WO2011010583A1 - Method for screening for oligonucleotide, and oligonucleotide library - Google Patents

Abstract

Disclosed is a method for screening for an oligonucleotide, which can be employed for highly efficiently obtaining an oligonucleotide having biological or chemical activities including an activity of inhibiting the expression of a target gene. Specifically disclosed is a method for screening for an oligonucleotide, which comprises: a step S₂ of preparing at least two oligonucleotide pools; and a step S₃ of identifying an oligonucleotide pool that comprises an oligonucleotide having a biological or chemical activity among from the oligonucleotide pools. In the step S₂, each of the oligonucleotide pools comprises an oligonucleotide composed of a 7-mer base sequence comprising a nucleic acid analogue and one or two components which are bound to the base sequence and are independently selected from a nucleic acid analogue and a nucleic acid, wherein the nucleic acid analogue is selected from adenine analogues, guanine analogues, thymine analogues and cytosine analogues and the nucleic acid is selected from adenine, guanine, thymine and cytosine, and wherein base sequences of the oligonucleotide pools are different from each other.

Description

Oligonucleotide screening method and oligonucleotide library

The present invention relates to an oligonucleotide screening method and an oligonucleotide library. More specifically, the present invention relates to an oligonucleotide screening method for obtaining a target oligonucleotide exhibiting a desired biological activity or chemical activity, an oligonucleotide library used in the screening method, and the like.

Oligonucleotides in which several to several tens of nucleotides are arranged in a chain by phosphodiester bonds via phosphoric acid are formed between base pairs with respect to a nucleotide chain having a complementary sequence. Are bonded by hydrogen bonds. By utilizing this characteristic, oligonucleotides have been widely used as research reagents and gene diagnostic reagents.

Oligonucleotides as research reagents are used, for example, as primers for polymerase chain reaction (PCR) and used to amplify specific gene sequences. Alternatively, it is also used as a probe for detecting the amplified gene sequence. Moreover, as a genetic diagnostic reagent, it is used as a probe immobilized on a DNA chip or a DNA array.

In recent years, oligonucleotides have been developed as pharmaceuticals called nucleic acid pharmaceuticals. As an example, an antisense method that selectively inhibits translation of a sense strand into a protein by introducing an oligonucleotide (antisense oligonucleotide) complementary to a partial sequence of mRNA (sense strand) of a target gene into a cell. There is. In the antisense method, by binding an antisense oligonucleotide to the mRNA of the target gene and inhibiting the binding of the translation factor complex to the mRNA, the translation of the mRNA into the protein is inhibited and the expression of the target gene product is suppressed. can do.

As an example of an oligonucleotide as a nucleic acid drug, “decoy oligonucleotide” that binds to a specific transcription factor as “bait” and inhibits the function of the transcription factor can also be mentioned. Transcription factors bind to transcriptional control regions such as promoters and enhancers on the genome to control transcription of genes into mRNA. The decoy oligonucleotide binds to this transcription factor as a decoy and competitively inhibits the transcription factor from binding to the transcription control region, thereby inhibiting the transcription of the target gene mRNA and suppressing the expression of the target gene product.

Here, “locked nucleic acid (LNA)” is described in relation to the present invention. LNA is also referred to as “bridged nucleic acid (BNA: Bridged Nucleic Acid)”. In the present invention, LNA and BNA are used synonymously.

Nucleosides in natural nucleic acids (DNA and RNA) have two conformations, N-type and S-type. Because of this “fluctuation” between conformations, the double strands formed between DNA-DNA, RNA-RNA strands, and DNA-RNA are not necessarily thermodynamically stable.

2'-O, 4'-C-methano-bridged nucleic acid (2 ', 4'-BNA) bridges the 2' and 4 'positions of ribose (sugar) with "-O-CH2-" It is an artificial nucleic acid whose conformation is fixed to N-type. Since 2 ', 4'-BNA has no fluctuation between conformations, oligonucleotides synthesized with several units of 2', 4'-BNA are compared to oligonucleotides synthesized with conventional natural nucleic acids. It has extremely high binding power and sequence specificity for RNA and DNA, and exhibits excellent heat resistance and nuclease resistance. So far, about 10 types of LNAs have been developed in addition to 2 ′, 4′-BNA (see Patent Documents 1 to 4).

Special Table 2002-521310 Japanese Patent Laid-Open No. 10-304889 JP 10-195098 A International Publication No. 2005/021570

In order to obtain an antisense oligonucleotide or a decoy oligonucleotide that functions as a nucleic acid drug, it is necessary to synthesize an oligonucleotide having a sequence complementary to a binding region of a translation factor complex or a binding region of a transcription factor. Therefore, the antisense method and the decoy method target only the genes for which the translation factor complex and transcription factor binding region sequences (hereinafter also referred to as “target sequences”) have been clarified. Cannot target unknown genes.

Furthermore, even an oligonucleotide synthesized as a sequence complementary to the binding region of a translation factor complex or a transcription factor may not necessarily show the expected activity. Therefore, for example, in the antisense method, a plurality of oligonucleotides are synthesized for one translation factor complex-binding region on the target gene mRNA, or a large number of translation factor complex-binding regions on the mRNA are synthesized. It is necessary to synthesize oligonucleotides to find oligonucleotides exhibiting a desired activity by screening.

Such restrictions on the target gene and the cost for designing and screening a plurality of oligonucleotides for each target gene are major obstacles in the development of nucleic acid drugs.

Therefore, the main object of the present invention is to provide a method for screening an oligonucleotide for efficiently obtaining an oligonucleotide exhibiting biological activity or chemical activity such as target gene expression-suppressing activity.

In order to solve the above problems, the present invention provides a 7mer basic sequence comprising any nucleic acid analog, any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or adenine, guanine, Prepare two or more oligonucleotide pools composed of oligonucleotides in which one or two nucleic acids selected from thymine or cytosine are bound so that the basic sequences differ between the oligonucleotide pools. An oligonucleotide screening method comprising: a procedure; and a procedure for identifying an oligonucleotide pool containing a target oligonucleotide from the oligonucleotide pool.
The screening method may further comprise a procedure for obtaining the target oligonucleotide from the identified oligonucleotide pool.
In this screening method, when an oligonucleotide pool is prepared by binding one nucleic acid analog or nucleic acid to a basic sequence, an adenine analog, guanine analog, thymine is added to either the 5 ′ end or the 3 ′ end of the basic sequence. Two oligonucleotide pools composed of four kinds of oligonucleotides, each of which binds any one nucleic acid analog selected from analogs or cytosine analogs, or any one nucleic acid selected from adenine, guanine, thymine, or cytosine, respectively. Prepare as above.
In this screening method, when preparing an oligonucleotide pool by binding two nucleic acid analogs or nucleic acids to a basic sequence, for example, an adenine analog, a guanine analog, An oligonucleotide pool consisting of 16 types of oligonucleotides, each of which binds any one nucleic acid analog selected from thymine analogs or cytosine analogs, or any one nucleic acid selected from adenine, guanine, thymine or cytosine Prepare two or more.
This screening method preferably includes a procedure for preparing 16384 (4 7) basic sequences consisting of all combinations of seven nucleic acid analogs. By preparing 16384 oligonucleotide pools using 16384 basic sequences, it is theoretically possible to prepare oligonucleotides that can specifically bind to nucleotide chains of any sequence.
In the screening method according to the present invention, for example, in the presence and absence of an oligonucleotide pool, the mRNA is contacted with an mRNA binding protein that binds to the mRNA, and the amount of binding between the mRNA and the mRNA binding protein is measured. Thus, it is possible to identify an oligonucleotide pool containing oligonucleotides that exhibit an activity of inhibiting the binding between mRNA and mRNA-binding protein.
In addition, for example, by measuring the expression level of a protein in a cell into which an oligonucleotide pool has been introduced and a cell into which the oligonucleotide pool has not been introduced, an oligonucleotide pool containing an oligonucleotide exhibiting an activity of enhancing the expression of the protein can be identified it can.

In addition, the present invention is an oligonucleotide library provided for the above-described oligonucleotide screening method, wherein a 7mer basic sequence comprising any nucleic acid analog is added to an adenine analog, guanine analog, thymine analog or cytosine analog. It is composed of two or more oligonucleotide pools composed of one or two oligonucleotides bound to any one nucleic acid analog selected, or any one nucleic acid selected from adenine, guanine, thymine, or cytosine. The present invention also provides an oligonucleotide library in which the basic sequences are different between the oligonucleotide pools.
This oligonucleotide library preferably has 16384 basic sequences consisting of all combinations of seven nucleic acid analogs.

Furthermore, the present invention also provides an oligonucleotide exhibiting an activity that inhibits the binding between tumor necrosis factor α mRNA and RC3H1, and an oligonucleotide exhibiting an activity that enhances the expression of a low density lipoprotein receptor.

Here, the definition of terms in the present invention will be described.

A “nucleic acid analog” is an artificial nucleic acid obtained by artificially modifying the chemical structure of a ribose or phosphodiester bond of a natural nucleic acid (DNA and RNA), and includes at least one in the oligonucleotide sequence. By being included, the binding affinity and sequence specificity for the complementary strand of the oligonucleotide can be increased as compared to the oligonucleotide consisting only of the natural nucleic acid. Specifically, the “nucleic acid analog” obtained by modifying the chemical structure of ribose includes at least the above-described bridged nucleic acid (BNA) or locked nucleic acid (LNA). As LNA, 2'-O, 4'-C-methano-bridged nucleic acid (2 ', 4'-BNA) and 3', 4'-BNA, 3'-amino- Conventionally known LNA such as 2 ′, 4′-BNA and 5′-amino-3 ′, 4′-BNA are included. “Nucleic acid analogs” in which the chemical structure of the phosphodiester bond is modified include phosphorothioate-type artificial nucleic acids (S-oligo) in which the oxygen atom of the phosphate group is replaced with a sulfur atom.

The present invention provides an oligonucleotide screening method for efficiently obtaining a target oligonucleotide exhibiting biological activity or chemical activity such as target gene expression suppression activity.

It is a figure explaining the oligonucleotide library which concerns on 1st embodiment of this invention. It is a figure explaining the oligonucleotide library which concerns on 2nd embodiment of this invention. It is a flowchart explaining the procedure of the screening method of the oligonucleotide which concerns on this invention. It is a figure which shows the result of the Western blot which identified the oligonucleotide pool which shows the binding inhibitory activity of TNF- (alpha) * mRNA and RC3H1 (Example 1). It is a figure which shows the result of the Western blot which evaluated the LDLR expression increase activity of an oligonucleotide pool (Example 2). It is a figure which shows the result of the Western blot which evaluated the binding inhibitory activity of oligonucleotide of ZFP36L1 and LDLR (TM) mRNA (Example 3).

In general, the binding affinity and sequence specificity for the complementary strand of an oligonucleotide decreases as the length of the oligonucleotide decreases. Conventional oligonucleotides for research, genetic diagnostic reagents, and nucleic acid pharmaceuticals usually have a binding affinity and sequence specificity for complementary strands of 10 to 40 mer in normal cases, and in most cases around 20 mer. It is said to be long. Since the length of the oligonucleotide is short, the desired activity of the oligonucleotide cannot be obtained unless sufficient binding affinity and sequence specificity for the complementary strand are exhibited.

On the other hand, the longer the length of the oligonucleotide, the higher the cost for synthesis. Moreover, when the length of the oligonucleotide becomes too long, nonspecific binding to a nucleotide chain having a partially complementary sequence (non-complementary chain) occurs. Therefore, it is desirable that the oligonucleotide be synthesized as short as possible on condition that sufficient binding affinity and sequence specificity for the complementary strand are exhibited.

Furthermore, when trying to construct a library of oligonucleotides, the increase in synthesis cost due to the length of the oligonucleotide requires the synthesis of four types of oligonucleotides for every 1 mer. It will be functional. Therefore, in order to construct an oligonucleotide library at an economically feasible cost, it is desirable that the length of the oligonucleotide be as short as possible.

As a result of studying the minimum oligonucleotide length for obtaining an oligonucleotide exhibiting biological activity or chemical activity, the present inventors added a 1-mer or 2-mer nucleic acid analog or nucleic acid to a nucleic acid analog 7mer. It was found that a total of 8 mer or 9 mer oligonucleotides synthesized in this manner exhibit necessary and sufficient binding affinity and sequence specificity and can express a desired biological activity or chemical activity.

The present invention has been completed by the inventors based on this finding, and uses an oligonucleotide library for efficiently obtaining a target oligonucleotide exhibiting a desired biological activity or chemical activity, and this library. The present invention provides a screening method for oligonucleotides.

Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

1. Oligonucleotide Library The oligonucleotide library according to the present invention is composed of two or more oligonucleotide pools. Each oligonucleotide pool has a 7-mer “basic sequence” consisting of any nucleic acid analog, any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or adenine, guanine, thymine or It consists of an oligonucleotide in which one or two nucleic acids selected from cytosine are linked.

(1-1) 8-mer oligonucleotide library FIG. 1 schematically shows an oligonucleotide library according to the first embodiment of the present invention. The figure shows an oligonucleotide library L in which each oligonucleotide pool is composed of oligonucleotides in which one nucleic acid analog or nucleic acid is linked to a 7mer basic sequence.

In the figure, “A” represents an adenine analog, “G” represents a guanine analog, “T” thymine analog, “C” represents a cytosine analog, “a” represents adenine, “g” represents guanine, “t” thymine, “ “c” represents cytosine. "N" represents any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog, or cytosine analog, and "n" represents any one nucleic acid selected from adenine, guanine, thymine, or cytosine. Represents.

Oligonucleotide library L is composed of a total of K types of oligonucleotide libraries from pool 1 to pool K.

Pool 1 includes four types of oligonucleotides 11, 12, 13, and 14. Oligonucleotides 11, 12, 13, and 14 have a basic sequence 1 represented by “AGTCAGGT” in common, and have a sequence in which A, G, T, and C are linked to the 3 ′ end of the basic sequence 1, respectively. ing. In the pool 1, it is desirable that the oligonucleotides 11, 12, 13, and 14 are contained in the same number of moles, but the concentration of each oligonucleotide is not particularly limited. Hereinafter, the same applies to the pool 2, the pool 3, and the pool K.

Pool 2 includes four types of oligonucleotides 21, 22, 23, and 24, and oligonucleotides 21, 22, 23, and 24 have a common basic sequence 2 represented by “AGTGGAGT”. The basic sequence 2 has a sequence in which A, G, T, and C are bonded to the 3 ′ end. The basic sequence 1 of the oligonucleotides 11, 12, 13, and 14 included in the pool 1 and the basic sequence 2 of the oligonucleotides 21, 22, 23, and 24 included in the pool 2 include the fourth nucleic acid analog from the 5 ′ end. “C” and “G” are different.

Pool 3 includes four types of oligonucleotides 31, 32, 33, and 34, and oligonucleotides 31, 32, 33, and 34 have a basic sequence 3 represented by “AGTTAGT” in common. The basic sequence 3 has a sequence in which A, G, T, and C are bonded to the 3 ′ end. The basic sequence 1 of the oligonucleotides 11, 12, 13, and 14 included in the pool 1 and the basic sequence 3 of the oligonucleotides 31, 32, 33, and 34 included in the pool 3 include the fourth nucleic acid analog from the 5 ′ end. “C” and “T” are different. The basic sequence 2 of the oligonucleotides 21, 22, 23, and 24 included in the pool 2 and the basic sequence 3 of the oligonucleotides 31, 32, 33, and 34 included in the pool 3 are the fourth nucleic acid from the 5 ′ end. The analogs are different for “G” and “T”, respectively.

The pool K (K is an integer of 2 to 16,384) includes four types of oligonucleotides K1, K2, K3, and K4. The oligonucleotides K1, K2, K3, and K4 are indicated by “NNNNNNNN”. The basic sequence K is commonly used, and A, G, T, and C are connected to the 3 ′ end of the basic sequence K, respectively. The basic sequences K of the oligonucleotides K1, K2, K3, and K4 included in the pool K are different from the basic sequences 1 to K-1 of the oligonucleotides included in the pool 1 to the pool (K-1).

As described above, the oligonucleotide library L includes two or more oligonucleotide pools composed of four types of oligonucleotides in which a nucleic acid analog of A, G, T, or C is bound to a basic sequence that is different between pools. Has been.

FIG. 1 illustrates the case where the oligonucleotides K1, K2, K3, and K4 constituting the oligonucleotide pool K are sequences in which A, G, T, and C are bonded to the 3 ′ end of the basic sequence K, respectively. Each nucleic acid analog may be bound to either the 3 ′ end or the 5 ′ end of the basic sequence K, and the oligonucleotides K1, K2, K3, and K4 are respectively A and G at the 5 ′ end of the basic sequence K. , T, and C may be combined.

FIG. 1 illustrates the case where oligonucleotides K1, K2, K3, and K4 constituting the oligonucleotide pool K are sequences in which the nucleic acid analogs A, G, T, and C are bonded to the ends of the basic sequence K, respectively. did. Either the nucleic acid analog or the nucleic acid may be bound to the end of the basic sequence. The oligonucleotides K1, K2, K3, and K4 have the nucleic acids a, g, t, and c at the ends of the basic sequence K, respectively. A combined sequence may also be used.

(1-2) 9-mer oligonucleotide library FIG. 2 schematically shows an oligonucleotide library according to the second embodiment of the present invention. The figure shows an oligonucleotide library L in which each oligonucleotide pool is composed of oligonucleotides in which two nucleic acid analogs or two nucleic acids are linked to a 7mer basic sequence.

Oligonucleotide library L is composed of a total of K types of oligonucleotide libraries from pool 1 to pool K.

Pool 1 contains 16 types of oligonucleotides 101-116. Oligonucleotides 101 to 116 have a basic sequence 1 represented by “AGTCCAGT” in common, and any one of A, G, T, and C is linked to the 5 ′ end and the 3 ′ end of this basic sequence 1, respectively. It is an array. In the pool 1, it is desirable that the oligonucleotides 101 to 116 are contained in the same number of moles, but the concentration of each oligonucleotide is not particularly limited. Hereinafter, the same applies to the pool 2, the pool 3, and the pool K.

The pool 2 includes 16 types of oligonucleotides 201 to 216, and the oligonucleotides 201 to 216 have the basic sequence 2 indicated by “AGTGAGT” in common, and 5 ′ of the basic sequence 2 It is set as the arrangement | sequence which combined either A, G, T, or C to the terminal and 3 'terminal. In the basic sequence 1 of the oligonucleotides 101 to 116 included in the pool 1 and the basic sequence 2 of the oligonucleotides 201 to 216 included in the pool 2, the fourth nucleic acid analog from the 5 ′ end is “C” and “G”, respectively. And is different.

The pool 3 includes 16 types of oligonucleotides 301 to 316, and the oligonucleotides 301 to 316 have a basic sequence 3 represented by “AGTTAGT” in common. It is set as the arrangement | sequence which combined either A, G, T, or C to the terminal and 3 'terminal. In the basic sequence 1 of the oligonucleotides 101 to 116 included in the pool 1 and the basic sequence 3 of the oligonucleotides 301 to 316 included in the pool 3, the fourth nucleic acid analogs from the 5 ′ end are “C” and “T”, respectively. And is different. In addition, in the basic sequence 2 of the oligonucleotides 201 to 216 included in the pool 2 and the basic sequence 3 of the oligonucleotides 301 to 316 included in the pool 3, the fourth nucleic acid analog from the 5 ′ end is “G” and “ T ”.

The pool K (K is an integer of 2 to 16384) includes 16 types of oligonucleotides K01 to K16, and the oligonucleotides K01 to K16 share the basic sequence K represented by “NNNNNNNN”. The basic sequence K has a sequence in which any one of A, G, T, and C is linked to the 5 ′ end and 3 ′ end. The basic sequences K of the oligonucleotides K01 to K16 included in the pool K are different from the basic sequences 1 to K-1 of the oligonucleotides included in the pools 1 to (K-1).

As described above, the oligonucleotide library L includes two or more oligonucleotide pools composed of 16 kinds of oligonucleotides in which a nucleic acid analog of A, G, T, or C is bound to a basic sequence that is different between pools. Has been.

In FIG. 2, the oligonucleotides K01 to K16 constituting the oligonucleotide pool K are divided into one of A, G, T, C at the 5 ′ end of the basic sequence K, and A, G, T, at the 3 ′ end. The case where any one of Cs is a sequence in which one is combined has been described. Two nucleic acid analogs may be bound to the 5 ′ end and / or 3 ′ end of the basic sequence K, and both nucleic acid analogs are bound to the 5 ′ end or the 3 ′ end to form 16 types of oligonucleotides. Also good.

FIG. 2 illustrates the case where the oligonucleotides K01 to K16 constituting the oligonucleotide pool K have a sequence in which two nucleic acid analogs A, G, T, and C are bonded to the ends of the basic sequence K, respectively. Either the nucleic acid analog or the nucleic acid may be bound to the end of the basic sequence. The oligonucleotides K1, K2, K3, and K4 have the nucleic acids a, g, t, and c at the ends of the basic sequence K, respectively. Two sequences may be combined, or one nucleic acid analog and one nucleic acid may be combined.

(1-3) Number of oligonucleotide pools The oligonucleotide library L shown in FIG. 1 and FIG. 2 contains A, G, T, or C nucleic acid analogs or nucleic acids in a 7mer basic sequence that is different between pools. It comprises two or more oligonucleotide pools consisting of linked 8mer or 9mer oligonucleotides. As described above, a total of 8mer or 9mer oligonucleotide synthesized by adding 1mer or 2mer nucleic acid analog or nucleic acid to nucleic acid analog 7mer such as 2 ', 4'-BNA has the necessary and sufficient binding affinity and It has been shown that it can exhibit sequence specificity and express a desired biological or chemical activity.

Therefore, when the oligonucleotide library L is composed of 16384 (4 to the 7th power) oligonucleotide pools, which are all combinations of nucleic acid analogs of the basic sequence 7mer, it binds specifically to nucleotide chains of any sequence. An oligonucleotide library can be constructed such that possible 8mer or 9mer oligonucleotides are always included in either oligonucleotide pool.

2. Oligonucleotide Screening Method Next, an oligonucleotide screening method according to the present invention will be described. In this screening method, an oligonucleotide exhibiting a desired biological activity or chemical activity is obtained using the oligonucleotide library L described above.

FIG. 3 is a flowchart showing the procedure of the oligonucleotide screening method according to the present invention.

(2-1) Synthesis of Basic Sequence In FIG. 3, symbol S ₁ is a procedure for preparing a 7mer basic sequence consisting of an arbitrary nucleic acid analog.

The basic sequence can be synthesized by combining nucleic acid analogs using a conventional known method. The basic sequence is synthesized using, for example, a known DNA synthesizer. The synthesized basic sequence can be confirmed by purifying using a reverse phase column and then analyzing by reverse phase HPLC or MALDI-TOF-MS. In addition, the basic sequence can also be obtained by using a custom oligonucleotide synthesis service.

Two or more basic arrays are prepared as different arrays. Preferably, the base sequence is prepared as 16384 (4 to the 7th power) of all combinations of 7-mer nucleic acid analogs. In Step S ₂ described below, using the basic sequence of 16384 ways, by preparing an oligonucleotide pool 16384 types, theoretically, an oligonucleotide capable of specifically binding to the nucleotide strand of any sequence Can be prepared.

(2-2) in Preparation view third oligonucleotide pool, the letter S ₂ designates each basic sequence prepared in Step S ₁ one nucleic acid analogs or nucleic acid, or two bonds to, the procedure for preparing oligonucleotide pool It is.

Specifically, when the oligonucleotide library L is constituted by oligonucleotides in which one nucleic acid analog or nucleic acid is bound to a 7mer basic sequence, for example, oligonucleotides included in pool 1 to pool (K-1) A, G, T, and C are respectively linked to the 3 ′ end of the basic sequence K “NNNNNNNN” that is different from the basic sequences 1 to K-1. Thereby, four types of oligonucleotides K1, K2, K3, and K4 are synthesized, and an oligonucleotide pool K (K is an integer of 2 to 16384) is prepared (see FIG. 1).

When the oligonucleotide library L is composed of oligonucleotides in which two nucleic acid analogs or two nucleic acids are linked to a 7mer basic sequence, for example, the basic sequence 1 of oligonucleotides contained in pool 1 to pool (K-1) A, G, T, and C are respectively linked to the 5 ′ end and 3 ′ end of the basic sequence K “NNNNNNNN” that is different from the sequence of K−1. Thus, 16 types of oligonucleotides K01 to K16 are synthesized, and an oligonucleotide pool K (K is an integer of 2 to 16384) is prepared (see FIG. 2).

Oligonucleotide pools are prepared as two or more pools based on two or more basic sequences having different sequences. Preferably, the oligonucleotide pool is prepared as 16384 (4 to the 7th power) pools of all combinations of 7mer nucleic acid analogs. By preparing 16384 oligonucleotide pools, it is possible to prepare an oligonucleotide library L that always contains oligonucleotides that can specifically bind to nucleotide chains of any sequence.

(2-3) Identification of oligonucleotide pool In FIG. 3, reference numeral S ₃ denotes an oligonucleotide containing an oligonucleotide exhibiting a desired biological activity or chemical activity from among the oligonucleotide pools constituting the oligonucleotide library L. Procedure for identifying nucleotide pools.

Each oligonucleotide pool contains 4 types or 16 types of oligonucleotides. In this procedure, each of the oligonucleotide pools is subjected to various assays in the presence and absence thereof to identify an oligonucleotide pool containing the oligonucleotide exhibiting the desired activity (target oligonucleotide pool).

Here, the “biological activity or chemical activity” of an oligonucleotide means the amount of organism that can be quantified by experiment, and specifically means the activity of the following oligonucleotide, for example.

(1) Antisense activity Activity that inhibits the binding of the translation factor complex to the mRNA by inhibiting the binding of the translation factor complex to the mRNA by binding to the mRNA of the target gene in a complementary manner. Activity that inhibits translation into protein and suppresses expression of target gene product.

This activity inhibits the binding of mRNA binding proteins other than translation factor complexes to mRNA, or by inhibiting the binding of mRNA binding proteins to mRNA, resulting in decreased or increased expression of the target gene product. It may be an activity. One example is the activity of stabilizing the mRNA and increasing the expression of the target gene product by inhibiting the binding of the cis element binding factor to the mRNA. Furthermore, this antisense activity may be an activity that binds complementarily to non-coding RNA such as micro-RNA (miRNA) and inhibits or enhances its function.

The following briefly describes the “cis element binding factor”. “Cis element binding factors” bind to specific sequences called “cis-elements” present in the 5 ′ and 3 ′ untranslated regions of DNA and RNA. A cis element is involved in regulation of the expression of a gene encoded by its DNA strand or RNA strand. A cis-element binding factor functions as a “trans-acting factor” that binds to a cis-element and positively or negatively controls gene expression.

The cis element present in mRNA and the cis element binding factor that binds to this cis element mainly negatively regulates the stability of mRNA and the amount of translation into protein (“AU-rich elements and associated factors: are there unifying principles? ”Nucleic Acids Research, 2005, Vol.33, No.22, p.7138-7150,“ AU-rich element-mediated translational control: complexity and multiple activities of trans-activating factors. ”Biochemical Society Transactions, 2002, Vol.30, part 6, p.952-958.) One of the typical mRNA cis elements is “AU-rich element (AU-Rich Element; ARE)”. ARE is a base sequence of about 10 to 150 bps rich in adenosine and uridine, and is abundant in the 3 ′ untranslated region (3 ′ UTR) of mRNA. ARE was initially found as a region where the nucleotide sequence of “AUUUA” frequently overlaps in the 3 ′ UTR of cytokines and lymphokines. ARE is currently estimated to be present in 5-8% of all genes, and ARE is considered to be present in many genes involved in the maintenance of homeostasis (“ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. ”Nucleic Acids Research, 2001, Vol.29, No.1, p.246-254.).

(2) Decoy activity Binding to a transcription factor as a decoy, inhibiting the transcription factor from binding to the transcriptional regulatory region on the genome, or inhibiting the transcription factor from binding to the transcriptional regulatory region on the genome. Inhibits the transcription of the target gene mRNA and suppresses the expression of the target gene product. TATA boxes, CAT boxes, Sp1 and other transcriptional regulatory regions present on the genome are cis elements present in DNA.

This activity binds to DNA-binding proteins or RNA-binding proteins other than transcription factors as decoys, or binds to DNA-binding proteins or RNA-binding proteins as a decoy, resulting in decreased or increased expression of the target gene product It may be an activity. One example is the activity of stabilizing mRNA and increasing the expression of the target gene product by binding as a decoy to the cis element binding factor and inhibiting the binding of the cis element binding factor to the mRNA.

(3) Aptamer activity Activity that specifically binds to a specific molecule and inhibits or enhances the function of the molecule by the three-dimensional structure created by the oligonucleotide. So far, oligonucleotides exhibiting aptamer activity for growth factors, enzymes, receptors, membrane proteins, viral proteins, etc. are known.

The biological activity or chemical activity of oligonucleotides can be evaluated by conducting various assays in the presence and absence of each oligonucleotide pool.

For example, when evaluating the activity of the oligonucleotide that binds complementarily to the mRNA of the target gene and inhibits the binding of the translation factor complex to the mRNA, the following assay is performed. First, in the presence of each oligonucleotide pool, the mRNA of a target gene is brought into contact with an mRNA binding protein (here, a translation factor) that binds to the mRNA, and the amount of binding between the mRNA and the mRNA binding protein is measured. Further, in the absence of each oligonucleotide pool, the target mRNA is brought into contact with the mRNA binding protein that binds to the mRNA, and the amount of binding between the mRNA and the mRNA binding protein is measured. Then, the amount of binding in the presence of the oligonucleotide pool is compared with the amount of binding in the absence of the oligonucleotide pool, and the oligonucleotide pool containing the oligonucleotide that exhibits the activity of inhibiting the binding between the mRNA and the mRNA binding protein is identified. . The amount of binding between mRNA and mRNA binding protein can be evaluated, for example, by a pull-down assay using mRNA bait.

By this assay, it is possible to identify a target oligonucleotide pool containing an antisense oligonucleotide that binds complementarily to the mRNA of the target gene and inhibits the binding of the translation factor complex to the mRNA.

Also, for example, when the activity of the oligonucleotide is evaluated by binding to a DNA-binding protein or RNA-binding protein as a decoy and consequently increasing the expression of the target gene product, the following assay is performed. First, the expression level of the target protein is measured for the cells into which each oligonucleotide pool has been introduced. Moreover, the expression level of protein is measured about the cell which has not introduce | transduced each oligonucleotide pool. Then, the expression level of the cells into which the oligonucleotide pool has been introduced is compared with the expression level of the cells into which the oligonucleotide pool has not been introduced, and an oligonucleotide pool containing oligonucleotides that exhibit the activity of increasing the expression of the target gene product is identified. The expression level of the target protein can be evaluated, for example, by Western blot.

This assay can identify a target oligonucleotide pool containing decoy oligonucleotides that have the activity of binding to DNA or RNA binding proteins as decoys and increasing the expression of target gene products.

The assay is performed for each of two or more oligonucleotide pools constituting the oligonucleotide library L. At this time, 16384 kinds of oligonucleotide pools are prepared, and an oligonucleotide library L containing oligonucleotides that can specifically bind to nucleotide chains of any sequence is always prepared. Thus, one or more oligonucleotide pools exhibiting the desired activity can be identified.

(2-4) Acquisition of oligonucleotide In FIG. 3, symbol S ₄ is a procedure for acquiring an oligonucleotide exhibiting a desired biological activity or chemical activity from the oligonucleotide pool identified in step S _3. is there.

The oligonucleotide pools identified in step S _3, contains four or 16 different oligonucleotides. In this procedure, an oligonucleotide exhibiting a desired activity (target oligonucleotide) is identified from these oligonucleotides.

The purpose oligonucleotides can be identified by performing for each of the four or 16 kinds of oligonucleotides were synthesized by the same method as described in Step S _2, the various assays in a manner similar to that described in step S _3.

The screening method of an oligonucleotide according to the present invention performs the identification of the object oligonucleotide pool in step S _3, performing a two-step screening procedure of identifying the target oligonucleotide from the object pool of oligonucleotides further in this procedure. Thereby, in the oligonucleotide screening method according to the present invention, an oligonucleotide exhibiting a desired activity can be efficiently obtained.

In the oligonucleotide screening method according to the present invention, by preparing 16384 oligonucleotide pools, oligonucleotides that can specifically bind to nucleotide chains of any sequence must be in any one or more oligonucleotide pools. Since it can be included, it is possible to obtain an oligonucleotide exhibiting an antisense activity, a decoy activity, etc. even for a target gene for which the target sequence cannot be specified.

In the oligonucleotide screening method according to the present invention, it is not always necessary to prepare 16384 oligonucleotide pools. The greater the number of oligonucleotide pools, the higher the accuracy with which the target oligonucleotide can be obtained by screening. However, screening can be carried out using a minimum of two or more oligonucleotide pools.

3. Oligonucleotide An oligonucleotide obtained by the oligonucleotide screening method of the present invention exhibits a desired biological activity or chemical activity such as the above-described antisense activity or decoy activity. Therefore, this oligonucleotide can be developed as a nucleic acid medicine, for example.

Specifically, an oligonucleotide exhibiting antisense activity or decoy activity can be used as a nucleic acid drug for enhancing or suppressing the expression of a predetermined target gene. Since the oligonucleotide synthesized by LNA has high heat resistance and excellent nuclease resistance, the obtained oligonucleotide exhibits high stability when used as a nucleic acid pharmaceutical.

For example, when the expression of a target gene is controlled using the obtained oligonucleotide, the oligonucleotide is added to the cell culture medium and taken into the cell to bind to the target gene mRNA in the cell. Alternatively, the obtained oligonucleotide is introduced into the cell by lipofection or microinjection to bind to the target gene mRNA in the cell. In addition, when the target of gene expression is a living organ or a living body, the oligonucleotide is administered in vivo by an administration route such as oral route, rectal route, nasal route, vascular route, or by direct local administration to the target organ. Alternatively, it is introduced into a living organ and taken into a cell.

For the development as a nucleic acid drug, the base sequence and length of the obtained oligonucleotide, the chemical structure of ribose or the chemical structure of the phosphodiester bond, etc. can be appropriately changed and optimized. By optimizing the base sequence and the like, it is possible to develop an oligonucleotide exhibiting higher activity or an oligonucleotide having lower toxicity and side effects.

Furthermore, by transfecting a vector capable of expressing the oligonucleotide, the oligonucleotide can be expressed in the cell and bound to the target gene mRNA. As the vector, plasmids derived from Escherichia coli, Bacillus subtilis or yeast, animal viruses such as bacteriophages, retroviruses, vaccinia viruses, baculoviruses, or those obtained by fusing them with liposomes can be used. The expression vector can be constructed by a known genetic engineering technique.

Oligonucleotide or expression vector capable of expressing it is a unit dose required for the practice of a generally accepted formulation together with a pharmaceutically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc. It can be produced as a nucleic acid medicine by mixing in the form. This pharmaceutical composition is sterilized, for example, orally as tablets, capsules, elixirs, microcapsules or the like with sugar coating as necessary, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections such as solutions or suspensions. Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry.

As will be described later in detail in the Examples, the present inventors show that the oligonucleotide represented by the base sequence “CGGAAACA” binds to tumor necrosis factor α (Tumor Necrosis Factor α: TNF-α) mRNA and this mRNA. It was found to show an activity of inhibiting the binding to the known transcription factor RC3H1.

Therefore, this oligonucleotide or an expression vector capable of expressing this oligonucleotide can be used to regulate the expression of TNF-α, and can be used as an immunoactivator or anticancer agent.

It was also found that the oligonucleotide represented by the base sequence “ATGAATAAA” has an activity of increasing the expression of a low-density lipoprotein receptor (Low-） Density Lipoprotein Receptor: LDLR).

Therefore, this oligonucleotide or an expression vector capable of expressing this oligonucleotide can be used as an LDLR expression enhancer.

LDLR has a function of taking LDL cholesterol in plasma into cells by receptor-mediated endocytosis. In particular, intracellular uptake of LDL cholesterol by LDLR expressed in the liver plays an important role in the clearance of plasma LDL cholesterol. LDL cholesterol is responsible for cholesterol transport from the liver to peripheral tissues. However, in a hyperlipidemic state where the LDL cholesterol in the blood has increased beyond normal levels, LDL cholesterol causes vascular injury due to deposition on the inner wall of the blood vessel, causing arteriosclerosis, hypertension, cerebral infarction, myocardial infarction, narrowing. Causes heart disease.

Since the above-mentioned oligonucleotide can enhance the expression of LDLR, according to the pharmaceutical composition comprising this oligonucleotide or the like as an active ingredient, plasma LDL cholesterol can be applied by enhancing the expression of LDLR, particularly when applied to the liver. It becomes possible to prevent, ameliorate or treat hyperlipidemia and hyperlipidemia-related diseases. Note that “prevention” here includes not only prevention in the previous stage of disease, but also prevention against recurrence after treatment of the disease. In addition to arteriosclerosis, hypertension, cerebral infarction, myocardial infarction, and angina, hyperlipidemia-related diseases include diabetes, obesity, and cancer. It is not excluded.

Regarding LDLR, it has been reported that the genetic polymorphism is associated with the risk of developing Alzheimer's disease (“Function of beta-amyloid in cholesterol transport: a lead to neurotoxicity.” FASEB J., 2002, Oct; 16 (12): 1677-9. See Epub 2002, Aug 21. It has also been suggested that elevated cholesterol levels are involved in the pathology of Alzheimer's disease (“Genetic study evaluating LDLR polymorphisms and Alzheimer's disease.” Neurobiol Aging., 2008, Jun; 29 (6): 848-55 (See Epub 2007 (Jan 18)).

Therefore, the pharmaceutical composition containing the above-mentioned oligonucleotide or the like as an active ingredient has the potential to be applied to prevent, ameliorate, or treat Alzheimer's disease by enhancing the expression of LDLR and controlling the level of plasma LDL cholesterol. There is also.

<Example 1>
1. Screening of oligonucleotides having binding inhibitory activity between TNF-α mRNA and transcription factor RC3H1 By using the screening method according to the present invention, the present inventors have made a tumor necrosis factor α (TNF-α) mRNA An attempt was made to obtain an oligonucleotide exhibiting an activity of inhibiting the binding between the transcription factor RC3H1 that was revealed to bind to TNF-α mRNA.

(1) Synthesis of basic sequence Using the BNA synthesis service of Gene Design Co., Ltd., oligonucleotides having the basic sequences of the following two different sequences were synthesized.

(2) Preparation of oligonucleotide pools The oligonucleotide pools 1N and 2N were composed of 16 types of oligonucleotides in which any one of A, G, T, and C was linked to the 5 'end and 3' end of the basic sequences 1 and 2, respectively. In addition, 16 types of oligonucleotides in which any of a, g, t, and c were bonded to the 5 ′ end and 3 ′ end of the basic sequences 1 and 2, respectively, were designated as oligonucleotide pools 1n and 2n. Here, “A” represents an adenine analog, “G” represents a guanine analog, “T” thymine analog, “C” represents a cytosine analog, “a” represents adenine, “g” represents guanine, “t” thymine, “ “c” represents cytosine. "N" represents any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog, or cytosine analog, and "n" represents any one nucleic acid selected from adenine, guanine, thymine, or cytosine. Represents.

(3) Identification of oligonucleotide pool Among the oligonucleotide pools 1N, 1n, 2N, and 2n, an oligonucleotide pool showing an activity that inhibits the binding between TNF-α mRNA and RC3H1 was identified.

TNF-α mRNA was synthesized by in vitro translation. MEGAscriptT7 kit by amplifying the 869-1652th nucleotide sequence from the 5 ′ end of TNF-α mRNA (GenBank Accession No.NM_000594.2, SEQ ID NO: 1) by PCR using a primer having T7 promoter sequence at the 5 ′ end (Ambion) was used to synthesize RNA according to the attached protocol. A reaction for covalently binding Flag-hydrazide to the 3 ′ end of the synthesized TNF-α mRNA was performed, and the 3 ′ end of the TNF-α mRNA was labeled with Flag. The labeled mRNA was purified using RNeasy Mini Kit from Qiagen. In addition, mRNA flag labeling is performed according to a known method (see “Programmable ribozymes for mischarging tRNA with nonnatural amino acids and their applications to translation.” Methods, 2005, Vol, 36, No.3, p.239-244). It was.

The purified Flag-labeled TNF-α mRNA (hereinafter referred to as “TNF-α mRNA-Flag”) is mixed with cell-extracted protein extracted from 293T cells, and oligonucleotide pools 1N, 1n, 2N, or 2n are mixed with each oligonucleotide. The reaction was carried out by adding the final concentration to 100 nM. After immunoprecipitation, the eluted sample was subjected to Western blotting using an anti-RC3H1 antibody (Bethyl Laboratories).

Western blot results are shown in “FIG. 4”. RC3H1 was detected in samples (lanes 3 and 4) obtained by mixing the cell extract protein with 10 pmol of TNF-α mRNA-Flag and oligonucleotide pool 1N or 1n and immunoprecipitating with anti-Flag antibody. In lane 1, the cell extract protein itself is mixed. In lane 2, the cell extract protein is mixed with 10 pmol TNF-α mRNA-Flag and oligonucleotide solvent (water), and a sample obtained by immunoprecipitation with anti-Flag antibody is obtained. It is flowing.

In contrast, RC3H1 was detected in samples (lanes 5 and 6) obtained by mixing the cell extract protein with 10 pmol of TNF-α mRNA-Flag and oligonucleotide pool 2N or 2n and immunoprecipitating with anti-Flag antibody. It was revealed that the binding of RC3H1 to TNF-α mRNA-Flag was inhibited.

(4) Obtaining Oligonucleotides Oligonucleotides showing activity to inhibit the binding between TNF-α mRNA and RC3H1 were obtained from the oligonucleotide pools 2N and 2n.

Immunoprecipitation and Western blotting were performed on 16 kinds of oligonucleotides constituting the oligonucleotide pool 2N or 2n by the same procedure as described above.

As a result, the following oligonucleotides 2N1 and 2n1 were obtained from each of the oligonucleotide pools 2N and 2n as oligonucleotides exhibiting binding inhibitory activity between TNF-α mRNA and RC3H1.

In this example, each screening step was performed with the oligonucleotide library as 2 ( oligonucleotide pools 1N and 2N, or oligonucleotide pools 1n and 2n), which is the minimum number of oligonucleotide pools. As a result, the oligonucleotides shown in “Table 3” were obtained as oligonucleotides showing the binding inhibitory activity between the target TNF-α mRNA and RC3H1.

From this result, it is possible that the 9mer oligonucleotide in which two nucleic acid analogs or nucleic acids are bound to the basic sequence of the nucleic acid analog 7mer can exhibit a desired activity (in this case, the binding inhibitory activity between TNF-α mRNA and RC3H1). It was revealed. This result shows that oligonucleotides that can specifically bind to nucleotide chains of any sequence are prepared by preparing 16384 (4 to the 7th power) oligonucleotide pools that are all combinations of 7-mer nucleic acid analogs. It is shown that it is possible to construct an oligonucleotide library that always contains an oligosaccharide that exhibits a desired activity such as an antisense activity or a decoy activity even for a target gene for which a target sequence cannot be specified by using this library. It shows that nucleotides can be obtained.

<Example 2>
2. Screening for oligonucleotides having an activity of inhibiting the binding of ZFP36L1 to LDLR mRNA and increasing the expression of LDLR In this example, a 9-mer oligonucleotide in which two nucleic acid analogs or nucleic acids were bound to the basic sequence of nucleic acid analog 7-mer, It has been clarified that the activity of increasing the expression of a low-density lipoprotein receptor (LDLR) is exhibited.

(1) Synthesis of basic sequence Using the BNA synthesis service of Gene Design Co., Ltd., an oligonucleotide having the following basic sequence was synthesized.

(2) Preparation of oligonucleotide pool Sixteen types of oligonucleotides in which A, G, T, and C were bonded to the 5 ′ end and 3 ′ end of basic sequence 3 were designated as oligonucleotide pool 3N. In addition, 16 types of oligonucleotides in which a, g, t, and c were bonded to the 5 ′ end and 3 ′ end of the basic sequence 3, respectively, were designated as an oligonucleotide pool 3n.

(3) Evaluation of activity of oligonucleotide pool It was evaluated whether the oligonucleotide pools 3N and 3n have an activity to increase the expression of LDLR.

Hela cells were seeded on a 12-well plate at 1 × 10 ⁵ cells / well and cultured using DMEM medium containing 10% FBS. After 24 hours in culture, oligonucleotide pools 3N or 3n were transfected into cells by lipofection (Darmafect 4, Thermo Scientific). Oligonucleotide pools 3N and 3n were used after being diluted so that the final concentration of each oligonucleotide was 40 nM.

Cells 24 hours after transfection were collected, and protein extraction was performed by a conventional method. 5 μg of the extracted protein was separated by SDS-PAGE and blotted on a membrane. Western blotting was performed by a conventional method to evaluate the expression level of LDLR. As an anti-LDLR antibody, an Abcam antibody (Cat. No. ab52818) was used.

Western blot results are shown in “FIG. 5”. An increase in LDLR expression level was confirmed in cells transfected with oligonucleotide pools 3N and 3n (lanes 2 and 3), compared to cells not transfected with the oligonucleotide pool (lane 1). Note that β-actin was used as an internal standard for expression level evaluation. The expression level of β-actin was not significantly changed regardless of the presence or absence of transfection.

(4) Acquisition of oligonucleotides Oligonucleotides having activity to increase the expression of LDLR were acquired from the oligonucleotide pools 3N and 3n.

For 16 types of oligonucleotides constituting the oligonucleotide pool 3N or 3n, transfection and Western blotting were performed by the same procedure as described above.

As a result, from the oligonucleotide pools 3N and 3n, the following oligonucleotides 3N1 and 3n1 were obtained as oligonucleotides showing LDLR expression increasing activity.

<Example 3>
3. In this example, the mechanism by which the oligonucleotides 3N1, 3n1 obtained in Example 2 increase the expression of LDLR was examined. As the mechanism of action of oligonucleotide 3N1, 3n1, it is considered that oligonucleotide 3N1, 3n1 inhibits the binding of mRNA-binding protein to mRNA, resulting in decreased or increased expression of the target gene product. . Therefore, in this example, mRNA binding proteins that bind to LDLR mRNA were identified, and it was clarified that oligonucleotides 3N1, 3n1 inhibit the binding of this mRNA binding protein to LDLR mRNA.

(1) Identification of mRNA-binding protein that binds to LDLR mRNA First, for the purpose of identifying mRNA-binding protein that binds to LDLR mRNA, it is covered by immunoprecipitation using LDLR mRNA as a bait and proteome analysis using a mass spectrometer. Analysis was performed.

In accordance with the method described in Example 1, LDLR mRNA was synthesized by in vitro translation. Amplification of the 2677-3585bp region of LDLR mRNA (SEQ ID NO: 2, GenBank Accession No.NM_000527) was performed by PCR using a primer having a T7 promoter sequence at the 5 'end, and MEGAscript T7 kit (Cat.No.1333, Ambion) Was used to synthesize RNA. A reaction for covalently binding Flag-hydrazide to the 3 ′ end of the synthesized LDLR mRNA was performed, and the 3 ′ end of the LDLR mRNA was labeled with Flag. The labeled mRNA was purified using RNeasy Mini Kit (Cat. No. 74106) manufactured by Qiagen.

After purification, 10 pmol of Flag-labeled LDLR mRNA was mixed with anti-Flag antibody beads (Cat. No. F2426, Sigma) and reacted at 4 ° C for 1 hour. Thereafter, 3 mg of cell-extracted protein extracted from 293T cells cultured in DMEM medium containing 10% FBS 培 地 was added, and further reacted at 4 ° C for 1 hour. After washing away unbound protein, RNA and RNA binding protein were eluted with Flag peptide. Samples obtained by elution were treated with lysyl endopeptidase, and the LC-MS / MS method (“A direct 手法 nanoflowliquid chromatography-tandem mass spectrometry system for interaction proteomics.” Analytical Chemistry, 2002, Vol. 74, No.18, p.4725-4733). As the mass spectrometer, QSTAR バ イ オ XL (Applied Biosystem) was used.

“ZFP36L1” and “ZFP36L2” were identified by LC-MS / MS. ZFP36L1 and ZFP36L2 form one family of ARE binding factors (hereinafter referred to as “ZFP36 family”) together with ZFP36 (also known as TTP). ZFP36 family has been reported to bind to ARE and destabilize mRNA and function to promote degradation (“Tristetraprolin and its family members can promote the cell-free deadenylation of AU-rich element-containing mRNAs by poly (A) ribonuclease. ”Molecular Cell Biology, 2003, Vol.23, No.11, p.3798-812).

(2) Binding inhibition experiment with oligonucleotides 3N1 and 3n1 Next, it was examined whether oligonucleotides 3N1 and 3n1 could inhibit the binding of ZFP36L1 and ZFP36L2 to LDLR mRNA.

LDLR mRNA was synthesized by in vitro translation and labeled with Flag peptide. For 3′UTR, 2677-3585 bps from the 5 ′ end of LDLR mRNA (see SEQ ID NO: 2) was used.

Purified Flag-labeled LDLR mRNA (hereinafter referred to as "LDLR mRNA -Flag") was mixed with cell-extracted protein extracted from 293T cells, and oligonucleotide 3N1 or 3n1 was added at a final concentration of 100 nM for reaction. After immunoprecipitation, the eluted sample was subjected to Western blotting using an anti-ZFP36L1 antibody (Cell signaling).

Western blot results are shown in “FIG. 6”. ZFP36L1 was detected in a sample (lane 2) obtained by mixing cell extract protein containing ZFP36L1 / ZFP36L2 with LDLR mRNA-Flag and immunoprecipitating with anti-Flag antibody. In lane 1, the cell extracted protein itself flows.

This indicates that ZFP36L1 can bind to LDLR mRNA-Flag. Although not shown, the same result is obtained for ZFP36L2 (the same applies to the present embodiment hereinafter).

In the lanes 3 and 4 where the cell extract protein and LDLRLD mRNA-Flag were reacted in the presence of the oligonucleotide 3N1 or 3n1, the detection signal of ZFP36L1 was significantly reduced. The intensity of the detection band in lanes 8-13 was less than or equal to the intensity of the detection band obtained by the reaction with Actin 3′UTR-Flag shown in lane 2. On the other hand, in Lanes 4-7 where the reaction was carried out with the addition of control LNA4, 5, no decrease in the detection signals of ZFP36L1 and ZFP36L2 was observed.

From this, it was revealed that the oligonucleotides 3N1, 3n1 can remarkably inhibit the binding of ZFP36L1 and ZFP36L2 to LDLR mRNA.

The result of this example is that the 9mer oligonucleotide in which two nucleic acid analogs or nucleic acids are bound to the base sequence of the nucleic acid analog 7mer inhibits the binding of the desired activity (here, ZFP36L1 and ZFP36L2 to LDLR mRNA) As a result, it is clarified that the activity of increasing the expression of LDLR can be exhibited.

Z ZFP36 family has been reported to bind to ARE, destabilize mRNA, and function to promote degradation. Therefore, from the results of this example, the oligonucleotides 3N1, 3n1 inhibit the binding of ZFP36L1 and ZFP36L2 to LDLR mRNA, thereby inhibiting the mRNA degradation promoting function of ZFP36L1 and ZFP36L2 and stabilizing LDLR mRNA. It was strongly suggested that the expression of LDLR mRNA was increased by promoting. This is very significant in that it is revealed for the first time that ZFP36L1 and ZFP36L2 are involved in the expression control mechanism of LDLR mRNA in vivo. Thus, the oligonucleotide screening method according to the present invention can contribute to elucidating a new control mechanism in the living body, starting from the obtained oligonucleotide.

Claims (17)

1. Any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or any one selected from adenine, guanine, thymine or cytosine in a 7mer basic sequence consisting of any nucleic acid analog A procedure of preparing two or more oligonucleotide pools composed of oligonucleotides obtained by binding one or two nucleic acids, such that the basic sequences differ between the oligonucleotide pools;
   A procedure for identifying an oligonucleotide pool containing the target oligonucleotide from the oligonucleotide pool;
   A method for screening oligonucleotides.
2. At either the 5 ′ end or 3 ′ end of the basic sequence,
   Any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or
   The oligonucleotide screening method according to claim 1, wherein two or more oligonucleotide pools each comprising four kinds of oligonucleotides each bound to any one nucleic acid selected from adenine, guanine, thymine, or cytosine are prepared.
3. At the 5 ′ end and 3 ′ end of the basic sequence,
   Any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or
   The oligonucleotide screening method according to claim 1, wherein two or more oligonucleotide pools each comprising 16 kinds of oligonucleotides each bound with any one nucleic acid selected from adenine, guanine, thymine, or cytosine are prepared.
4. The method for screening an antisense oligonucleotide according to any one of claims 1 to 3, comprising a procedure for preparing 16384 basic sequences comprising all combinations of seven nucleic acid analogs.
5. 5. The nucleic acid analog according to claim 4, wherein at least one of the nucleic acid analogs is included in the sequence of the oligonucleotide, thereby increasing the binding affinity for the complementary strand of the oligonucleotide as compared to the oligonucleotide consisting of only the nucleic acid. Oligonucleotide screening method.
6. The oligonucleotide screening method according to claim 5, wherein a bridged nucleic acid or a locked nucleic acid is used as the nucleic acid analog.
7. In the presence and absence of the oligonucleotide pool, the mRNA is contacted with the mRNA binding protein that binds to the mRNA, and the amount of binding between the mRNA and the mRNA binding protein is measured. The method for screening an oligonucleotide according to claim 1, wherein an oligonucleotide pool containing an oligonucleotide having an activity of inhibiting binding is identified.
8. The oligonucleotide screening method according to claim 7, wherein the mRNA is tumor necrosis factor α 因子 mRNA, and the mRNA binding protein is RC3H1.
9. The oligonucleotide pool containing the oligonucleotide which shows the activity which enhances the expression of this protein is identified by measuring the expression level of protein about the cell which introduce | transduced the said oligonucleotide pool, and the cell which is not introduce | transduced. Oligonucleotide screening method.
10. The oligonucleotide screening method according to claim 9, wherein the protein is a low-density lipoprotein receptor.
11. The method for screening an oligonucleotide according to claim 1, comprising a procedure for obtaining a target oligonucleotide from the identified oligonucleotide pool.
12. An oligonucleotide library provided for the oligonucleotide screening method according to claim 1,
    Any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or any one selected from adenine, guanine, thymine or cytosine in a 7mer basic sequence consisting of any nucleic acid analog Consisting of two or more oligonucleotide pools consisting of oligonucleotides linked with one or two nucleic acids,
    An oligonucleotide library in which the basic sequences are different from each other between each oligonucleotide pool.
13. The oligonucleotide library according to claim 12, wherein the basic sequence is 16384 consisting of all combinations of seven nucleic acid analogs.
14. An antisense oligonucleotide represented by the base sequence CGGAAACA obtained by the oligonucleotide screening method according to claim 8.
15. A tumor necrosis factor α expression regulator containing an oligonucleotide represented by the base sequence CGGAAACA or an expression vector capable of expressing the oligonucleotide.
16. An antisense oligonucleotide represented by the base sequence ATGAATAAA obtained by the oligonucleotide screening method according to claim 10.
17. A low-density lipoprotein receptor expression enhancer containing an oligonucleotide represented by the base sequence ATGAATAAA or an expression vector capable of expressing the oligonucleotide.

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