (Qiagen), and sequence with the capillary sequencer CEQ2000XL DNA analyzer (Beckman) by dye termination method using DTCS cycle sequence reaction kit (Beckman) It was determined. KY— 1354, KY— 1355, KY— 1357, a polymer with a mixture of three single-stranded DNAs. One clone (EHB # 7: SEQ ID NO: 5), KY— 13 54, KY— 1355 FIG. 4 shows three clones (EHBX1 to EHBX3: SEQ ID NOs: 6, 7, and 8) obtained from the polymer power obtained by mixing four kinds of single-stranded DNAs of KY-1356 and KY-1357. As shown in FIG. 4, an artificial gene population in which DNA containing 3 or 4 kinds of restriction enzyme recognition sequences was randomly polymerized at an arbitrary ratio could be obtained.

Example 2

[0034] [Preparation of artificial protein population (A) by random polymerization of BH1-BH4 motif sequences present in apoptosis-regulated proteins]

(Selection of apoptosis control motif BH1- BH4)

As a natural protein that regulates programmed spontaneous cell death (apoptosis), Bcl-

2 family protein families are known. BcHd, a member of the family, has been shown to be important in inhibiting apoptosis, and Noxa is important in promoting apoptosis. Here, the partial sequence of BH1, BH2, and BH4 motifs in human BcHd protein and the partial sequence of BH3 motif in hu man Noxa protein are used as motif sequences for artificial protein population creation. Tried. Part encoding the following peptide sequence Minutes were selected from each BH1-BH4 motif. BH1: ELFRDGVN, BH2: ENGG WDTF, BH3: LRRFGDKLN ゝ BH4: RELWDFL.

[0035] (BH1--design of single-stranded DNA encoding BH4 motif sequence: A)

In order to create an artificial protein group A composed of BH1-BH4 motifs, a multi-functional nucleotide sequence design method invented by Shiba et al. — Designed by 352990). Here, another reading frame of the gene encoding the BH1-BH4 motif includes four types of single-stranded chains that are a-helix-like and highly peptide-encoded to help stabilize the artificial protein. DNA was designed (Figure 5). Here, KY-1372 (SEQ ID NO: 9), KY-1377 (SEQ ID NO: 10), KY-1375 (SEQ ID NO: 11), KY-1374 (SEQ ID NO: 12) are the restriction enzyme recognition sequences GTCGAC (Sail), It was designed to include AAGCTT (HindIII), AGATCT (BglII), and GAATTC (EcoRI) (Figure 5, Design A, underlined).

[0036] In addition, KY— 1372 3 ′ end 7 bases (5,-GGCGGGG-3,), KY— 1377, KY— 1375, KY-1374 ^ end 7 bases (5 '-CCCCGCC-3') It was designed to form a complementary base pair. In addition, adenine (A) and cytosine (C) were inserted so that no interaction of 7 base pairs or more was formed at the 9th base from the ^ terminal and ^ terminal of KY-1372. Furthermore, when designing these four types of single-stranded DNA, the length (40-45), GC base content (55-65%), intramolecular hairpin formation ability, and Tm are close to each other. Designed as follows.

[0037] (BH1-Random polymerization of single-stranded DNA containing BH4 motif)

First, 50 L of the single-stranded DNA random polymerization reaction solution was prepared. The composition of this reaction solution is 5 μL of 10 X ThermoPol Reaction Buffer (same as Example 1), 2.6 L of 350 μM dNTP ゝ Vent DNA polymerases (2 units / μL, NEW ENGLAND BioLabs), and Four types of single-stranded DNA (KY-1372, KY-1377, KY-1375, KY-1374) were mixed at various ratios, and the same DNA random polymerization reaction as in Example 1 was performed. As a pretreatment for the polymerization reaction, a reaction was carried out at 94 ° C for 10 minutes and at 69 ° C for 10 minutes. The polymerization reaction temperature was set to 72 ° C. The polymerization reaction cycle was performed at 94 ° C for 10 seconds and at 55 ° C for 60 seconds, and 40 cycles of the reaction proceeded. [0038] After completion of the polymerization reaction, an elongation termination reaction was carried out at 69 ° C for 7 minutes. The DNA polymerization product was electrophoresed at 100 V for 15 minutes using 1.0% TAE agarose gel (Agarose ME Iwai Chemical Tokyo) and Mupid electrophoresis apparatus (Cosmo Bio, Tokyo) to confirm the polymerization reaction (Fig. 6). . In addition, as a result of optimizing the reaction so that single-stranded DNAs encoding the BH1-BH4 motif each randomly polymerize with a similar frequency, KY-1374: KY-1375: KY-1377: ΚΥ— 1372 The ratio of single-stranded DNA was set as follows: 2: 1: 100: 100. By this reaction, DNA force of several kilobase pairs or more was confirmed to be polymerized by this method.

[0039] (Confirmation of DNA random polymerization containing BH1-ΒΗ4 motif by restriction enzyme reaction)

In order to confirm the random polymerization of single-stranded DNA containing the BH1-—4 motif sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. 50 μL of five reaction solutions containing different restriction enzymes were prepared as follows. 5 μL of DNA polymerization reaction solution, 10 × restriction enzyme buffer H (SalI, or EcoRI), or 5 μL of M (Hindm, or Bglll) (Roche Diagnostics, Basel), restriction enzyme (Rochedai (Agnostic, Basel) has (1) no enzyme, (2) Sail (40units / μL) ^ 2 μ (3) HindIII (10units Z μL) in 2 μm for each of the five reaction solutions. L, (4) EcoRl (10 units / μL) was added at 2 μL, and (5) Bglll (10 units / μL) was added at 2 μL. The cleavage reaction was allowed to proceed at 37 ° C for 90 minutes. In order to confirm the DNA cleavage reaction, 5 L was extracted from the 50 L reaction solution and subjected to electrophoresis (100 V, 15 minutes) on a 1.0% TAE agarose gel (FIG. 7). As shown in Fig. 7, it was found that a DNA polymer composed of single-stranded DNA containing the BH1-BH4 motif sequence was partially cleaved by all the restriction enzymes used. From these facts, a random polymerization reaction between four types of single-stranded DNA was confirmed.

[0040] (Transformation to E. coli, screening, and sequencing of DNA polymerization product)

The DNA polymerization reaction product containing the BH1-BH4 motif was cloned and sequenced using a commercially available pcDNA3.1Directiona 1 TOPO Expression Kit (Invitrogen, CA). Here, an artificial gene was ligated into a mammalian expression vector (pcDNA3.1D / V5-His-TOPO, Invitrogen) for screening functional artificial proteins in mammalian cell systems. The ligation reaction is performed on a 6 μL scale. Polymerization reaction product l / L, Salt Solution 1 L, TOPO vector 1 L, ultrapure water 3 L force S are included. After the reaction solution was reacted at room temperature for 30 minutes, the 2 / ^ reaction solution was mixed with Ding 0-10 ^ 11 (1 nvitrogen), allowed to stand on ice for 30 minutes, and transformed. From the screening PCR, 10 types of clones containing inserts were selected, and the plasmids were purified with QIAGEN mini kit (Qiagen). The sequence was determined with (Beckman) (SEQ ID NO: 13-42).

[0041] The gene sequence and amino acid sequence of the obtained artificial protein are shown in Fig. 8 (SEQ ID NOs: 13-42). Figure 8 showed that an artificial protein population in which artificial DNA encoding the BH1-BH4 motif was randomly inserted could be created. In addition, in “Design of single-stranded DNA containing BH1-BH4 motif sequence A”, in order to create a library with higher sequence diversity, the motif depends on the frame shift that occurs during the DNA polymerization reaction. The gene was designed to be introduced (the reading frame would be shifted if no frame shift occurred) o Many reading frame arrangements different from the reading frame encoding the BH1-BH4 motif appeared as shown in Fig. 8. . Thus, in this method, a library can be created using multiple motif sequences, but sequence diversity can be increased by using sequences in different translation reading frames.

[0042] (Confirmation of expression of artificial protein in mammalian cells)

In order to confirm the expression of the above-described sequenced artificial protein in mammalian cells, a plasmid encoding the gene for MxA6 (see Fig. 8, a, b, c) was used as one of the representative human breast cancer cell lines, MCF-7 The gene was introduced into. As a control, V, pcDNA vector (Invitrogen) that does not encode an artificial protein was introduced into cells. MCF-7 was fixed with methanol 24 hours after gene introduction, and the expression of histidine-tag and artificial protein was confirmed by immunostaining with Alexa-His-antibody (Qiagen) (FIG. 9). As shown in Fig. 9, the expression was not observed in the control, whereas in the cells into which the MxA6 gene was introduced, the expression of the artificial protein was observed and the localization was consistent with the mitochondria. Using such a mammalian cell system or E. coli expression system, expression of an artificial protein population and screening of functional artificial proteins can be performed. Example 3

[0043] [Preparation of artificial protein population (B) by random polymerization of BH1-BH4 motif sequence existing in apoptosis-regulated protein]

In the production of the artificial protein population (A) of Example 2, KY-1 372 encoding a BH4 motif single-stranded DNA encoding a BH3, BH2 or BH1 motif (KY-1377, KY-1375, KY-1374) It was designed to form a complementary base pair with (1: 3 correspondence). The human protein population (B) is designed to form a complementary base pair with the single-stranded DNA encoding the BH2 or BH1 motif (2: 2 correspondence) (Figure 5). Thus, the ratio of single-stranded DNA forming complementary base pairs can be freely adjusted. Other methods are basically the same as the production of artificial protein population (A).

[0044] (BH1--design of single-stranded DNA containing BH4 motif sequence: B)

Four types of single-stranded DNAKY—1372, KY—1379 (BH3 motif, SEQ ID NO: 43), KY—1 375, and KY—1374 were used to create artificial protein population B composed of BH1—BH4 motifs. It was designed by the multi-functional nucleotide sequence design method (Japanese Patent Laid-Open No. 2001-352990) invented by Shiba et al. Here, KY-1372, KY-1379, KY-1375, and KY-1374 were designed to contain restriction enzyme recognition sequences GTCGAC (Sail), CTCGAG (XhoI), AGATCT (Bglll), and GAATTC (EcoRI), respectively. (Figure 5 Design B, underlined). Also, the 3 'end 7 bases of KY-1372 or KY-1379 (5,-GGCGGGG-3,) are complementary to the 7 end bases of KY-1375 and KY-137 4 (5' -CCCCGCC-3 ') It was designed to form a target base pair. In addition, the 9th base from the 3 'end and the 3' end of KY-1372 and KY-1379 does not form an interaction of 7 base pairs or more, so that adenine (A) and cytosine (C) are added. Each inserted.

[0045] (BH1-Random polymerization of single-stranded DNA containing BH4 motif)

First, 50 L of the single-stranded DNA random polymerization reaction solution was prepared. Adjustments were made in the same way as for the production of artificial protein population (A), except that Xhol was used to study the polymerization reaction of KY—1379, which encodes Xhol. The polymerization reaction was confirmed by 1% agarose gel. [0046] (Figure 6). In addition, as a result of the optimal reaction of the reaction so that the BH1-BH4 motifs randomly polymerize at similar frequencies, KY— 1374: KY— 1375: KY— 1379: KY— 1372 = 4: 1: 10: 1 The BH1-BH4 amount ratio was set as follows.

[0047] (Confirmation of DNA random polymerization containing BH1-—4 motif by restriction enzyme reaction)

In order to confirm the random polymerization of single-stranded DNA containing the BH1-—4 motif sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. The reaction is the same as in the section on the production of artificial protein population (A), except that Xhol is carotenized as a restriction enzyme. (1) No enzyme, (2) 3 _& 11 (40 3 2) (3) XhoI (lOunits / μL) 2 μL (4) EcoRI (10 units / μL) ) Was added at 2 μL, and (5) BgllKlOunits / μL) was added at 2 / z L. The cleavage reaction was allowed to proceed at 37 ° C for 90 minutes. To confirm the DNA cleavage reaction, 5 L was extracted from the 50 L reaction solution, and subjected to electrophoresis (100 V, 15 minutes) on a 1.0% TAE agarose gel. (Figure 7). As shown in Fig. 7, it was found that a DNA polymer composed of single-stranded DNA containing the BH1-BH4 motif sequence was partially cleaved by all the restriction enzymes used. From this, a random polymerization reaction of four types of single-stranded DNA was confirmed.

[0048] (Transformation to E. coli, screening, and sequencing of DNA polymerization product)

Transformation and sequencing were performed in the same manner as in the section on production of artificial protein population (A). The gene sequence and amino acid sequence of human protein is shown in FIG. 10 (SEQ ID NO: 4453). Example 4

[0049] [Preparation of artificial protein population (C) by random polymerization of BH1-BH4 motif sequence existing in apoptosis-regulated protein]

In the production of artificial protein populations (A, B) in Example 2 and Example 3, the BH1-BH4 motif is an artificial protein depending on the frame shift caused by random base deletion or insertion that occurs during the DNA polymerization reaction. Randomly inserted into the population. For this reason, the appearance frequency of the BH1—BH4 motif was relatively low (a lot of different translation reading frames appear). Here, the design of the single-stranded DNA was modified so that the BH1-BH4 motif was randomly polymerized without depending on the frame shift. The other methods are basically the same as the production of the artificial protein population (A). More BH1—BH4 modules without relying on frameshift It is expected in Example 4 that the chief appears in the artificial protein population.

[0050] (BH1--design of single-stranded DNA containing BH4 motif sequence: C)

Four types of single-stranded DNAs KY— 1372, KY— 1389 (SEQ ID NO: 54), KY— 1390 (SEQ ID NO: 55), KY — 1391 (SEQ ID NO: 56) was designed (FIG. 11, KY— 1372: BH4 motif, KY— 1389: BH3 motif, KY— 1390: BH1 motif, KY— 1391: BH2 motif). KY-1389, KY-1390, and KY-1391 are sequences with CC attached to the ^ end of KY-1377, KY-1374, and KY-1375, respectively. As a result, the randomly polymerized BH1-BH4 motif appears in the artificial protein population without depending on the frame shift. KY— 1372, KY— 1389, KY— 1390, and KY— 1391 were designed to include the restriction enzyme recognition sequences GTCGAC (Sail), AAGCTT (Hindlll), GAATTC (EcoRI), and AGATCT (Bglll), respectively. C, underlined). In addition, KY— 1372 3 ′ end 7 bases (5, -GG CGGGG-3,) force KY— 1389— KY— 1391 3 ′ end 7 bases (5,-CCCCGCC-3,) and complementary base pair Designed to form. In addition, adenine (A) and cytosine (C) were inserted so that no interaction of 7 base pairs or more was formed at the ^ terminal of KY-1372 and the ninth base from the ^ terminal.

[0051] (BH1-Random polymerization of single-stranded DNA containing BH4 motif)

50 μL of the single-stranded DNA random polymerization reaction solution was prepared in the same manner as in the section on the production of artificial protein population (Α). The single-stranded DNA to be added was added to the polymerization reaction system in the same amount of single-stranded DNA as follows: 1-1372: ΚΥ-1389: ΚΥ-1390: ΚΥ-1391 = 1: 1: 1: 1: 1. The polymerization reaction was carried out at 72 ° C and 45 cycles, and the polymerization was confirmed by 1% agarose gel (Fig. 11).

[0052] (Confirmation of DNA random polymerization containing BH1-BH4 motif by restriction enzyme reaction)

In order to confirm the random polymerization of single-stranded DNA containing the BH1-BH4 motif sequence, the DNA polymer was cleaved using an arbitrary restriction enzyme. The reaction conditions are the same as in the section on production of artificial protein population (A). (5) HindllKlOunits / μL) 2 μL, (3) EcoRI (10units / μL) 2 μL, (4) BglII (l) OunitsZ L) was adjusted under the condition of 2 L. As shown in Figure 12, BH1—BH4 motif arrangement It was found that a DNA polymer composed of single-stranded DNA containing a sequence was partially cleaved by all the restriction enzymes used. From this, a random polymerization reaction of four types of single-stranded DNA was confirmed.

[0053] (Transformation to E. coli, screening, and sequencing of DNA polymerization product)

Transformation and sequencing were performed in the same manner as in the section on production of artificial protein population (A). The gene and amino acid sequence of the artificial protein obtained by Design C are shown in FIGS. 13a and b (SEQ ID NOs: 57-74). As shown in Fig. 13, we succeeded in obtaining an artificial protein population in which four BH1-BH4 motif sequences appear frequently.

Example 5

[0054] [Preparation of artificial protein population (D) by random polymerization of BH1-BH4 motif sequence existing in apoptosis-regulated protein]

As a result of the production of the artificial protein population (C) of Example 4, it was found that the obtained protein population had a relatively low frequency of appearance of the BH3 motif. Therefore, in Example 5, the amount ratio of the single-stranded DNA added to the DNA polymerization reaction was KY— 1372: KY— 1389: KY— 139 0: KY— 1391 = 2: 2: 0.2.4: 0.3. Thus, the proportions of KY-1 372 and KY-1389 encoding コード 4 and ΒΗ3 motifs were increased and added to the polymerization reaction system. All the reaction conditions were the same as in the production of the artificial protein population (C) in Example 4. FIG. 11 shows an electrophoretogram of the random polymer, FIG. 12 shows the digestion with restriction enzymes, and FIGS. 14a, b and c show the gene and amino acid sequences of the human protein obtained (SEQ ID NOs: 75 to 100). As shown in Fig. 14, we succeeded in creating an artificial protein population in which four motifs appeared frequently, and in particular, the BH4—BH3 motif appeared frequently. In this way, by changing the amount ratio of single-stranded DNA to be mixed during the DNA polymerization reaction, the ratio of the motif contained in the artificial protein population can be controlled. Figure 15 compares the frequency of BH1-BH3 motifs in the artificial protein population (C) and the artificial protein population (D). Example 6

[0055] [Expression and localization diversity of artificial proteins in human cancer cells]

Forty-one clones were randomly selected from the artificial protein populations A, B, C, and D prepared in Examples 2, 3, 4, and 5, and the genes were introduced into the human breast cancer cell line MCF-7. Relic MCF-7 was fixed with methanol 24 hours after introduction of the gene, and the expression of the artificial protein with Myc epitope tag (EQKLIS EEDL; SEQ ID NO: 101) was confirmed by immunostaining with Myc antibody. As a result, 28 out of 41 clones (68%), the effective expression of the protein could be observed in human cells. In addition, as a result of examining the intracellular localization, it was found that there were those that were present in the cytoplasm (11 clones), those that were present in the nucleus (5 clones), those that were present in mitochondria (5 clones), and others (7 clones). However, it was very diverse (Fig. 16). In this way, the artificial protein obtained by this method was effectively expressed in mammalian cells and was found to have the ability to localize to various organelles in cells.

Example 7

[0056] [Creation of functional artificial protein that induces cell death in cancer cells]

In Example 6, 28 clone genes whose protein expression was confirmed were introduced into human breast cancer cell line MCF-7, and the effect on the growth of MCF-7 was examined. A 96-well cell culture plate was seeded with lxlO ⁴ MCF-7 per well and cultured at 37 degrees for 24 hours. Thereafter, plasmid 0.2 / zg encoding the gene of 28 clones was introduced into MCF-7 using Lipofectamine (20 OO lnvitrogen). 48 hours after gene introduction, the proliferation activity was quantified using tetrazorium salt WST-1 which is an indicator of mitochondrial metabolic activity (Roche). Do not code a control protein! Compared to the growth activity when an empty vector was introduced, one clone out of 28 clones has a D29 force that significantly inhibits the growth of MCF-7. (Approximately 40% growth inhibitory effect over control).

[0057] In order to examine whether this proliferation inhibitory activity was due to programmed spontaneous cell death (apoptosis), TUNEL staining, an index for detecting apoptotic cells, was performed (Roche). In TUNEL staining, fragmented DNA in the early stage of apoptosis can be detected with fluorescently labeled nucleotides. As a result, D29 was able to induce apoptosis in MCF-7 to the same extent as the natural cell death-inducing protein Bax (Fig. 17). In contrast, A10, one of the clones in which cell growth inhibition was not observed by WST-1, was found to be unable to induce apoptosis in MCF-7. In addition, as a result of confocal research and detailed examination of the state of cells that crawl and express artificial proteins, there was a clear correlation between cells that express D29 and TUNEL-positive cells (Fig. 18). this Thus, the artificial protein population produced by random polymerization of the apoptosis-control motif sequence by this method was found to contain functional artificial proteins capable of inducing apoptosis in breast cancer cells.

Example 8

[0058] [Creation of cell death-suppressing functional artificial protein using the same protein population]

The clone whose expression was confirmed in Example 6 contained a cell death inhibitory BH4 motif as well as a cell death promoting BH3 motif. Therefore, the possibility of obtaining a cell death-suppressing functional protein having a function opposite to that of the artificial cell death promoting protein of Example 7 from these clones was examined. The genes of 28 clones whose protein expression was confirmed and the natural cell death-promoting protein BIM were introduced into the human cervical cancer cell line HeLa, and the effects of the clones on the growth inhibitory activity of BIM were examined. The cell culture plates 96 Ueru were seeded with 0. 5xl0 ⁴ of HeLa per Ueru were incubated at 37 ° for 24 hours. Thereafter, 100 ng of a plasmid encoding the gene of 28 clones and 50 ng of a plasmid encoding BIM were introduced into HeLa using Lipofectamine 2000 (Invitrogen). Forty-eight hours after the introduction of the gene, the proliferation activity was quantified with tetrazolium salt WST-1 (Roche). As a result of comparison with the growth activity when a vector encoding GFP, which is a control, was introduced, two clones A10 and D16 of 28 clones were found to significantly inhibit the growth inhibition by BIM (Fig. 19). Such a growth inhibition inhibitory effect could be observed even in cells into which a plasmid encoding the gene of Bel-xL, a natural apoptosis inhibitory protein, was introduced. Furthermore, it was found that the growth was inhibited by HaLa cells into which A10 and D16 had been inhibited by two types of anticancer drugs, etoposide (VP-16) and staurosporine (STS) (Fig. 19).

[0059] In order to examine whether this cell death inhibitory activity was due to inhibition of apoptosis, TUNEL staining was performed (Roche). About lxlO ⁵ HeLa cells were transfected with an artificial protein-encoding plasmid 0.4 / zg, and after 24 hours, the medium was replaced with a medium containing 125 nM STS and further incubated for 23 hours. As a result, it was found that apoptosis induced by STS was significantly suppressed in HeLa cells into which A10 or D16 had been introduced (Fig. 20). In contrast, almost all cells introduced with the empty vector pcDNA or plasmid encoding D29 TUNEL was positive. Furthermore, as a result of detailed examination of the state of cells expressing the artificial protein using a confocal microscope, a clear correlation between D16-expressing cells and TUNEL-negative cells was observed (FIG. 21). This apoptosis-inhibiting effect was also observed in cells expressing Bel-xL, a natural apoptosis-inhibiting protein. In this way, the artificial protein population produced by this method has been shown to be able to create not only an apoptosis-inducing type but also a suppressive functional artificial protein.

Industrial applicability

[0060] The artificial gene population production method of the present invention has made it possible to produce an artificial gene population in which a large number of motif sequences are randomly polymerized, which was not possible with the conventional artificial gene population production method. Moreover, it has become possible to produce an artificial protein population encoded by the artificial gene population using the artificial gene population. According to the method for producing an artificial gene population of the present invention, it is possible to randomly polymerize a plurality of types of DNAs, provided that the motif sequence is encoded by at least a part of the gene group, provided that the similarity is high. In addition, by randomly polymerizing single-stranded DNA that avoids the appearance of stop codons, DNA sequences with long translation reading frames (ORF) can be randomly polymerized. Furthermore, an arbitrary motif sequence is changed based on a sequence such as a functional motif sequence, a structural motif sequence, an artificial peptide sequence obtained by evolutionary molecular engineering, or a sequence designed based on protein engineering knowledge. By constructing, arbitrarily selecting many types of motif sequences, polymerizing randomly, and creating artificial gene populations and artificial protein populations, we add rational predictability in terms of functionality, It became possible to create artificial gene populations and artificial protein populations in which multiple motif sequences were randomly polymerized.

[0061] From this, it is possible to create a powerful artificial gene diversity population (one library) that is the key to success in the evolutionary molecular engineering of artificial proteins with unusual functions that do not exist in nature Sexually increased. Furthermore, in the present invention, by selecting the functions of multiple species of motif sequences, adjusting the combinations of complementary base pairs of various single-stranded DNAs and the respective concentrations of Z or multiple single-stranded DNAs, It is possible to control the appearance frequency of motif sequences used in random polymerization reactions, and when creating an artificial gene cluster, a certain degree of function is predicted and the random polymerization reaction is regulated, and artificial genetics are controlled. It became possible to create child populations.

Claims

The scope of the claims

[1] (1) Construct at least three kinds of DNA sequences of single-stranded DNA containing a sequence encoding an arbitrary motif sequence, (2) Part of the terminal sequence of the single-stranded DNA sequence Introduce complementary base sequences so that they can form complementary base pairs with each other, and (3) allow DNA polymerase to act on a reaction mixture in which various single-stranded DNAs constructed by the method are mixed in an arbitrary ratio Thus, a method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized, wherein a random polymerization reaction is allowed to proceed between DNAs encoding the motif sequences.

[2] At least 4 types of single-stranded DNA containing sequences encoding arbitrary motif sequences

A DNA sequence is constructed, and a DNA polymerase is allowed to act on a reaction solution in which the single-stranded DNA is mixed at an arbitrary ratio, thereby causing a random polymerization reaction between DNAs encoding motif sequences. A method for producing an artificial gene population in which the multiple motif sequences according to claim 1 are randomly polymerized.

[3] The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to claim 1 or 2, wherein a complementary base sequence is introduced at the 3 ′ end.

[4] The method for producing an artificial gene population in which multiple motif sequences are randomly polymerized according to any one of claims 1 to 3, wherein the number of complementary base pairs is 6 or more.

[5] The multiple motif sequences according to any one of claims 1 to 4, wherein one or more bases are present at both ends of the complementary base pair without pairing with the partner base. A method for producing a synthetic artificial gene population.

[6] The multiple motif sequence according to claim 5, wherein a base that does not pair with the partner base is present at both ends of the complementary base pair in any number of 1 to 3 base groups. A method for producing a polymerized artificial gene population.

[7] Control the frequency of appearance of motif sequences used in random polymerization reactions by adjusting the combination of complementary base pairs of various single-stranded DNAs and the concentration of each of Z or various single-stranded DNAs. A method for producing an artificial gene population in which the multiple motif sequences according to any one of claims 1 to 6 are randomly polymerized.

[8] Concentration power of various single-stranded DNA that can be used in random polymerization reaction system 0.2 μ Μ to 20 μ 8. The method for producing an artificial gene population in which the multiple motif sequences are randomly polymerized according to claim 7, wherein the artificial gene population varies in a range of M.

[9] Mix the various single-stranded DNAs that have been constructed in the reaction mixture, and use the complementary base sequence of the introduced single-stranded DNA as a saddle shape to exert a 3 'end force in the 5' end direction. The multi-motif according to any one of claims 1 to 8, wherein a random polymerization reaction is caused to proceed between DNAs encoding the motif sequence by acting a thermostable DNA polymerase including Method for producing artificial gene population with randomly polymerized sequences

[10] Single-stranded DNA force containing sequences encoding arbitrary motif sequences Based on functional motif sequences, structural motif sequences, artificial peptide sequences obtained from evolutionary molecular engineering, or protein engineering knowledge! 9. The method for producing an artificial gene population in which a large number of motif sequences are randomly polymerized according to any one of claims 1 to 8, wherein the gene is a microgene constructed on the basis of the designed sequence.

[11] The multi-motif sequence according to claim 10, wherein a stop codon is excluded in advance in each translation reading frame in the single-stranded DNA sequence to be synthesized when constructing the microgene. A method for producing a population of artificial genes polymerized.

[12] Single-stranded DNA strength containing sequences encoding arbitrary motif sequences Motifs used in random polymerization reactions by adding restriction enzyme recognition sequences to single-stranded DNA encoding multiple motif sequences The artificial gene population in which multiple types of motif sequences according to any one of claims 1 to 11 are randomly polymerized, wherein the frequency of appearance of the sequences is constructed so that it can be directly monitored by a restriction enzyme reaction. Manufacturing method.

[13] An artificial gene population produced by the production method according to any one of claims 1 to 12 and having a multi-motif sequence force S randomly polymerized.

[14] A vector incorporating a gene produced by the method for producing an artificial gene population in which the multiple motif sequences according to any one of claims 1 to 12 are randomly polymerized is introduced into a host cell and expressed. A method for producing an artificial protein population in which various characteristic motif sequences are randomly inserted.

[15] Do not insert nucleotide mutations or deletions in single-stranded DNA encoding multiple motif sequences. 15. A variety of motif sequences according to claim 14 are obtained by randomly polymerizing to produce an artificial gene population, and translating the artificial gene population to obtain a diverse molecular population independent of frame shifts. Of artificial protein population inserted in

[16] A single-stranded DNA encoding multiple types of motif sequences is polymerized into a random sequence with base mutations and deletions inserted, and an artificial gene population is created, and the artificial gene population is translated to depend on the frame shift. 15. The method for producing an artificial protein population in which various motif sequences are randomly inserted according to claim 14, characterized in that a diverse molecule population is obtained.

[17] An artificial protein population produced by the method according to any one of claims 14 to 16 and into which various motif sequences are randomly inserted.

[18] A functional gene or function for an artificial gene population in which the multiple motif sequences according to claim 13 are randomly polymerized, or an artificial protein population into which various motif sequences according to claim 17 are randomly inserted. A method for creating a functional artificial gene or a functional artificial protein, characterized by screening a sex protein.

1/19

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Surface 1]

Motif Motif B Motif C Motif D Design artificial gene encoding motif sequence

1

Base pair is formed by 3 'end sequence (black square).

Introduced mismatched bases (bold characters) at both ends of the base pair.

5'-CCGCGCTGCA-3 ⁱ

3'-AGCGCGACGC-5 '

I Random polymerization / translation From multiple motif sequences

3 '

¾¾¾¾¾¾¾ Configured library

, 3 '

: Different reading frames

Garden 2]

¾13S5 3 ^¾ HGe 30 kG®S

Dew, 3 G © ¾¾y s¾ So-called

'-I ^¾ -AC≤ieC € ¾T0¾¾ <3M © 0-5 ³

Replacement paper (MA¾¾26) 2/19

WO 2006/011589 PCT / JP2005 / 013913

Picture

Picture

EcoRI recognition sequence: GAATTC '

Hind m recognition sequence: AAGCTT

Bgl II recognition sequence: AGATCT

Xba l recognition: TCTAGA

EHB # 7. EHBX # 2 EHBX # 3

GGGGAATTCGGCGGGTAQAICrcCC GGGGGATTCGGCGGGTAOArCICCC GGGGAATTCGGCGGGTAQATCECCC GGGGAATTCGGCGGGTAAGCTTCCC GGGGAATTCGGCGGGTAQ ECECCC GGGGAATTCGACGGGTAAQATCICCC GGGGAATTCGGCGGGTAQATdCCC GGGGAATTCGGCGGGTAQAIETCCC GGGGAATTCGGCGGGTAAGC7 CCC -GGGAATTCGGCGGGTAAGCTTCCC GGGGAATTCGGCGGGTAAGCT CCC GGGGAATTCGGCGGGTA .GC7TCCC GGGGAATTCGGCGGGTAAGCTTCCC

GGGGAATTCGGCGGGTAG ^ CICCC GGGGAATTCGGCGGGTAGAATCTCCC GGGGAATTCGGCGGGTXCmiACCC GGGGAATTCGGCGGGTAAGC7TCCC GGGGAATTCGGCGGGTIQI ^ GACCC GGGGAATTCGGCCSGGTy GCrrCCC GGGGAATTCGGCGGGTAQAECICCC GGGGAATTCGGCGGGTEglAOACCC GGGGAATTCGGCGGGTICIAGACCC GGGGAATTCGGCGGGTAGAG -GGGAATTCGGCCGGGTTAG ^ ECCC GGGGAATTCGGCGGGTTCIAGACCC

GGGGAATTCGGCGGGTA ^ E ICCC GGGGAATTCGGCGGGTAQ ^ CCXCCCA

EHBX # 1 GGGGCATATTCGGCGGGTI ^ G ^ CCC GGGGAATTCGGCGGGTICIAgACCC

GGGGAATTCGGCGGGTAAGTCT---GGGGAAT CGGCGGGTAi rCICCC

GGGGAATTCAGGCGGGTAAGCT---

GGGGAATTCGGCGGGT ^ AGCT CCC GGGGAATTCGGCGGGTAGAICrCCC

GGGGAATTCGGCGGGTAQArSCCC

CGGGGAATTCGGCGGG

Replacement paper (Rule 26) 3/19

WO 2006/011589 PCT / JP2005 / 13913

[Figure 5]

Design A

~ Base pairing site

圆 6]

· ^^^

1: Artificial gene polymer A

2: Artificial gene polymer B

6/19

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[08c]

A15-gene A15

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELWDF .RRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC LRRFGDKLNKLP

CACC ^ TGCGCGAGCTTGTCGTCGACTT C CCGGCGGGGC HHARACRRLSPA

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC GPAEIRRQAQQA CACCAm GCGAGC G CG CGACT TCTCCGGCGGGGC SPPCASLSSTFS

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC GGACGDSATSSTSF

A16-gene A16

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELWD LRRG CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGGCGGGGCTTG LRRFGDKLNKLPGGC CGCGAGCTTATCGTCGACTTTC CCGGCGGGGC ASLSSTFSGGAC

"-'·' ■ ·-, ■ ':', ΛΛ: [: (-Re: ΛΛ (AAGCTTCCC AKPATNSSFPRA

GCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC CRRLSPAGRALQ

GAGCTCTTCAGGGACGGCGTCAACGAATTCCC- GRRQRIPHHARA

CACCATGCGCGAGC TGTCGTCGACT TCTCCGGCGGGGC CRRLSPAGRERR

GAGAACGGCGGATGGGACACC TCAGATCTCCC MGHLQISPPCAS

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC LSSTFSGGACGD

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC SATSSTSF

Alf gene A17

CACCATGCGCGAGCTT-TCGTCGACTTTCTCCGGCGGGGC MRELSSTFSGGA

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC CGDSATSSTSFPP

-ACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC CASLSSTFSGGAC

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC GDSATSSTSFPT

CACCA GCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MREL DFLRRG

CTGCGGAGATTCGGCGACA¾GCTCAACAAGCTTCCC LRRFGDKLNKLP

A18-gene A18

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MREL DFLRRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC LRRFGDKLNKLP

CACCATGCGCGAGCTTGTCT ^ TCGACTTTCTCCGGCGGGGC HHARACRRLSPA

CTGCGGAGATTCGGCGACAAGCTC7VACAAGCTTCCC GPAEIRRQAQQA

CACCATG GCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC SPPCASLSSTFS

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC GGACGDSATSSTSF

A19-gene A19

CACCA GCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELVV & FLRRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC LRRFGDKLNKLP

CACCATGCGCGAGC TG CGTCGACTTTC CCGGCGGGGC HHARACRRLSPAG CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC PAEIRRQAQQAS

-ACCATGCGCGAGCTTGTCGTCGACTT-CTCCGGCGGGGC HHARACRRLLRR

CTGTGGAGATTCGGCGACAGCTCTCAACAAGCTTCCC GLWRFGDSSQQAS

A20-gene A20

CACCATGCGCGAGCTTGTCGTCGACTTTC CCGGCGGGGC RELVVDFI.RRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGGCGGGGC LRRFGDKLNKLP

r i ^ ノ: :: ;; (: AGGA ''.: AAGGTTCCCGGCGGGGC GGACA PATNSS

ATGCGCGAGCTTGTCGTCGACTT CTCCGGCGGGGC FPAGHARACRRLSPA

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCC GPAEIRRQAQQAS 7/19

WO 2006/011589 PCT / JP2005 / 013913 圆 9]

pcDNA A6

Nuclear £ 卜 Chondria

Alexa-His Merge 卜

*

8/19

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[Figure 10]

B3 B3

CACCATGCGCGAGCTTG CGTCGACT'rTCTCCGGCGGGGC MRELVVD 'LRRG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCCC ELFRDGVNEFPH

CACCA GCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC HARACRRLSPAG

GAGCTCTTCAGGGACGGCGTC7VACGAATTCCC RALQGRRQRIPH

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCG HARACRRLSPAL CTCGAGGTTGAGCT G CO;-\: ^: x.-': CCATGGTG EVELVAESPQHG

B6 B6

CACCATGCTGCGGAGATTCGGCGACAAGCTCAACCTCGAGCGGCGGGGC MLRRFGDKLNLERRG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCC ELFRDGV EFPT

CACCATGCGCGAG TTCTCGTCGACTTTCTCCGGCGGGGC MKELVVDP'I..RRG

GAGC CTTCAGGGACGGCGTCAACGAATTCCC ELFRDGVNEFPT

CACCATGCGCGAGCTTGTCG CGACTTTCTCCGGCGGGGC MRELWDFLRRG

GAGA.ACGGCGGATGGGACACCTTCAGATCTCCC ENGG DTFRSPH

CACCATGCGCGAGCTTGTCG CGAC'r TCTCCGCCG HARACRRLSPPLEVE

CTCGAGGTT ί Γ A CATGGTG LVAESPQHG

Β8 B8

CACCATGCGCGAGCTTGTCGTCGACT CTCCGGCG MREL D.FLRRS

CTCGAGGTTGAGC ^; rTG ::;;: ϊ 0 'AGCATGGTG RLSLSPNLRSMV

Β9 B9

CACCATGCTGCGGAGATTCGGCGACAAGCTCAACCTCGAGCGGCGGGGC MLRRFGDKLNLERRG

GAG ^ CGGCGGATGGGACACCTTCAGATCTCCC ENGGWDTFRSPHHAR

CACCATGCGCGAGCTTGTCGTCGACT TCTCCGGCGGGGC AC RLSPAGRALQGR

GAGC'rCTTCAGGGACGGCGTCAACGAATTCCC RQRIPHHARACRRLS

CACCATGC (X GAGCT'TGTCG'T ^, CGACTT ^; i ¹ C CCGGCGGGGC PAGRALQGRRQRIPH

GAGCTCTTCAGGGACGGCGTCAACGAATTCCC ARACR

CATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC RLSPAGRALQGRRQR GAGCTCTTCAGGGACGGCGTCAACGAATT IHHARACRRLSPALE CACCA GCGCGAGCTTGTCGTCGACT CTCCGGCGC VELVAESPQHG TCGAGGTCC v ^: V: ': A? :; AGCATGGTG

Bl l

Bl l MLRRFGDKLNLERH

CACCCGACAAGCTCAACCTCGAGCGG HAAEIRRQAQPRAA CACCATGCTGCGGAGATTCGGCGACAAGCTCAACCTCGAGCGG PCCGDSATSSTSSGT CACCATGCTGCGGAGATTCGGCGACAAGCTCAACCTCGAGCGG MLRRFGDKL LER CACCATGCTGCGGAGATTCGGCGACAAGCTCAACCTCGAGCGG HHAAEIRRQAQPR CACCATGCTGCGGAGATTCGGCGACAAGCTCAACCTCGAGCGG AAGRERRMGHLQIS CACCATGCTGCGGAGATTCGGCGACAAGCTCAACCTCGAGCGGCGGGGC G AG .¾ CGGCGGATGGGACACCTTCAGATCTCCC 9/19

WO 2006/011589 PCT / JP2005 / 013913 Sono 11]

Design C and D

圆 12]

Design C Design D

Hind III + +

EcoR I ++

Bgl II + +

10/19 O 2006/011589 PCT / JP2005 / 013913 3a]

C2 C2

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELVVDFT.RRG

G.AGAACGGCGGATGGGACACCTTCAGATCTCCCGG ENGG DTFRSPGT

CACCATGCGCGAGCTTGTCGTCGAC C CCGGCGGGGC MRBJLWDFLRRG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCCCGG ELFRDGVNEFPGT

CACCATGCGCGAGCT'T ^; G CGTCGACTTTC CTGGCGGGGC MRELVVDF'LWRG

GAG. ^ CGGCGGATGGGACACCTTCAGATCTCCCG- ENGGWDTFRSPAP

CACCATGCGCGAGCTTGTCGTCGAC T CTCCGGCGGGGC CASLSSTFSGGA

GAGAACGGCGGATGGGACACCTTCAGATCTCCCGG RTADGTPSDLPAP

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC CASLSSTFSGGA

GAGCTCT CAGGGACGGCGTCAACGAATTCCCCGG SSSGTASTNSPAP

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC CASLSSTFSGGA

GAG.¾ACGGCGGATGGGACACCTTCAGATCTCCCGG RTADGTPSDLPAP

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC CASLSSTFSGGA

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG CGDSATSSTSFP

C5 C5

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELWDFLRRG

G-GAACGGCGGATGGGACACCT CAGATCTCCC GTADGTPSDLPT

CACCATGCGCGAGCTTGTCGTCGACT TCTCCGGCGGGGC MRET ^ WDFI-RRG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCCCGG ELFRDGVNEFPGT

CACCATGCGCGAGCT GTCG CGAC TTC CCGGCGGGGC MRELWDFLRRG

GAGAACGGCGGATGGGACACCTTCAGATCTCCCGG ENGGWDTFRSPGT

CACCATGCGCGAGCTTGTCGTCGAC TCTCCGGCGGGGC MRELWDFLRRG

GAGAACGGCGGATGGGACACCTTCAGATCTCCCGG ENGGWDTFRSPGT

CACCATGCGCG-AC: TTGTCGTCGACTTTCTCCGGCGGGGC MRELWDFLRRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG LRRFGDKL KLP

C6 C6

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELWDFLRRG

GAGAACGGCGGATGGGACACCTTCAGATCTCCCGG ENGGWDTFRSPGT

CACCATGC A: GAGCTTGTCGTCGACT TC ^; rCCGGCGGGGC MRELWDFLRRG

GAG ^ CGGCGGATGGGACACCTTCAGATCTCCCGG ENGGWDTFRSPGT

CACCATGCGCGAGCTTGTCGTCGAC CTCCGGCGGGGC MRELWDFLRRG

GAGAACGGCGGA GGGACACC TCAGATCTCCCGG ENGGWD FRSP

CIO CIO

CACCATGCGCGAGCTTG CGTCGACTT CTCCGGCGGGGC MRELWDFLRRG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCCCGG ELFRDGVNEFPGT

CACCATGCGCGAGCTTGTCGTCGACTT'T'C CCGGCGGGGC MRELWDFLRRG

GAGAACGGCGGATGGGACACCTTCAGATCTCCCGG ENGGWDTFRSPGT

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELWDFLRRG

G GAACGGCGGATGGGACACC TCAGATCTCCCGG ENGGWDTFRSPGT

CACCATGCGCGAGCTTCn'CGTCGACTTTCTCCGGCGGGGC MRELWDFLRRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG LRRFGDKLNKLP n //: 6nosooI £ / O 68Ϊ09001AV

[Fix] ¾ΐ 0ν i

. XH vasd.LQV_

a: GVf: SD〇) lv3] _: 5 "":.;>,

[; [

. ¾H ΠΛΛΏΗΗ

Re∞ Re D¾ Re DS¾3: Sr;

: i¾; cQAu [lGvlx, \]),

¾¾08Οοο¾ο3: δΜ: ¾δ¾. .Ι. v. , εν-: ¾o, co¾ys

DOOO Hi DOOOD. VD,,: vo

D0¾0¾o¾u: Y-]

Ds_LdHLai¾-, f] [o. ¾v. "¾. ,; 12/19 O 2006/011589 PCT / JP2005 / 013913 4a]

D4 D4

CACCATGCGCGAGC TGTCGTCGAC T CTCCGGCGGGGC HRELWDFL RG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCC ELFRDGVNEFPT

CACCATGCGCGAGCTTG CGTCGACTTTCTCCGGCGGGGC MRELVW '-LRRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG LRRFGDKLNKLP GCGCGAGCT CTCGTCGAC'IT C CCGGCGGGGC GREi— 'i LRRG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCC ELFRDGVNEFPT

CACCATGCGCGAGCTTG'rCGTCGACTTTCTCCGGCGGGGC MRELWDFLRRG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCG LRRFGDKLNKLP

CACCATGCGCGAGCTTGTCGTCGACTTTC CCGGCGGGGC APCASLSSTFSG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCCC GASSSGTASTNS

D5 D5

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC MRELWDFI ^ RRG

GAGCTCTTCAGGGACGGCGTCAACGAATTCCCCGGA ELFRDGVNEFP

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC GHHARACRRLSP

GAGA¾CGGCGGATGGGACACCTTCAGATCTCCCGG AGRERRMGHLQ

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC ISRHHARACRRL

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG SPAGPAEIRRQ

CACCATGCGCGAGC GTCGTCGACTT CTCCGGCGGGGC AQQASRHHARAC

GAGC CTTCAGGGACGGCGTCAACGAATTCCC RRLSPAGRALQ

CACCATGC (; CGAGCTTGTCGTCGACTTTCTCCGGCGGGGC GRRQRIPHHARA

GAGAACGGCGGATGGGACACCTTCAGATCTCC CR LSPAGRER

CACCATGCGCGAGCTTGTCGTCGACTTTCTCCGGCGGGGC RMGHLQISHHAR

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG ACRRLSPAGPA

EIRRQAQQASR

D6

CACCATGCGCC ^ AGCTTGTCGTCGACTTTCTCCGGCGGGGC D6

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG MRELWDFLRRG

CACCATGCGCGAGCT GTCGTCGACT TCTCCGGCGGGGC LRRFGDKLNKLPGT

GAGAACGGCGGATGGGACACCTTCAGATCTCCCGG MRELWDFT, RRG

CACCATGCGCGAGCTTGTCG CGACTTTCTCCGGCGGGGC ENGGWDTFRSPGT

GAGCTCTT-AGGGACGGCGTCAACGAATTCCCC MRELVVDFLjmG

GGGCGCGAGCTTGTCGTCGACTTTC CCGGCGGGGC ELLGTAST SPG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG ASLSSTFSGGAC

GDSATSSTSFP

Dl l

CACCATGCGCGAGCTTG CGTCGACTTTC CCGGCGGGGC Dl l

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG MRELWDFLRRG

CACCATGCGCGA.GCTTG CGTCGACTTTCTCCGGCGGGGC LRRFGDKLNKLPGT

GAGCTCTTCAGGGACGGCGTCAACGAATTCCCCGG MRELWDFLRRG

CACCATGCGCG C T ^ X rCG C ^ ¾C TTCT CGGCGGGGC ELFRDGV EFPGT

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG MRELVVD LRRG GCGCGAGCTTGTCGTCGACT TCTCCGGCGGGGC LRRFGDKLNKLPG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG-ELVVDFIi¾RG

LRRFGDKLNKLP

LWLRHG.PF

CACCMrrcT Q8 IHRAASR Q8 PAEIHRAAS 14/19

WO 2006/011589 PCT / JP2005 / 013913

[Figure 14c]

D21 D21

CACCA GCGCGAGCTTGTCG ^r rCG.ACT rC CCGGCGGGGC M ELWDFLRRG

CTGCGGAGATTCGGCGACA AACAAGCT CCCGG LRRFGDKQASR

CACC ^ TGCGCGAGCT G CGTCGAC TTC -CCGGCGGGGC HHARACRRLSPA

GACiAACGGCGGATGGGACACCTTCAGATCTCCCGG GRERRMGHLQISR

CACCATGCGCGAGCTTGT'CG'rCGACTTTCTCCGGCGGGGC HHARACRRLSPA

CTC ^ GGAGA TCGGCGACAAGCTCAACAAGC TCCCGG GPAEIRRQAQQASR

D23 D23

CACCA GCGCGAGCTTGTCG CGACTTTC CCGGCGGGGC MRE WDFLRRG

CT∞GGAGAT CGGCGACAAGCTCAACAAGCTTCCCGG LRRFGDKL KLPGT

Ci ^ CA GCGCGAGCT G CGTCGAC'T TCTCCGGCGGGGC MRELVV FLJ RG

GAGCTCT CAGGGACGGCGTCAACGAATTCCC ELFRDGVNEFPT

CACCATGCGCGAGCTTGTCGTCGACT TCTCCGGCGGGGC ΜΗΓ and WifLRRG

GAGAACGGCGGATGGGACACCTTCAGATCTCCCGG E GG FRSPGT

CACCATGCGi: GAGCT'rGTCGTCGAC'X'TTCTCCGGCGGGGC MRELWOriJ ^ RG

CTGCGGAGATTCGGCGACAAGC CAACAAGCTTCCCGG LR FGDKLNKLP

D26 D26

CACCATGCGCGAGCTTGTCG CGAC 'rTCTCCGGCGGGGC MREL DFLRRG

GAGAACGGCGGATGGGACACCT CAGATCTCCC E GGV¾) FRSPHH

CACCATGCGCGAGC TG CGTCGACTTTCTCCGGCGGGGC ARACRRLSPAGR

'.:.' .: C] T :; ''; ;;,.;

CACCATGCGCGAGCTl¾ CG CGACT'rTCrCCGGCGGGGC ARACRRLSPAGR

GAGC / CT CAGGGACGGCGTCAACGAATTCCCCGG ALQGRRQRIPRPC

—— CCATGCGCGAGCT G CGTCGAC TTCTCCGGCGGGGC ASLSSTFSGGAS

GAGCTC'I ^{: r} rCAGGGACGGCGTCAACGAA CCCCGG SSGTASTNSPAPC

CACCATGCGCGAGCTTGTCG CGACTTTC CGGCGGGGC ASLSSTFSGGAR

GAGAACGGCGGA GGGACACCTTCAGATCTCC TA GTPSDLPPCA

CACCATGCGCGAGCTTf ^ CGTCGACTT C CCGGCGGGGC SLSSTFSGGACG

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG DSATSSTSFP

D2 D29

CACCATGCGCGAGCTTG CGTCGACTL'TCL'CCGGCGGGGC MRELxA / DF Jt G

C GCGGAGATTCGGCGACAAGCTCAACAAGCT CCCGG LRRFGDKLNKLPGT

CACCA GCI:; CGAGCTTGTCG CGACTT ^; RCTCCGGCGGGGC MRE: LVVDFiJ¾RG

CTGCGGAGATTCGGCGACAAGCTCAACAAGC TCCCGG LRRFGDKLNKLPGT

^{CACCATGCGC: GAGCTTGTCGTCGA.CTT C:: RCCGGCGGGGC M} ELVV F1.JIRG

GAGCTCT CAGGGACGGCGTCAACGAA TCCCCGG ELFRDGWEFPGT

CACCATGCGCGAGCTTGTCGTCGAC TTCTCCGGCGGGGC M LVV ^ FLJ¾RG

GAGCTC CAGGGACGGCGTCAACGAATTCCCCGGC ELPRDGV EFPGHH

CACCATGCGCI ^ AGCTTGT-GTCG-"-~ TC C CCGGCGGGCC ARACWSPAGPA

CTGCGGAGATTCGGCGACAAGCTCAACAAGCT CCCGGCCCC EIRRQAQC¾ASRPPP

CACC ^ TO; GCGAGCT GTCGTCGA.C T CTCCGGCGGGGC CASLSSTFSGGA

GAGAACGGCGGATGGGACACC TCAGATCTCCCGG TAIX3TPSDLPAP

CACCA G: GCGAGC ^: R ^; I ^: GTCGTCGAC ^; I ^: TTCTCCGGCGGGGC CASLSSTFSGGA

CTGCGGAGATTCGGCGACAAGCTCAACAAGCTTCCCGG CGDSATSSTSFP 15/19

Nuclear core

16/19

WO 2006 Thigh 589 PCT / JP2005 / 013913

[Figure 17]

[Figure 18]

Myc TUNEL I Merge

17/19

O 2006/011589 PCT / JP2005 / 013913 0 πί

pcDNA Bcl-xL _a 10 dl6

pcDNA Bcl-xL a 10 dl6 18/19 006/011589 PCT / JP2005 / 013913

++ SSTTSS (125 nM)

ccoonntr hi Il pcDNA Bc and aio riifi ^

19/19 O 2006/011589

]

+ STS (l25nM)

pcD A

-d2B