WO2001021637A1

WO2001021637A1 - Light-responsive oligonucleotide

Info

Publication number: WO2001021637A1
Application number: PCT/JP2000/006415
Authority: WO
Inventors: Makoto Komiyama; Hiroyuki Asanuma; Takayuki Yoshida
Original assignee: Makoto Komiyama; Hiroyuki Asanuma; Takayuki Yoshida
Priority date: 1999-09-20
Filing date: 2000-09-20
Publication date: 2001-03-29
Also published as: AU7317300A

Abstract

An oligonucleotide to which an organic group undergoing structural isomerization depending on light irradiation is attached. The melting temperature of a double-strand formed by this oligonucleotide and another oligonucleotide complementary thereto reversibly changes depending on light irradiation. Thus, the synthesis of DNA, the expression of a gene, etc. can be controlled by a convenient procedure of light irradiation in the field of biotechnology.

Description

Description Photoresponsive oligonucleotide Technical field

The present invention relates to a light-responsive oligonucleotide, particularly to a light-responsive oligonucleotide used for controlling DNA synthesis and gene expression in the field of biotechnology. Background art

By controlling the transcription and translation of DNA, it is possible to artificially control gene expression. Various methods have been proposed to control gene expression using natural or chemically modified oligonucleotides. For example, in Biochemistry, 1991, Vol. 35, pp. 105-39, the use of the fact that the homo 'pyrimidine' and 'homo' purine sites in a DNA duplex can form a triplex is used. Thus, a method for controlling transcription has been proposed. The so-called “antisense method”, which inhibits the translation process by coexisting complementary oligonucleotides with messenger RNA generated by transcription, is also known as the “High Molecular Chemistry and Nucleic Acid Functional Design ”(published by the Institute of Technology, 1, 996). In addition, the DNA double strand dissociates into a single strand during transcription, and it is possible to inhibit the activity of transcriptase by adding an oligonucleotide complementary to this single-stranded portion from outside. . However, with these methods, gene expression can be inhibited, but not restored. That is, with these methods, it is virtually impossible to dissociate the oligonucleotide once it binds to the gene or messenger RNA that is the target. Therefore, it is impossible to reversibly control gene expression. Of course, it is possible to dissociate the oligonucleotide once bound by raising the temperature of the system or changing the pH or ionic strength. However, such a dramatic environmental change is not practical because it loses the activity of enzymes involved in gene expression such as polymerase. Gene expression Reversibly regulates duplex formation with DNA, triplex, and RNA with external stimuli, such as light irradiation, without changing pH, ionic strength, etc. A way was desired.

In the past, Yamana et al. Reported that azobenzene, a photoisomerized molecule, was introduced into the main chain of an oligonucleotide through a phosphodiester bond in Nucleosides Nucleotides, 1998, Vol. 17, p.233. Have been. They report that azobenzene introduced into the oligonucleotide backbone isomerically reversibly isomerized from the trans form to the cis form (or vice versa). However: No mention is made as to whether the stability of DNA changes based on the photoisomerization of azobenzene.

An object of the present invention is to obtain a photoresponsive oligonucleotide capable of irreversibly changing the melting temperature of the ability to form a duplex or a triplex by irradiation with light. Disclosure of the invention

The present invention relates to the following light-responsive oligonucleotide.

(1) a double strand of an oligonucleotide to which one or more organic groups that are structurally isomerized by irradiation with light of a specific wavelength are bonded and which is substantially complementary to the oligonucleotide; A photoresponsive oligonucleotide whose melting temperature in formation or triplex formation is reversibly changed by irradiation with the light.

(2) The photoresponsive oligonucleotide according to (1), wherein the rate of thermal isomerization of the photoresponsive oligonucleotide from one isomer to the other is 0.07 min ¹ or less at 50 C.

(3) The photoresponsive oligonucleotide according to (1) or (2), wherein the organic group is a residue of azobenzene or a derivative thereof, and is bonded to the oligonucleotide directly or via an intervening group.

(4) The following equation:

(In the formula, A and B each independently represent a hydrogen atom, a nucleotide or an oligonucleotide, X represents a residue of azobenzene or a derivative thereof, and R represents a hydrogen atom or a carbon number of 1 to 20, preferably 1 (3) representing a linear alkyl group of 1 to 4).

Wherein A and B each independently represent a hydrogen atom, a nucleotide or an oligonucleotide, Nb represents a nucleobase, and X represents a residue of azobenzene or a derivative thereof. Responsive oligonucleotide.

(6) The photoresponsive oligonucleotide of (4) or (5), wherein X is represented by the following formula, II or ill. -QR ¹ (I)

/

R (III)

(The i, in II and ill, RR, R ²¹ are each a direct bond; unsubstituted or halogen atom, a hydroxyl group, an amino group, a nitro group, carbon atom number of 1 to 20 substituted by a carboxyl group, etc., preferably 1 to 10, more preferably 1 to 4 alkylene groups, or 2 to 20, preferably 2 to 10, carbon atoms which are unsubstituted or substituted by a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxy group, or the like. More preferably, it is a 2-4 alkenylene group, Q is a direct bond, an oxygen atom, a mono-NH—CO— group or a —CO—NH— group, and R ^{2 to} R ⁷ , R ^s , I ¹ , R ^{12 to} R ¹⁷ , R ^1S , R ² R ^{22 to} R ²⁷ , R ²⁹ and R ³ ° are each independently unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, etc. Alkyl groups having 1 to 20, preferably 1 to 10, more preferably 1 to 4 carbon atoms Or an alkoxy group; unsubstituted or substituted with a halogen atom, hydroxyl group, amino group, nitro group, carboxyl group, etc., having 2 to 20, preferably 2 to 10, more preferably 2 to 4 carbon atoms. Hydroxyl or alkynyl; hydroxyl; halogen; amino; nitro

-Δ- Or a carboxyl group. R ⁸ , R ¹⁸ , and R ²⁸ each independently represent 1 to 20, preferably 1 to 10, carbon atoms each of which is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, or the like. Preferably an alkyl or alkoxy group of 1 to 4; unsubstituted or substituted by a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, etc., having 2 to 20, preferably 2 to 10, more preferably 2 to 2 carbon atoms. Alkenyl group or alkynyl group to 4; a hydroxyl group; or a halogen atom). Preferably, R ⁷ , R ⁹ , R ¹⁷ , R ¹⁹ , H ²⁷ and R ²⁹ represent the above groups other than the nitro group. Also, preferably in Formula I, one Q—R ¹ — is an intervening group that does not form a resonance structure with azobenzene. For example, represents Q is oxygen atom in formula I, R ¹ is an unsubstituted or halogen atom, a hydroxyl group, an amino group, a nitro group, and 1 carbon atoms substituted with a carboxyl group or the like 20 ₃ preferably 1-10, More preferably, it represents 1-4 alkylene groups. In Formulas II and III, Q represents an oxygen atom, and R ¹ is an unsubstituted or substituted halogen atom, hydroxyl group, amino group, nitro group, carboxyl group, etc., having 1 to 20, preferably 1 to 1 carbon atoms. It is preferred that 10, more preferably represents 1 to 4 alkylene groups, or one Q—: ^1— represents one NH—CO— or one CO—NH—.

(7) In the above formulas, II, ill, I ¹ , R ¹¹ and; ²¹ represent a direct bond, and R ² to R ¹⁰ , H ^{12 to} R ² ° and R ²² to R ³ ° represent a hydrogen atom or (6) a light-responsive oligo represented by the formula (6), which represents a linear alkyl group having 1 to 20, preferably 1 to 10, and especially 1 to 4 carbon atoms, and Q represents -NH-CO- or -CO-NH- nucleotide.

(8) In the above formulas ι, ιι, ill, IT, R ¹¹ and R ²¹ represent a direct bond, and R ² to R ^I0 _S R ^{12 to} R ² ° and R ^{22 to} R ³ ° represent a hydrogen atom. (6) The photoresponsive oligonucleotide according to (6), wherein Q represents —NH—CO— or —CO—NH—.

(9) The formula iota, represents Iotaiota, in ill, I ^1, R ¹¹ and R ²¹ is from 1 to 20 carbon atoms, preferably rather 1-10, more preferably 1-4 alkylene group, R ² ~R ^10, R ¹² ~: ^2G and R ²² ~! (6) The photoresponsive oligonucleotide according to (6), wherein ³ ° represents a hydrogen atom or a linear alkyl group having 1 to 20, preferably 1 to 10, particularly 1 to 4 carbon atoms, and Q represents an oxygen atom.

(10) In the above formulas Ι, ΙΙ, ill, R ^] , 11 ¹¹ and 1 are preferably those having 1 to 20 carbon atoms. Properly 1 to 10, represents a more preferably 1 to 4 alkylene ^{^{^{group, R 2 ~R 1Q, R 1}}} 2 ~: R 2 ° and R ²² to R ³ ° represents a hydrogen atom, Q is an oxygen atom The photoresponsive oligonucleotide of (6).

Further, the present invention uses the above-mentioned photoresponsive oligonucleotide having a sequence substantially complementary to at least a part of DNA which is a type II of DNA extension reaction, and irradiates light having a wavelength within a specific range. Isomerization of the organic group of the photoresponsive oligonucleotide from one isomer to another isomer by irradiation with light having a wavelength within another specific range, thereby obtaining a double-stranded or The present invention relates to a method for controlling a DNA extension reaction, including reversibly forming and dissociating a triplex.

Furthermore, the present invention uses the above-described photoresponsive oligonucleotide having a sequence substantially complementary to at least a part of mRNA to be controlled, and irradiates the photoresponsive oligonucleotide by irradiation with light having a wavelength within a specific range. Isomerization from one isomer to another isomer and the opposite isomerization by irradiation with light having a wavelength within another specific range, whereby the light-responsive oligonucleotide and mRNA can be converted into two. The present invention relates to a method for controlling gene expression including reversibly forming and dissociating a heavy chain.

Further, the present invention provides the following compound for synthesizing a photoresponsive oligonucleotide.

(Where A 'represents a protecting group, for example, a dimethoxytrityl group or a monomethoxytrityl group, and B, is a reactive group for the phosphodiester bond at the 5' position of the oligonucleotide, for example,

NC

0 / ^P \

N

Man X represents a residue of azobenzene or a derivative thereof, and I represents a hydrogen atom or an unsubstituted or substituted carbon atom having 1 to 20, preferably 1 to 10, more preferably 1 to 4 linear alkyl. Represents a group, and Nb represents a nucleobase. )

X may represent the groups described above for the photoresponsive oligonucleotide as X and also described below.

More preferably, X is of the following formula I:

(Wherein Q represents an oxygen atom, R ¹ represents an alkylene group having 1 to 20, preferably 1 to 10, and more preferably 1 to 4 carbon atoms, and ² ° is a hydrogen atom or a carbon atom. A linear alkyl group having 1 to 20, preferably 1 to 10, and more preferably 1 to 4 atoms) represented by the following formula II:

Wherein Q represents an oxygen atom and R ¹¹ represents an alkylene group having 1 to 20, preferably 1 to 10, more preferably 1 to 4 carbon atoms, or Q represents -ΝΗ—CO— or — Represents CO—NH— and R ¹ represents a direct bond, and! ^ ~! ^^ is a hydrogen atom or a linear alkyl having 1 to 20, preferably 1 to 10, and more preferably 1 to 4 carbon atoms. Which represents a group). BEST MODE FOR CARRYING OUT THE INVENTION

(Light-responsive oligonucleotide)

In the present specification, the “light-responsive oligonucleotide” has a structure One or more organic groups that are isomerized (hereafter, sometimes referred to as photoresponsive organic groups) are bonded to each other to reversibly convert a substantially complementary oligonucleotide and double or triple strand by irradiation with light. Is an oligonucleotide that can be formed at

The photoresponsive oligonucleotide may be an oligomer or polymer of ribonucleic acid, an oligomer or polymer of deoxyribonucleic acid, or an oligomer or polymer in which both are mixed. The oligonucleotide in which the photoresponsive oligonucleotide of the present invention forms a double-strand may be any of an oligomer or polymer of ribonucleic acid, an oligomer or polymer of deoxyribonucleic acid, or an oligomer or polymer in which both are mixed. Good. Further, the photoresponsive oligonucleotides of the present invention may form a double chain with each other. When a double or triple strand is assembled, the sequences of the nucleobases of the oligonucleotides may be at least 50% or more complementary, but preferably at least 70% of the sequences are complementary. As used herein, “substantially complementary” means that at least a portion of the target DNA or mRNA under the conditions under which the experiment is performed when the organic group assumes a steric structure that stabilizes the duplex. It is complementary to the extent that it can hybridize to

The photoresponsive organic group may have any number of molecules as long as it has at least one molecule in the oligonucleotide. The ratio of one molecule to four to 50 bases is preferable, and the ratio of one molecule to five to 16 bases is more preferable.

In order to reversibly control the duplex and triplex forming ability with light, the rate of thermal isomerization of the photoresponsive organic group is preferably 0.07 min ¹ or less at 50 ° C. the good Mashiku is 0. 0 3 min ¹ below. The thermal isomerization rate as used herein indicates a value measured for an optically responsive organic group introduced into an oligonucleotide in an aqueous solution having a pH of 6.0 to 8.0.

Further, the photoresponsive organic group is preferably introduced as a side chain into the oligonucleotide. When a photoresponsive organic group is introduced into the main chain of the oligonucleotide, the structural difference from the natural oligonucleotide is increased, and the formation of a duplex or a triplex with the corresponding complementary strand is greatly inhibited. is there. In the present specification, “introduced as a side chain” refers to an oligonucleotide or an intervening group such as an alkylene group interposed between an oligonucleotide and a photoresponsive organic group, and one photoresponsive organic group. It means that they are bound by a covalent bond. Therefore, in the present specification, the state where a photoresponsive organic group is bonded to the 5, 5 or 3 'end of the oligonucleotide also means that it is introduced as a side chain.

For example, the photoresponsive organic group of the present invention may be bound to the 5 'end of the oligonucleotide or may be introduced at the 2-position of ribose or deoxyribose. Further, the photoresponsive organic group of the present invention may be covalently bonded to an ethylene chain or trimethylene chain side chain, and this may be introduced into the oligonucleotide. Further, the photoresponsive organic group of the present invention may be introduced into a phosphate group at the 5 ′ end or forming a phosphodiester bond.

In the present invention, the “photoresponsive organic group” is preferably a three-dimensional structure capable of stabilizing a double-chain or triple-chain bond by light of a specific wavelength to a three-dimensional structure destabilizing a double-chain bond. It may be an organic group that is structurally isomerized, preferably reversibly, from a substantially planar structure to a non-planar structure, for example, from a trans form to a cis form, from a merocyanine form to a svirobilane form, or vice versa. As such, the organic group whose steric structure can be reversibly changed by irradiation with light of a specific wavelength can be, for example, the residue of azobenzene, ssubiropyran, stilbene, and derivatives thereof, but preferably azobenzene or a derivative thereof. Derivative residue.

The photoresponsive organic group such as the residue of azobenzene or a derivative thereof is directly or an appropriate intervening group, for example, an alkylene group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, for example, an ethylene group or trimethylene group. Can be bound to the oligonucleotide via Further, the light-responsive organic group may be introduced into the phosphoric acid moiety be introduced into 2 ^3-position of the ribose or Dokishiribosu, it may also be introduced at the 5 'or 3' end.

The photoresponsive oligonucleotide of the present invention is, for example, one represented by the following formula:

SIWO / OOdT / IDd LZ9UIIQ OAV In the above formula, X is a photoresponsive organic group, and A and B each represent hydrogen, nucleotide or oligonucleotide. Nb indicates a nucleobase (adenine, cytosine, guanine, thymine or peracil). n is an integer of 1 or more, preferably 1 or more and 10 or less, more preferably 1 or more and 6 or less. R represents an alkyl group or alkoxy group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms, which is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, or the like. Si group; unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, etc., having 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 4 alkenyl groups or alkynyl. A hydroxyl group; a halogen atom; an amino group; a nitro group; or a carboxyl group.

X is preferably azobenzene or a derivative thereof. In this case, any substituent may be present on the benzene ring, as long as it does not impair the function of reversibly changing the melting temperature of oligonucleotide duplex formation or triplex formation by isomerization by light irradiation. Further, any intervening group may be present at the bonding portion with the oligonucleotide. However, the substituent and intervening group at the p-position of azobenzene are preferably groups that do not take a resonance structure with the benzene ring. For example, substituents such as carboxyl, amino and nitro at the p-position and amide bonds at the p-position are not preferred. This is because when a substituent or an intervening group having a resonance structure is bonded to the p-position, azobenzene is easily thermally isomerized from a cis-form to a trans-form. The substituent at the m-position is desirably a group other than the two-terminal group.

Is represented, for example, by the following formulas I, I and III.

/

In the formulas i, II and ill, Q: iTR represents the meaning defined above for the formulas i, II and ill. The above R ^{2 to} R ^{1 ()} and R ^{12 to} R ² . In R ²² to R ^3C), unsubstituted or substituted alkyl group, alkenyl group, alkynyl group and alkoxy group is preferably straight-chain.

More specific examples of X are shown in the following formulas _c ,

ゝ N

In the formula, Z ¹ is any group other than a group having a resonance structure with a benzene ring, such as a nitro group, an amino group, and a carboxyl group.For example, each independently represents an unsubstituted or halogen atom, a hydroxyl group, an amino group, a nitro group An alkyl or alkoxy group having 1 to 20, preferably 1 to 10, more preferably 1 to 4 carbon atoms substituted with a carboxyl group or the like; unsubstituted or halogen atom, hydroxyl group, amino group, nitro group A alkenyl group or alkynyl group having 2 to 20, preferably 2 to 10, more preferably 2 to 4 carbon atoms substituted with a carboxyl group or the like; a hydroxyl group; or a halogen atom. Z ² and Z ³ are arbitrary groups other than a nitro group, and each independently represents, for example, 1 to 2 carbon atoms that are unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, an nitro group, a carboxyl group, or the like. 0, preferably 1 to 10 and more preferably 1 to 4 alkyl groups or alkoxy groups; 2 to 2 carbon atoms which are unsubstituted or substituted by halogen atoms, hydroxyl groups, amino groups, nitrite groups, carboxyl groups, etc. 0, preferably 2 to 10, more preferably 2 to 4 alkenyl or alkynyl; hydroxyl; halogen; amino; The unsubstituted or substituted alkyl group, alkenyl group, alkynyl group and alkoxy group are preferably straight-chain.

(Synthesis of photoresponsive oligonucleotide)

As a method for introducing a photoresponsive organic group into an oligonucleotide, a polymethylene chain which is unsubstituted or substituted with an alkyl group (for example, one having 2 to 10 carbon atoms, preferably one having 2 to 5 carbon atoms) After the covalent introduction into the side chain of (2), a method of converting this into a phosphoramidite monomer and introducing it into an oligonucleotide using an existing DNA synthesizer can be used. The synthesis of phosphoramidite monomers is described in The Journal of Organic 'Chemistry.

The method described in Organic Chemistry (1992), Vol. 62, No. 62, page 846 may be used. In this case, polymethylene chains of various lengths can be used, but ethylene chains or trimethylene chains which are unsubstituted or substituted with an alkyl group are preferred. In this case, the photoresponsive organic group is preferably introduced covalently to any carbon atom in the case of an ethylene chain, or to the central carbon atom in the case of a trimethylene chain. The photoresponsive organic group is located at the 2 position of ribose or deoxyribose. It may be introduced at the phosphate site, or may be introduced at the 5 or 3 terminal.

Irradiation light for structural isomerization of a photoresponsive organic group can be light of any wavelength from the ultraviolet region to the infrared region as long as the organic group can be isomerized. It is preferably 300 nm or more. For example, by irradiating 300 to 400 nm light (UV light), one isomer is structurally isomerized to another isomer, and by irradiating light (visible light) of 400 nm or more, the opposite is true. Can change.

The change in melting temperature due to light irradiation may be as small as possible as long as it is a significant change exceeding the range of error, but is preferably 1.0 ° C or higher, more preferably 2.0 ° C or higher. Melting temperature measurements were made using the bioconjugate 'Bioconjugate Chemistry', Volume 8, page 3 et seq. 1997, and the journal 'Ob the American K. 7J Le Society' (Journal of the American Chemical Society). ) Magazine, 1997, Vol. 119, p. 263 et seq. The melting temperature can be determined in various ways, but is preferably determined as the temperature that gives the maximum of the first derivative of the melting temperature curve.

The photoresponsive oligonucleotide of the present invention can be used in any of in vivo and in vitro systems. The photoresponsive oligonucleotide of the present invention may be applied to the light control of a DNA or RNA extension reaction by DNA polymerase or RNA polymerase. In addition, the photoresponsive oligonucleotide of the present invention can be used for a protein or an enzyme that binds to DNA or RNA. In addition, the photoresponsive oligonucleotide of the present invention can be used in Ribozyme DNA Enzyme. Furthermore, the photoresponsive oligonucleotide of the present invention may be enzymatically introduced into natural DNA or may be enzymatically introduced into natural RNA. Accordingly, the present invention also provides a method for treating a human or non-human animal, comprising introducing the photoresponsive oligonucleotide into the DNA of a human or non-human animal.

When used for the extension reaction of DNA polymerase, the photoresponsive oligonucleotide (DNA) of the present invention can be hybridized with DNA used as a template in the middle. For trans form, template; strong against DNA As a result, the elongation reaction by the polymerase stops before the photoresponsive oligonucleotide. On the other hand, when the cis-isomer isomerized, the photoresponsive oligonucleotide dissociates from the template DNA, and the DNA extension reaction proceeds to the end of the template strand. Therefore, the present invention also provides a method for controlling a DNA extension reaction using the above-described photoresponsive oligonucleotide.

When used for the elongation reaction of RNA polymerase, the photoresponsive DNA of the present invention in which azobenzene is introduced into a part of the promoter or in the vicinity thereof may be used. Transcription by RNA polymerase occurs when the promoter forms a double strand (ie, trans form), but does not occur when the double strand is dissociated (ie, cis form). . Therefore, the present invention also provides a method for controlling an RNA extension reaction using the above-mentioned photoresponsive oligonucleotide.

Further, by using the photoresponsive oligonucleotide of the present invention for antisense DNA, protein synthesis can be light-controlled. That is, when the photoresponsive oligonucleotide of the present invention as antisense DNA strongly hybridizes with mRNA (ie, in the case of trans form), translation is inhibited, but when dissociated from mRNA (ie, In the case of cis form), translation is performed. Therefore, the present invention also provides an antisense control method using the above-mentioned photoresponsive oligonucleotide as antisense DNA. Example

Example 1: Synthesis of phosphoramidite monomer

First, Tetrahedron, 1992, Vol. 48, p. 2223, Nucleic Acids Research, Nucleic Acids Research, 1987, Vol. 15, p. 6131, and The Journal of Organics. According to the method described in The Journal of Organic Chemistry, Vol. 54, pp. 2321, 1989, the hydroxyl groups of 5, 5 and 3 of peridine are 1,3-dichloro-1,1,3,3- Protected with tetraisopropyldisiloxane, followed by benzyl chloride protection at the N 3 position. Next, follow the method described in Macromolecules, Vol. W

The 4-bromomethylazobenzene synthesized above was added to the protected peridine and dissolved in dimethylformamide. This was cooled to 120 ° C and azobenzene was introduced at the 2'-position by slowly adding sodium hydride dispersed in dimethylformamide. Thereafter, the protecting group was removed in accordance with a standard method, and 2,10- (p-phenylazobenzyl) peridine was synthesized (Uazo). Then, according to the method described in The Journal of Organic Chemistry, Vol. 62, p. 846, 1997, the fifth place was obtained as shown in the following formula. Phosphoramidite monomer (Amidite-Uazo) was synthesized by adding a dimethyltrityl group and a phosphoramidide at the 3-position.

Example 2: Synthesis of phosphoramidite monomer

The following phosphoramidite monomers were synthesized for the purpose of introducing them to the 5 'end of the oligonucleotide. 4 Dissolve bromoazobenzene and ethylene glycol in dimethylformamide, and add sodium hydride dropwise at 120 ° C to give 2- (p-phenylazo) benzyloxyethanol (XI in the following formula). Synthesized. Similarly, 3- (p-phenylazo) benzyloxy 1-propanol (X2 in the following formula) was synthesized. Phosphoramidite monomers (Amidite-Xl and Amidite-X2 in the following formula) having phosphoramidide added thereto were synthesized in the same manner as described above.

One 1 foo ° ^OH

X1

Amidtto-X1

Ο Q

C

X2 Amidite-X2

Example 3 Synthesis of Photoresponsive Oligonucleotide

Next, using a DNA synthesizer, the photoresponsive oligonucleotide of the present invention having various sequences was synthesized using the above-described phosphoramidite monomer. For comparison, the oligonucleotide having no photoresponsive organic group of the present invention was synthesized in the same manner using a DNA synthesizer. Table 1 shows the synthesized oligonucleotides.

Hereinafter, Uazo 'represents a group represented by the following formula.

Hereinafter, Uazo ′ ′ represents a group represented by the following formula ₍

XI ′ represents a group of the following formula _c

X2 ′ represents a group of the following formula <

table 1

Sequence number Symbol Sequence

1 (Uazo ') 5'-(Uazo ') TTTTTTT-3' The present invention

2 (Uazo '') A ₄ 5 '-AAA (Uazo'') AAAA-3

_{3 T 3 (Uazo ',)} T 4 5' -TTT (Uazo '') TTTT-3 ' present invention

4 (Uazo ') 5'-(Uazo ') AAAAAAAA-3, the present invention 5 (XI ') Ae 5'-(XI,) ΑΑΑΑΑΑΑΑ-3 'The present invention

6 (Χ2 ') As 5'-(Χ2 ') ΑΑΑΑΑΑΑΑ-3' The present invention

7 (XI ') Τπ 5'-(χι ') ΤΤΤΤΤΤΤΤΤΤΤ-3'

8 (XI ') ΤΗ 5'-(χΐ ') ττττττττττττττ-3'

9 (Χ2 ') Tu 5,-(Χ2 *)' -3 'The present invention

10 (Χ2 ') Τΐ4 5,-(Χ2') τΊ ΤΤΤΤΤΤΤΤΤΤΤ-3 'today

11 As 5 '-AAAAAAAA-3' Comparative example

12 Τβ 5 '-ΤΤΤΤΤΤΤΤ-3' Comparative example

13 ΤΑ 3 5 '-ΤΤΤΤΑΤΤΤ-3, Comparative example

14 Ύ 、 Ά 5 '-ΤΤΤΤΤΤΤΑ-3' Comparative example

15 Τπ 5 '~ * τ τ τ τ τ τ τ τ τ τ ττ ~~ * ^' Comparative example

16 T 5 '"ΤΤ'ΤΤ'ΤΤΤΤ'ΤΤΤΤ'ΤΤΤΤ'Τ"-, Comparative example Test example 1: Measurement of thermal isomerization rate

Each of the photoresponsive oligonucleotides of the present invention of SEQ ID NOS: 1 to 10 synthesized as described above was adjusted to ρΗ 7.0 with 1 Ommo 1Z liter of a buffer, and the salt concentration was adjusted with sodium chloride. It was dissolved in an aqueous solution adjusted to 1 mo 1Z liter. This was irradiated with light (UV light) having a wavelength of 30 Onm or more and 400 nm or less through a UVD-36C filter using a xenon lamp for 10 minutes. As a result, 90% of the added photoresponsive oligonucleotide was isomerized to cis form. This is 50 in a thermostat. The thermal isomerization rate constant k was determined by keeping the temperature at C and tracking the process from the cis-form to the trans-form thermally by UV-Vis spectra. The results are shown in Table 2. Table 2

Sequence number Symbol Rate constant k (min—

1 (Uazo *) Τ7 0.01 or less

2 As iUazo '') A «0.01 or less

3 T ₃ (Uazo '') T ₄ 0.01 or less

4 (Uazo ') A, 0.01 or less 5 (XI ') A ₈ 0 01 or less

6 (X2 ') AB 0 01 or less

7 (XI ') T _1: 0 01 or less

8 (XI ') TI <0 01 or less

9 (X2,) TIJ 0 01 or less

10 (X2 ′) T ″ 001 and below As shown in Table 2, it was found that the photoresponsive oligonucleotide of the present invention had a sufficiently low rate of thermal isomerization from a cis-form to a trans-form.

Test example 2: Measurement of melting temperature

The corresponding complementary strands were added to the oligonucleotides of SEQ ID NOs: 1, 2, 3, 4, and 12, and the melting temperature was measured to evaluate the stability of the duplex. The conditions for measuring the melting temperature are shown below.

p H (10 mm o 1 / Li Tsu torr N a ₂ HP 0 ₄ buffer 1): 7.0

Oligonucleotides: 50 m o 1 liter

Sodium chloride: 1 m 01 / liter

Measurement wavelength: 260 nm

Temperature sweep speed: _ 1.0 ° CZmin

Measurement temperature range: 6 CTC ^ ^ — 5 ° C

Table 3 shows the results. Before UV irradiation, more than 80% of the azobenzene residues were in the trans form. UV irradiation was performed by irradiating the sample containing the oligonucleotide with light of 300 nm or more and 400 nm or less through a UVD-36C filter using a xenon lamp for 10 minutes. As a result, more than 80% changed to cis form. The melting temperature was determined as the temperature giving the maximum value of the first derivative of the melting temperature curve. Table 3

Melting temperature (° c)

SEQ ID No.Complementary strand U Before irradiation After UV irradiation

1 (Uazo ') Ίι Αβ 30.5 21.9 The present invention 2 A3 (Uszo '4AT3 21.4 0.4 The present invention

3 T ₃ (UaZO '''AS 13.4 8.4 The present invention

4 (Uazo ') Ai T7A 30.9 25.3 The present invention

12 Ts As 23.7 23.6 Comparative Example As described above, when an oligonucleotide bound to an azobenzene derivative residue is hybridized with the complementary strand of Furumatsu, the azobenzene is converted from a trans form to a cis form by irradiation with UV light. It was found that the melting temperature changed by more than 5 ° C. Even more surprisingly, the oligonucleotide of the present invention of SEQ ID NO: 1 has a melting temperature higher than that of the corresponding conventional oligonucleotide (SEQ ID NO: 12) by 5 ° C. or more, and forms a duplex by introduction of azobenzene. It can be seen that the performance is getting higher. On the other hand, as shown in the comparative example, the melting temperature of the conventional unmodified oligonucleotide hardly changed before and after UV irradiation.

After that, visible light of 420 nm or more was irradiated for 10 minutes by passing through a L-42 filter using a xenon lamp, and 80% or more returned to the transformer. When the melting temperature was determined under the same conditions as above, it was consistent with the value before UV irradiation within the error range. Thus, it was revealed that the melting temperature changes reversibly. The above operation was repeated five times, and the azobenzene residue was reversibly isomerized without any particular deterioration. The melting temperature also reversibly changed.

Test Example 3: Measurement of melting temperature of triple chain

The melting temperature of the triplex was measured using a duplex having the sequence shown below. Was.

5 'One GCCACGAAATTTTTTTTTTTTTTAAACCGACG-3' (SEQ ID NO: 17)

5 ¹ -CGTCGGTTTAAAAAAAAAAAAAATTTCGTGGC-3 '(system!) Number 18

A triple strand was formed by the duplex formed by these oligonucleotides and the oligonucleotides having the sequences of SEQ ID NOs: 7, 9, 8, 15, and 16, and the melting temperature was measured. Table 4 shows the results. The conditions for measuring the melting temperature of the triplex are shown below.

pH (10 mmo1 / liter little buffer): 7.0

Oligonucleotide of SEQ ID NO: 17: 2 mo1 / liter Oligonucleotide of SEQ ID NO: 18: 2.2 mo 1 / liter

Light-responsive oligonucleotide: 2.4 mol / liter

Magnesium chloride: 0.1 or 0.4 mol / liter

Measurement wavelength: 280 nm

Temperature sweep speed: 0.5. C / m i n

Measurement temperature range: 60 ° Celsius to one 5 ^e C Table 4

Melting temperature ()

SEQ ID No.Before UV irradiation After UV irradiation

7 (XI ') Τπ 25 3 6.

9 (Χ2 ') Τπ 25 6 10 9

8 (XI ') Tn 23 1 10 6

10 (X2,) T 25 8 1 1 4

Magnesium chloride concentration: 0.4 mol トル torr for SEQ ID NOs: 7,9,15

As for SEQ ID NOs: 8, 10, and 16, 0 1 mol liter As described above, it was revealed that the use of the photoresponsive oligonucleotide of the present invention changed the melting temperature by 1 o ° c or more by light irradiation. . On the other hand, as shown in the comparative examples, the melting temperature of the conventional unmodified oligonucleotide hardly changed before and after UV irradiation.

After that, when a visible light of 420 nm or more was irradiated for 10 minutes by passing through an L-42 filter using a xenon lamp, 80% or more returned to the transformer. When the melting temperature was determined under the same conditions as above, it was consistent with the value before UV irradiation within the error range. Thus, it was revealed that the melting temperature changes reversibly. The above operation was repeated five times, and the azobenzene residue was reversibly isomerized without any particular deterioration. The melting temperature also reversibly changed. Example 4: Synthesis of phosphoramidite monomer

H

NC

V 0

N

Man

VI

DMT = Dimethoxytrityl group According to the method described in Tetrahedron, 1998, Vol. 39, pp. 9015 to 9018, azobenzene was introduced as a side chain of an oligonucleotide by the following method. First, 4-aminoazobenzene is converted to 2,2-bis (hydroxymethyl) propionic acid by reacting dicyclohexyl carbamide with dimethyl sulfoxide (DMF) in the presence of 1-hydroxybenzotriazole. And introduced by amide bond (IV). The obtained compound (IV) was purified by recrystallization from a mixed solvent of hexane and chloroform. Next, one hydroxyl group of compound (IV) is reacted with 4,4-dimethoxytrityl (DMT) chloride in the presence of 4-dimethylaminoviridine in a mixed solvent of pyridine and methylene chloride to convert one hydroxyl group of compound (IV) to a dimethoxytrityl group (DMT). ) Protected (V). The obtained compound (V) was separated and purified by silica gel chromatography using a mixed solvent of methylene chloride: triethylamine: methanol = 90: 5: 10 as a developing solvent. Thereafter, a phosphoramidide was added to the other hydroxyl group according to the method described in The Journal of Organic Chemistry, Vol. 62, p. 846, 1997, to give a phosphoramidite monomer (VI). Was synthesized.

Example 5

Using the phosphoramidite monomer synthesized in Example 4, photoresponsive oligonucleotides of the present invention having various sequences were synthesized by a DNA synthesizer. All of the resulting photoresponsive oligonucleotides have two diastereomers based on the asymmetric carbon of the phosphoramidite monomer (VI), which must be completely separated by liquid chromatography (HPLC) under the following conditions. Was completed.

Column: Merck LiChrospher 100 RP-18ie

Flow rate: l.Oml / min

Developing solvent: Acetonitrile Z water mixed solvent containing 50 mM ammonium formate

One h

The elution conditions were such that the concentration of acetonitrile increased linearly from 5% to 25% o in 25 minutes.

Under the above conditions, the diastereomer having the shorter retention time (eluting first) was used in the following experiment. One of the synthesized photoresponsive oligonucleotides (SEQ ID NO:

19) is shown in the following equation.

〇

5'-CCGAXGTCG- 3 'X two

(19)

Test example 4:

Using a DNA synthesizer, the oligonucleotide synthesized in Example 5 (oligonucleotide complementary to photoresponsive oligonucleotide (SEQ ID NO: 19): GGC TC AGC (SEQ ID NO: 20) was synthesized.

An aqueous solution was prepared by adding the photoresponsive oligonucleotide synthesized in Example 5 to a 1 OmM phosphate buffer (pH 7.1) containing 11 ^ & 〇1 to a concentration of 5 OiM. . The oligonucleotide (SEQ ID NO: 20) was added to a 1 OmM phosphate buffer (pH 7.1) containing 1 M NaCl to a concentration of 50 M to prepare an aqueous solution.

The aqueous solution of the photoresponsive oligonucleotide (SEQ ID NO: 19) prepared above and the aqueous solution of the above oligonucleotide (SEQ ID NO: 20) are mixed, and the azobenzene group is in a trans form by measuring the absorbance at 260 nm. The Tm of the case was determined. Next, each mixed solution was irradiated with ultraviolet rays (wavelength 300 to 400 nm) to make the structure of azobenzene a cis-form. Thereafter, Tm was determined by the same method as described above.

Tm when the azobenzene group is in the trans form is 35.0. In contrast to C, the Tm of the cis form was 25.0 ° C, and the difference was 10 ° C. Example 6: Synthesis of phosphoramidite monomer

According to the above reaction formula, a phosphoramidite monomer was synthesized.

First, 3--2-troazobenzene was synthesized by coupling 3-nitroazoline and nitrosobenzene in acetic acid ( _a ). Next, this was reduced with NaSH in a mixed solvent of ethanol and water to obtain 3-aminoazobenzene (b). Subsequently, 3-aminoazobenzene is converted to 2,2-bis (hydroxymethyl) propionic acid by reacting dicyclohexylcarbodiimide with 1-hydroxybenzotriazole in dimethyl sulfoxide (DMF). (C) This was separated and purified by column chromatography, and then mixed with 4,4 dimethyltrityl (DMT) in the presence of 4-dimethylaminoviridine in a pyridine-methylene chloride mixed solvent. The hydroxyl group was protected (d). This was separated and purified again by column chromatography, and then the other hydroxyl group was prepared according to the method described in The Journal of Organic Chemistry, 1997, Vol. 62, p. 846. A phosphoramidite monomer was synthesized by adding a phosphoramidide to the product.

Example 7: Synthesis of photoresponsive oligonucleotide

Using the phosphoramidite monomer synthesized in Example 6, a photoresponsive oligonucleotide shown in Table 5 below was synthesized by a DNII synthesizer. Table 5

Sequence number sequence

21 5'-ΑΑΑ (Χιη) ΑΑΑΑ-3 '(Ρ) 22 5'-AAA (XHI) AAAA-3 '(L)

23 5'-Α (Χια) ΑΑΑΑΑΑ-3 '(p)

24 5'-Α (ΧΠΙ) ΑΑΑΑΑΑ-3 '(l)

25 5 '-(Xm) TTTTTTTTTTT-3'

In the table, Xm represents a photoresponsive organic group represented by the following formula.

〇

ヽ

In all of the photoresponsive oligonucleotides obtained, there are two diastereomers based on the asymmetric carbon of the phosphoramidite monomer of formula VI above, except for the case where azobenzene is introduced at the 5, terminal (SEQ ID NO: 25). It could be separated by liquid chromatography (HPLC) under the following conditions.

(P) and (L) correspond to the shorter and longer diastereomers, respectively. Column: Merck Lichrospher 100RP-18 (e)

Passivity: 0.5ml / min

Developing solvent: Acetonitrile / water mixed solvent containing 50 itiM ammonium formate was eluted under elution conditions such that the concentration of acetonitrile increased directly from 5% to 25% in 25 minutes.

Test Example 5: Measurement of thermal isomerization rate

The pH of the photoresponsive oligonucleotide of the present invention synthesized in Example 7 was adjusted to 7.0 with 1 Ommo 1 / liter of a buffer, and the salt concentration was adjusted to 1 mol / liter with sodium chloride. It dissolved in the aqueous solution adjusted so that it might become. Using a xenon lamp, the UVD-36C filter is passed through the filter at a wavelength of 300 nm or more and 400 n Light (UV light) of 10 m or less was irradiated for 10 minutes. As a result, 90% of the added photoresponsive oligonucleotide was isomerized to cis form. 50 in a thermostat. The thermal isomerization rate constant k was determined by keeping the temperature at C and tracing the process from the cis-form to the trans-form by UV-Vis spectrum. Table 6 shows the results.

Table 6

Sequence number Rate constant k (min- ¹ )

21 0.01 or less

22 0.01 or less

23 0.01 or less

24 0.01 or less

25 0.01 or less

As shown in Table 6, the photoresponsive oligonucleotides of the present invention were found to have sufficiently low rates of thermal isomerization from cis to trans form. Test Example 6: Measurement of melting temperature

The corresponding complementary strand was added to each of the oligonucleotides of SEQ ID NOs: 21, 22, 23, and 24, and the melting temperature was measured, thereby evaluating the stability of the duplex. The conditions for melting temperature measurement are shown below.

p H (10 mmo 1 Z l N a ₂ HP 0 ₄ buffer): 7.0

Oligonucleotide: 50 m 01 / liter

Sodium chloride: 1 mo 1 / liter

Measurement wavelength: 260 nm

Temperature sweep speed: — 1.0 ° C / min

Measurement temperature range: Table 7 shows the melting temperatures measured under the conditions of 60 ° C to over 15 ° C. Before UV irradiation, more than 80% of the azobenzene residues were in trans form. UV irradiation was performed by irradiating the sample containing the oligonucleotide with light of 300 nm or more and 400 nm or less through a UVD-36C filter using a xenon lamp for 10 minutes. as a result, More than 80% changed to cis form. The melting temperature was determined as the temperature giving the maximum value of the first derivative of the melting temperature curve. Table 7

Melting temperature C)

SEQ ID NO Complementary strand U Before irradiation After UV irradiation

21 5'-ΤΤΤΤΤΤΤΤ-3 24.3 16.6

22 5 '-ΤΤΤΤΤΤΤΤ-3 20.5 15.1

23 5,-ΤΤΤΤΤΤΤΤ-3 '26.0 20.4

24 5'-ΤΤΤΤΤΤΤΤ-1 3 '22.7 19.4 After that, when irradiated with visible light of 420 nm or more for 10 minutes by passing through an L-42 filter using a xenon lamp, 80% or more returned to the transformer. When the melting temperature was determined under the same conditions as above, it was consistent with the value before UV irradiation within the error range. Thus, it was revealed that the melting temperature changes reversibly. The above operation was repeated five times, and the azobenzene residue was reversibly isomerized without any particular deterioration. The melting temperature also reversibly changed. Industrial applicability

By using the photoresponsive oligonucleotide of the present invention, it is possible to reversibly change the melting temperature of the duplex and the triplex by light irradiation. Therefore, it becomes possible to reversibly control gene expression, DNA extension reaction and the like by light irradiation. This makes it possible to perform DNA, RNA synthesis, amplification, transcription, and the like under the control of light, thereby improving the reliability and efficiency of experiments in various fields of biotechnology. In addition, by using the photoresponsive oligonucleotide of the present invention for controlling gene expression, it is possible to perform gene diagnosis and gene therapy efficiently.

Claims

The scope of the claims

1. A double strand of an oligonucleotide having one or more organic groups that are structurally isomerized by irradiation with light of a specific wavelength, and which is substantially complementary to the oligonucleotide. A photoresponsive oligonucleotide whose melting temperature in formation or triplex formation is reversibly changed by irradiation with the light.

2. The light according to claim ¹ , wherein the rate of thermal isomerization of the photoresponsive oligonucleotide from one isomer to the other is 0.07 min1 or less at 50 ° C. Responsive oligonucleotide.

3. The photoresponsive oligonucleotide according to claim 1, wherein the organic group is a residue of azobenzene or a derivative thereof, and is bonded to the oligonucleotide directly or via an intervening group.

4. The photoresponsive oligonucleotide according to claim 2, wherein the organic group is a residue of azobenzene or a derivative thereof, and is bonded to the oligonucleotide directly or via an intervening group.

5. The following formula:

(Wherein A and B each independently represent a hydrogen atom, a nucleotide or an oligonucleotide, X represents a residue of azobenzene or a derivative thereof, and R represents a hydrogen atom or a linear alkyl having 1 to 4 carbon atoms. The photoresponsive oligonucleotide according to claim 1, which is represented by the following formula: Next formula

A

Nb

O o

po o ll

o

H X NO B

(Wherein, A and B each independently represent a hydrogen atom, a nucleotide or an oligonucleotide, Nb represents a nucleobase, and X represents a residue of azobenzene or a derivative thereof). The photoresponsive oligonucleotide according to the above item.

7. The photoresponsive oligonucleotide according to claim 5, wherein X is represented by the following formula I, II or ill.

I

(In the above i, II and ill, RR ", R ²¹ are each a direct bond; unsubstituted or halogen atom, a hydroxyl group, an amino group, a nitro group, has been carbon atom number 1-2 0 substituted with a carboxyl group or the like An alkylene group or an unsubstituted or substituted alkenylene group having 2 to 20 carbon atoms such as a halogen atom, a hydroxyl group, an amino group, a nitro group or a propyloxyl group, wherein Q is a direct bond, an oxygen atom, NH—CO— group or one CO—NH— group, R ^{2 to} R ⁷ , R ^s , R ¹⁰ , R ¹² to: R ¹⁷ , R ¹⁹ , R ² , R ²² to R ² R ² R ^3D Is independently an alkyl or alkoxy group having 1 to 20 carbon atoms, which is unsubstituted or substituted by a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, etc .; an unsubstituted or halogen atom, a hydroxyl group, an amino group Carbon source substituted with a group, nitro group, carboxyl group, etc. . Alkenyl or aralkyl Kiniru group having 2-2 0, a hydroxyl group, a halogen atom; Amino group; a nitro group; represents a or carboxyl group R ^8, R ^1S,: ²⁸ each independently unsubstituted or halogen atom, An alkyl or alkoxy group having 1 to 20 carbon atoms substituted with a hydroxyl group, an amino group, a nitro group, a carboxyl group, etc .; unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, etc. An alkenyl or alkynyl group having 2 to 20 carbon atoms; a hydroxyl group; or a halogen atom).

8. The photoresponsive oligonucleotide according to claim 6, wherein X is represented by the following formula, II or ill. (I)

(II)

/

R

(In the above i, II and ill, I ¹ , R ¹¹ , and R ²¹ are each a direct bond; the number of carbon atoms is 1 to 10 A 20-alkylene group or an alkenylene group having 2 to 20 carbon atoms, which is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, etc., where Q is a direct bond or an oxygen atom , —NH—CO— group or one CO—NH— group, and: ^{2 to} R ⁷ , R ⁹ , R ¹⁰ , R ^{12 to} R ¹⁷ , R ″, R ^2. , R ²² to R ² R ² R ^3D is each independently an unsubstituted or halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group or the like, an alkyl or alkoxy group having 1 to 20 carbon atoms; an unsubstituted or halogen atom, a hydroxyl group, Carbon substituted with amino group, nitro group, carboxyl group, etc. Alkenyl or aralkyl Kiniru group atoms 2-2 0; hydroxyl; represents a group or a carboxyl group; a halogen atom; Amino group; a nitro group. ^8, R ^18, R ²⁸ are each independently unsubstituted or substituted with a halogen atom , A hydroxyl group, an amino group, a nitro group, a carboxyl group, etc. A alkenyl or alkynyl group having 2 to 20 carbon atoms, which is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, or the like; a hydroxyl group; or a halogen atom ).

9. In the above formulas Ι, ΙΙ, ill, RR ¹¹ and R ²¹ represent a direct bond, and R ² to R ^1D , R ^{12 to} R ^2D and R ^{22 to} R ³ ° are a hydrogen atom or a carbon atom having 1 to 8. The photoresponsive oligonucleotide according to claim 7, which represents 20 linear alkyl groups, and Q represents —NH—CO— or —CO—NH—.

1 0. The above equation Ι, ΙΙ, in ill, IT, R ¹¹ and R ²¹ represents a direct ^{^{bond, R 2 ~ R 10. 11}} 12 ~1 2 ° and 1 ²² to 1 ³ ° is a hydrogen atom or a C 9. The photoresponsive oligonucleotide according to claim 8, wherein the oligonucleotide represents a linear alkyl group having 1 to 20 atoms, and Q represents -NH-CO- or 1CO-NH-.

1 1. In the above formulas Ι, ΙΙ, ill, IT, R ¹¹ and R ²¹ represent a direct bond, R ² to R ^1Q , R ¹² to ² ° and R ^{22 to} R ^3Q represent a hydrogen atom. 8. The photoresponsive oligonucleotide according to claim 7, wherein Q represents -NH-CO- or -CO-NH-.

1 2. In the above formulas Ι, ΙΙ, ill, IT, R ¹¹ and R ²¹ represent a direct bond, R ² to R ¹⁰ , R ¹² to: ² ° and H ^{22 to} R ^3Q represent a hydrogen atom, 9. The photoresponsive oligonucleotide according to claim 8, wherein Q represents -NH-CO- or -CO-NH-.

1 3. above formula Ι, ΙΙ, in ill, RR ¹¹ and R ²¹ represent an alkylene group having 1 to 4 carbon ^{^{atoms, R 2 ~ R 1Q, R}} 12 ~R 2Q and R ²² to R ³ ° is 8. The photoresponsive oligonucleotide according to claim 7, wherein the photoresponsive oligonucleotide represents a hydrogen atom or a linear alkyl group having 1 to 20 carbon atoms, and Q represents an oxygen atom.

14. In the above formulas Ι, ΙΙ, ill, IT, R ¹¹ and R ²¹ represent an alkylene group having 1 to 4 carbon atoms, and R ^{2 to} R ^1Q , R ^{12 to} R ² ° and R ^{22 to} R ³ 9. The photoresponsive oligonucleotide according to claim 8, wherein ° represents a hydrogen atom or a linear alkyl group having 1 to 20 carbon atoms, and Q represents an oxygen atom.

1 5. In the above formulas ι, ιι, ill, ΙΓ, R ¹¹ and R ²¹ represent an alkylene group having 1 to 4 carbon atoms, R ² to: R ^1Q , R ¹² to: ² ° and R ²² to 8. The photoresponsive oligonucleotide according to claim 7, wherein R ³ ° represents a hydrogen atom, and Q represents an oxygen atom.

1 6. In the above formulas Ι, ΙΙ, ill, RR ¹¹ and R ²¹ are alkyls having 1 to 4 carbon atoms. Represents a len group, R ² ~! ^1D, R ¹² ~R ^2Q and R ²² to R ^3Q represents a hydrogen atom, Q is photoresponsive O Rigo nucleotide claims paragraph 8, wherein an oxygen atom.

17. The photoresponsive oligo according to any one of claims 1 to 16, which has a sequence that is substantially complementary to at least a part of DNA that forms type II of the DNA extension reaction. Using nucleotides, the organic group of the above-mentioned photoresponsive oligonucleotide is isomerized from one isomer to another by irradiation with light having a wavelength within a specific range, and is irradiated with light having a wavelength within another specific range. A method for controlling a DNA extension reaction comprising reversibly forming and dissociating a duplex or a triplex by isomerization in reverse.

18. Having a sequence substantially complementary to at least a part of mRNA to be controlled, using the photoresponsive oligonucleotide according to any one of claims 1 to 16 within a specific range. Isomerization of the organic group of the photoresponsive oligonucleotide from one isomer to another by irradiation with light having a wavelength within the range described above, and vice versa by irradiation with light having a wavelength within another specific range. A method for controlling gene expression including reversibly forming and dissociating a duplex between the photoresponsive oligonucleotide and mRNA.

19 Order

(In the formula, Α, represents a protecting group, B ′ represents a reactive group for a phosphodiester bond at the 5-position of the oligonucleotide, X represents the residue of azobenzene or a derivative thereof, and R represents hydrogen. 6. The compound for synthesizing a photoresponsive oligonucleotide for synthesizing a photoresponsive oligonucleotide according to claim 5, wherein the compound is a straight-chain alkyl group having 1 to 4 carbon atoms.

20. A ³ represents a dimethoxytrityl group or a monomethoxytrityl group, Β 'is

NC

0 par

Ν

20. The compound for synthesizing a photoresponsive oligonucleotide according to claim 19, which represents a group represented by human.

21. X is the following formula I:

(Wherein Q represents an oxygen atom, R ¹ represents an alkylene group having 1 to 4 carbon atoms, and R ^{2 to} R ^1Q represents a hydrogen atom or a straight-chain alkyl group having 1 to 20 carbon atoms). 21. The compound for synthesizing a photoresponsive oligonucleotide according to claim 20, which is a group represented by the formula.

22. X is the following formula II:

Wherein Q represents an oxygen atom and R ¹ represents an alkylene group having 1 to 4 carbon atoms, or Q represents —NH—CO— or 1 CO—NH— and R ¹ represents a direct bond. 21. The photoresponsive oligonucleotide synthesis according to claim 20, wherein R ² to R ^1Q represent a hydrogen atom or a linear alkyl group having 1 to 20 carbon atoms. Compound.

23. The following equation: b

O X

B 、

(Where A, represents a protecting group, B, represents a reactive group for the phosphodiester bond to the 5'-position of the oligonucleotide, Nb represents a nucleobase, and X represents a azobenzene or derivative thereof. 7. The compound for synthesizing a photoresponsive oligonucleotide for synthesizing a photoresponsive oligonucleotide according to claim 6, which is represented by the following formula: 24. A 'represents a dimethoxytrityl group or a monomethoxytrityl group, and B' represents the following formula:

0

NC

0 par

N A

Man

24. The compound for synthesizing a photoresponsive oligonucleotide according to claim 23, which represents a group represented by the formula:

25. X is the following formula I:

(I)

(Wherein Q represents an oxygen atom, R ¹ represents an alkylene group having 1 to 4 carbon atoms, and R ² to ^{1 (1} represents a hydrogen atom or a linear alkyl group having 1 to 20 carbon atoms.) 25. The compound for synthesizing a photoresponsive oligonucleotide according to claim 24, which is a group represented by the following formula:

26. X is the following formula II

(Wherein Q represents an oxygen atom and R ¹ represents an alkylene group having 1 to 4 carbon atoms, or Q represents —NH—CO— or 1 CO—NH— and H ¹ represents a direct bond. 25. The photoresponsive oligonucleotide according to claim 24, wherein: ^ ² to 1 ^ ° represents a hydrogen atom or a linear alkyl group having 1 to 20 carbon atoms. Compound for synthesis.