JP3103855B2 - Stabilized ribozyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stabilized ribozyme, and more particularly to a stabilized ribozyme by artificially modifying the ribozyme while maintaining the function of the ribozyme that specifically cleaves target RNA. Alternatively, the present invention relates to a stabilized ribozyme having a structure, which can be effectively used as a drug, an animal drug, a pesticide, a reagent, or the like.

[0002]

2. Description of the Related Art Conventionally, it was believed that all enzymes were composed of proteins. However, in 1981, an RNA molecule having enzyme activity, that is, a ribozyme was discovered, and the conventional concept of enzymes was broken. The landmark discovery was made by T. Chech and colleagues at the University of Colorado, where the protozoan tetrahymena ribosomal RNA (rRNA) precursor could transmit genetic information without the help of proteins. Unnecessary introns (IV
S) was proven to be removed by self-splicing (Cell, Vol. 31, p. 147-157 (1982); Nature, Vol. 308,
p.820-825 (1984)). Subsequently, RNA molecules having a catalytic function of self-splicing phosphodiester bonds have been found one after another, and RNA molecules (ribozymes) that cleave other RNA molecules have also been found.

Under these circumstances, recently, Haserov (J. Hase
Loff) and Jarrach (WLGerlach) focused on the nucleotide sequences that are commonly conserved among ribozymes of several plant viruses, and the size of the ribozyme molecule excluding the target RNA binding region was composed of only 24 bases. The short-chain ribozyme was successfully constructed using genetic engineering techniques and enzymatic techniques (Nature, Vol. 334, p. 585-591 (1988); Japanese Translation of PCT International Publication No. 3-502638). In addition, Otsuka et al.
Active short-chain ribozymes that catalyze the cleavage of molecules have been prepared by chemical synthesis (JP-A-2-195883).

FIG. 1 illustrates the structure and action site of a short-chain ribozyme. Ribozymes are the target RNA
Binding region C that recognizes the base sequence of a molecule and forms a base pair
And a region B (including a catalytically active site in which A, G or U is circled) having 24 specific base sequences, and the target RNA is an A region (GUC) in the target RNA.
At the position adjacent to (indicated by the arrow in FIG. 1).
It is known that the sequence of the A region is cleaved not only by GUC but also by other sequences.

[0005] The ribozyme is prepared by synthesizing a DNA encoding ribozyme RNA based on the ribozyme nucleic acid base sequence, inserting this into a plasmid, and transforming the recombinant plasmid with a suitable restriction enzyme. Cleavage is performed to obtain a DNA fragment encoding ribozyme RNA and transcribe the DNA fragment in vitro as a template, or by sequentially linking ribonucleotides by a chemical synthesis method. In the literature.

[0006]

RNases are widely distributed in nature. Ribozymes are easily degraded and deactivated by this RNase, and thus have a problem in stability. The present invention provides a stable modified ribozyme having resistance to RNase attack, that is, a stabilized ribozyme, while maintaining the specific ribozyme specific RNA degrading activity, that is, a drug, an animal drug, a pesticide or a reagent. It is intended to be used effectively for such purposes.

[0007]

Means for Solving the Problems In order to achieve the above object, the present inventors have studied various modification methods for stabilizing a ribozyme. As a result, the present inventors have found that a phosphate ester bond of a ribozyme [-OP (= O) (OH) -O-] is bonded to a thiophosphate ester bond [-OP (= S) (O
H) -O- or -OP (= O) (SH) -O-] or in addition thereto, the non-catalytic region of the molecule or structure constituting the ribozyme (in FIG. 1,
By substituting one or more of the constituting monoribonucleotides with the corresponding monodeoxyribonucleotides in the region other than the site where A, G or U is circled, the ribozyme is capable of its specific RNA The present inventors have found that a stable modified ribozyme having resistance to RNase attack while maintaining the degradation activity, that is, a stabilized ribozyme, has completed the present invention.

That is, the present invention provides the following (1) to (5)
It is about. (1) A stabilized ribozyme represented by the following formula (I).

Embedded image (In the formula (I), A, G, C, and U represent adenine monoribonucleotide, guanine monoribonucleotide, cytosine monoribonucleotide, and uracil monoribonucleotide, respectively; The symbol C represents adenine monodeoxyribonucleotide, guanine monodeoxyribonucleotide, or cytosine monodeoxyribonucleotide, respectively, and the shaded X represents any one of adenine monodeoxyribonucleotide, guanine monodeoxyribonucleotide, cytosine monodeoxyribonucleotide, and thymine monodeoxyribonucleotide. Represents a monodeoxyribonucleotide of the formula
Is a thiophosphate bond [-OP (= S) (OH)
-O- or -OP (= O) (SH) -O-],
m and n represent one or more integers which may be different from each other, and * represents a base pair between complementary nucleotides. )

(2) The active group and the 5'-hydroxyl group of the nucleobase are protected by a protecting group, and the 3'-hydroxyl group is converted from (a) from monodeoxyribonucleoside (solid phase) bound to the carrier via a cross-linking structure. ) After removing the protecting group for the 5'-hydroxyl group, the solid phase was washed, and (b) the active group of the nucleobase and 2 '
Ribonucleoside-3'-O- (β-cyanoethyl) phosphoramidite or a nucleobase active group in which-and 5'-hydroxyl groups are protected by a protecting group, and deoxyribonucleoside-3 in which 5'-hydroxyl group is protected by a protecting group '-O- (β
-Cyanoethyl) phosphoramidite, then washing the solid phase, (c) washing the solid phase after acetylation of unreacted 5'-hydroxyl group, and washing the solid phase after oxidation with an oxidizing agent Performing the steps in any order, and (d)
Hereinafter, (a) to (c) are repeated, (e) the solid phase is treated with an alkali to cut off the bond between the 3′-hydroxyl group and the carrier, and (f) other protective groups are removed. The method for producing a stabilized ribozyme according to the above (1).

(3) The method according to the above (2), wherein the oxidizing agent contains iodine and / or tetraethylthiuram disulfide.

(4) A method for cleaving or inactivating a target RNA, which comprises reacting the target RNA with the stabilized ribozyme of the above (1).

(5) A drug, animal drug, pesticide or reagent comprising the stabilized ribozyme of (1) as an active ingredient.

The stabilized ribozyme represented by the formula (I) of the present invention may be either a phosphorylated 3′- or 5′-terminal hydroxyl group or a free one. . The thiophosphate ester bond of the stabilized ribozyme represented by the formula (I) has an R-type and an S-type stereoisomer.

The method for producing the stabilized ribozyme of the present invention comprises the steps of:
It is based on the beta-cyanoethylphosphoramidite method of the so-called solid-phase chemical synthesis method, but when methylphosphoramidite is used, it is possible to produce stabilized ribozymes by simply adding a treatment with thiophenol. It is.

The active group of the nucleic acid base is, for example, an amino group, and the protecting group is, for example, a benzoyl group, anisoyl group, isobutyryl group and the like. 5'- from solid phase
To remove the hydroxyl-protecting group, a dichloromethane solution of dichloroacetic acid or trichloroacetic acid or the like can be used. Examples of the protecting group for the 2′-hydroxyl group include a t-butyldimethylsilyl group, and examples of the protective group for the 5′-hydroxyl group include a dimethoxytrityl group, a monomethoxytrityl group, and a trityl group.

Acetylation of unreacted 5'-hydroxyl group is 1-
The reaction can be carried out by acetic anhydride containing methylimidazole or dimethylaminopyridine. Washing of the solid phase is usually performed using acetonitrile.

As the oxidizing agent, a solution containing iodine, water, pyridine and tetrahydrofuran is used when a phosphate ester bond is formed, and tetraethylthiuram disulfide, 3H, 1,3 is used when a thiophosphate ester bond is formed. 2-benzodithiol-1,1-dioxide or the like is used.

To cleave the desired stabilized ribozyme from the solid phase, an alkaline solution such as 15 to 30% ammonia is used. The stabilized ribozyme synthesized in this manner is one of the methods commonly used for nucleic acid purification, such as reverse phase, column chromatography such as ion exchange, polyacrylamide gel electrophoresis, and agarose gel electrophoresis, or one of them. Purify by combination of

Since the stabilized ribozyme of the present invention cleaves or inactivates target RNA, it can be effectively used as a drug, an animal drug, a pesticide and the like. Examples of the target RNA include RNAs derived from various pathogenic viruses, RNAs derived from oncogenes, and RNAs derived from genes involved in cell growth and differentiation.
The reaction of cleavage or inactivation may be appropriately selected under suitable conditions. For example, in the presence of 5 to 500 mM magnesium or manganese ions, pH 8.0 ± 3.0, temperature 20 to 7
It is good to carry out at 0 ° C.

Examples of the medicament include a medicament for treating AIDS, leukemia, various malignant tumors, epidemic conjunctivitis and the like. Animal drugs include, for example, animal drugs for treating various viral diseases such as cows, pigs, chickens, dogs, and cats. Examples of pesticides include pesticides against various plant virus diseases such as tobacco mosaic disease, rice dwarf disease and cucumber mosaic disease. The reagent includes, for example, a biochemical reagent for research, such as for RNA analysis and RNA processing, and a diagnostic agent.

[0021]

The present invention will be described in detail with reference to the following examples. Example 1 Synthesis of stabilized ribozyme represented by the following formula (II)

Embedded image (In the formula (II), A, G, U, C, shaded A, shaded G, shaded C, s and * have the same meaning as in the formula (I).)

After a dC-CPG column (cytosine amount: 1 μmol) was attached to a DNA / RNA synthesizer (Model 380B, manufactured by Applied Biosystems), the following (1) to (1) to According to the procedure of (5), the stabilized ribozyme of the formula (II) was synthesized.

(1) A 2% (w / v) solution of trichloroacetic acid in dichloromethane is passed through the column at 1.6 ml / min for 50 seconds to remove the dimethoxytrityl group of the 5′-hydroxyl group. (2) After washing the column with acetonitrile, 5'-O
- dimethoxytrityl-deoxyguanosine -N ² - isobutyl -N, N-diisopropyl aminoethyl phosphoramidite in acetonitrile (77 / ml) and 4%
(W / v) 1: 1 solution of tetrazole in acetonitrile
(Volume ratio) The mixed solution was supplied at 1.5 ml / min for 10 seconds, held for 30 seconds, and a condensation reaction was caused to form a phosphite bond. (3) After washing the column with acetonitrile, 10% (v /
v) 1: a tetrahydrofuran solution containing acetic anhydride and 10% (v / v) 2,6-lutidine and a tetrahydrofuran solution containing 16% (v / v) 1-methylimidazole.
1 (volume ratio) mixture is flowed at 1.5 ml / min for 20 seconds to acetylate unreacted 5'-hydroxyl groups. (4) After washing with acetonitrile, acetonitrile solution containing 15% tetraethylthiuram disulfide was added to 1.
The solution is allowed to flow at 6 ml / min for 23 seconds and left for 15 minutes to oxidize the phosphite bond to a thiophosphate bond and wash again with acetonitrile. (5) The above steps (1) to (4) are repeated according to the third and subsequent nucleic acid base sequences from the 3 ′ side shown in the formula (II).

Here, the amidite reagent of the procedure (2) is
In the case of A, 5'-O-dimethoxytrityl-2'-O-
t-butyldimethylsilyl-adenosine-N ⁶ -benzoyl-N, N′-diisopropylaminocyanoethylphosphoramidite; in the case of G, 5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyl-guanosine-N ² -isobutyryl-N, N'-diisopropylaminocyanoethylphosphoramidite, in the case of C, 5'-
O-dimethoxytrityl-2′-Ot-butyldimethylsilyl-cytidine-N ⁴ -benzoyl-N, N′-diisopropylaminocyanoethylphosphoramidite, U
In the case of 5′-O-dimethoxytrityl-2′-Ot
- butyldimethylsilyl - uridine -N, N'-diisopropylamino cyanoethyl phosphoramidite, if the shaded A, 5'-O-dimethoxytrityl-deoxyadenosine -N ⁶ - benzoyl -N, N'-diisopropylamino cyanoethyl phosphoramidite If the shaded G, 5'-O-dimethoxytrityl-deoxyguanosine -N ² - isobutyryl -N, N'-diisopropylamino cyanoethyl phosphoramidite, if the hatching of C, 5'-O-dimethoxytrityl-deoxycytidine −
Using N ⁴ -benzoyl-N, N′-diisopropylaminocyanoethylphosphoramidite, when the sugar moiety of the amidite reagent is deoxyribose, the same conditions as described above are used. In the case of ribose, the amidite reagent in acetonitrile solution (77 mg / ml) is used. ) And a 1: 1 (volume ratio) mixture of 4% (w / v) tetrazole in acetonitrile was supplied at 1.5 ml / min for 10 seconds, and then reacted for 10 minutes. If a phosphite bond is formed instead of a thiophosphate bond, the procedure (4)
Instead of flowing an acetonitrile solution containing 15% tetraethylthiuram disulfide at 1.6 ml / min for 23 seconds and allowing it to stand for 15 minutes, instead of "3% (w / v) iodine, 20% (v / v) pyridine, And 2% (v / v) H
A tetrahydrofuran solution containing ₂ O was added at 1.6 ml / min.
Let flow for 5 seconds and let sit for 20 seconds "to oxidize the phosphite bonds to phosphate bonds.

After all the amidite reagents have been reacted, the 5′-hydroxyl protecting group is removed in the same manner as in step (1), and a 3: 1 (3: 1 by volume) mixture of ammonia and ethanol is added. After flowing for 18 seconds and holding for 15 minutes, the product was cut out of the column. This cutting operation was repeated three more times. Next, the synthesized product was treated at 55 ° C. for 8 hours to remove the protecting groups for the nucleic acid base and the phosphate group. After removing ammonia and ethanol under reduced pressure, they were dissolved in an appropriate amount of water, and the absorbance at a wavelength of 260 nm was measured. After removing the water under reduced pressure, 1 is adjusted so that the absorbance at a wavelength of 260 nm becomes 10.
M was dissolved in a tetrahydrofuran solution of tetrabutylammonium fluoride (hereinafter abbreviated as TBAF), and kept at room temperature for 12 hours to remove the 2'-hydroxyl protecting group. A 0.1 M triethylammonium-acetate buffer (hereinafter abbreviated as TEAA) in the same volume as the added TBAF was added and freeze-dried.

Next, the freeze-dried product was purified using an OPC column (manufactured by Applied Biosystems). That is, the column was washed first with 5 ml of acetonitrile, then with 5 ml of 2M TEAA, and
The lyophilized solution dissolved in 1 was flowed at a rate of 1 to 2 drops / second, and the flow-through was again flowed through the column to adsorb the target substance. After washing the column with 5 ml of TEAA and then 10 ml of deionized water, 1.8 ml of 50% (v / v) acetonitrile was flowed at 1-2 drops / second to elute the target substance.

The eluate was further purified by FPLC (Pharmacia). That is, the column is PepRPCH
R10 / 10, column temperature 40 ° C., flow rate 2 ml / m
The mobile phase was TEAA containing acetonitrile having an initial concentration of 10% (v / v). After loading the eluate on the column, the concentration of acetonitrile in the mobile phase was reduced to 1 over 1 hour.
The target substance was eluted with a linear increase from 0% (v / v) to 60% (v / v). The elution position of the target was 25
The absorbance at 4 nm was measured and monitored.

Example 2 Synthesis of stabilized ribozyme represented by the following formula (III)

Embedded image (In the formula (III), A, G, U, C, shaded A, shaded G, shaded C, s, and * have the same meaning as in the formula (I).)

In the same manner as in Example 1, a DNA / RNA synthesizer (380B manufactured by Applied Biosystems) was used.
Type) to dC-CPG column (cytosine amount: 1 μmol)
And using the version 1.34 program,
A stabilized ribozyme of the formula (V) was synthesized and purified by an OPC column and FPLC.

Comparative Example Synthesis of ribozyme represented by the following formula (IV)

Embedded image (In the formula (IV), A, G, U, C and * have the same meaning as in the formula (I).)

In the same manner as in Example 1, a DNA / RNA synthesizer (380B manufactured by Applied Biosystems) was used.
), A stabilized ribozyme of the formula (IV) was synthesized using a program of version 1.34, and purified by an OPC column and FPLC.

Test Example 1 Purity test of stabilized ribozyme The stabilized ribozyme synthesized in Examples 1 and 2 (formula (II)
And a ribozyme (compound of the formula (IV)) synthesized for comparison, respectively.
Take 3.0 μg each, and add 10 × Kation buffer (100 mM magnesium chloride and 100 mM
500 mM Tris-HC containing mercaptoethanol
buffer, pH 7.6) 1.0 μl, [γ- ³² P] AT
P (185 terabecquerels / 370 megabecquerels / ml /
0.5 μl of a 5 × -diluted solution (mmol), 0.5 μl of T4 polynucleotide kinase (Takara Shuzo) and purified water to make a total volume of 10 μl each, and reacted at 37 ° C. for 30 minutes. In this way, after phosphorylating the 5'-end and labeling with ³² P, heat treatment was performed at 70 ° C. for 10 minutes.
T4 polynucleotide kinase was inactivated. This is 2
It was electrophoresed in a 0% acrylamide gel at a voltage of 1800 volts for about 3 hours and analyzed by autoradiography (FIG. 2). It was found that both the stabilized ribozyme and the synthetic ribozyme labeled with ³² P corresponded to the size calculated from the nucleic acid base sequence, and contained almost no impurities.

Test Example 2 Stability test of stabilized ribozyme in serum The stabilized ribozyme synthesized in Examples 1 and 2 (formula (II)
And 2.4 μg each of a ribozyme (compound of formula (IV)) synthesized for comparison.
10 × Kation buffer (100m each)
500 mM Tris-HCl buffer containing M magnesium chloride and 100 mM mercaptoethanol, pH
7.6) 6 μl, [γ- ³² P] ATP (370 megabecquerel / ml and 185 terabecquerel / mmol)
3.0 μl, 1.0 μl of T4 polynucleotide kinase (Takara Shuzo) and purified water were added to make a total volume of 60 μl, and reacted at 37 ° C. for 30 minutes. In this way, the 5'-end was phosphorylated and labeled with ³² P, and then heat treated at 70 ° C. for 10 minutes to inactivate T4 polynucleotide kinase. Take 10 μl of each of the ³² P-labeled stabilized ribozyme or synthetic ribozyme solutions and add a reaction stop solution (9 M urea, 50 mM ED).
10 μl containing TA, 0.1% xylene cyanol and 0.1% bromophenol blue),
Sample. To 50 μl of each of the remaining stabilized ribozymes or ribozyme solutions, 5.5 μl of bovine serum diluted to a concentration of 1% with purified water was added, and the mixture was kept at 37 ° C. 5 minutes, 1 hour, 3 hours, and 6 hours after the start of heat retention
Each μl was sampled, added to the same amount of the reaction stop solution, and stored at −80 ° C. until analysis. At the time of analysis, these samples were thawed at room temperature, electrophoresed in a 20% polyacrylamide gel, and analyzed using an image analyzer (FIG. 3).

As a result, degradation of the synthetic ribozyme (comparison:
Shown by □. ) Is about 80% after 3 hours and about 90% after 6 hours, whereas the degradation of the stabilized ribozyme of formula (II) of the present invention (indicated by Δ) is about 3 hours after the reaction. 5
%, Only about 10% after 6 hours, and was found to be quite stable against bovine serum.

Test Example 3 Target RNA Cleaving Activity of Stabilized Ribozyme Target RNA (5′-GC labeled with ³² P at the 5 ′ end)
CGUCCCCCCG-3 ′) using 17.4 μM as a substrate,
In 20 μl of 50 mM Tris-HCl buffer (pH 8.0) containing 25 mM MgCl ₂ , the stabilized ribozyme (compound of formula (II) or (III)) synthesized in Example 1 or 2 or the ribozyme synthesized for comparison ( (Compound of formula (IV)) was reacted at a concentration of 0.788 μM at 37 ° C. After the start of the reaction, 5, 10 and 20 minutes after the start of the reaction, 5 μl of each sample was sampled, and a reaction stop solution (9 M urea, 5 M
5 μl containing 0 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) was added and stored at −80 ° C. until analysis. During analysis, these samples were thawed at room temperature and then electrophoresed in a 20% polyacrylamide gel (FIG. 4). From the results in FIG. 4, the stabilized ribozyme represented by the formula (II) of the present invention is:
It turns out that the target RNA cleavage ability is maintained.

[0036]

Industrial Applicability The present invention provides a method for preparing RNA from a conventional ribozyme.
A novel stabilized ribozyme having resistance to a degrading enzyme and the like is provided. In addition, the stabilized ribozyme of the present invention can cleave target RNA in a sequence-specific manner and inactivate the target RNA, so that the ribozyme is widely applied to drugs for treating diseases caused by RNA, veterinary drugs or agricultural chemicals. Furthermore, it can be applied as a reagent such as a diagnostic agent or a biochemical reagent as a restriction enzyme for cleaving RNA.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the secondary structure of a ribozyme and a cleavage site of a target RNA.

FIG. 2 is a polyacrylamide gel electrophoresis diagram of the stabilized ribozyme of the present invention (comparison: synthetic ribozyme).

FIG. 3 is a graph showing the time-dependent change of the stabilized ribozyme when the stabilized ribozyme of the present invention (comparative: synthetic ribozyme) was reacted with bovine serum.

FIG. 4 is a polyacrylamide gel electrophoresis diagram showing a time-dependent change of a reaction product when a stabilized ribozyme of the present invention is reacted with a target RNA.

[Explanation of symbols]

In FIG. 1, large letter A: G in target RNA
UC region Large letter B: A region having 24 specific base sequences including the catalytic active site in the ribozyme Large letter C: Binding region in the ribozyme that forms a base pair with the target RNA Arrow: Target RNA cleavage site A, G or U circled: catalytically active site In FIG. 2, II: compound of formula (II) III: compound of formula (III) IV: compound of formula (IV) ↓: electrophoresis direction diagram In 3, ３: compound of formula (II) ○: compound of formula (III) □: compound of formula (IV)

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hidekatsu Maeda 1-3-1 Higashi, Tsukuba City, Ibaraki Pref. Inside the Research Institute of Microbial Technology (72) Inventor Takashi Shimayama 2-1-1 Nishishinjuku, Shinjuku-ku, Tokyo No. 1 Inside Hitachi Chemical Co., Ltd. (72) Inventor Hiroshi Izutsu 48 Tsukuba, Ibaraki Prefecture, Tsukuba Development Laboratory, Hitachi Chemical Co., Ltd. (72) Inventor Atsushi Okawa 2--2 Muroya, Nishi-ku, Kobe City, Hyogo Prefecture No. 3 Nagase Sangyo R & D Center (72) Inventor Soichi Okabe 2-3-2 Muroya, Nishi-ku, Kobe City, Hyogo Prefecture Examiner, Nagase Sangyo R & D Center Mayumi Saito (58) Field (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 A01N 63/00 A61K 31/00-48/00 C07H 21/00-21/04 C12N 9/00-9/99 BIOSIS (DIALOG ) A (STN) MEDLINE (STN) REGISTRY (STN) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A stabilized ribozyme represented by the formula (I). Embedded image (In the formula (I), A, G, C, and U represent adenine monoribonucleotide, guanine monoribonucleotide, cytosine monoribonucleotide, and uracil monoribonucleotide, respectively; The symbol C represents adenine monodeoxyribonucleotide, guanine monodeoxyribonucleotide, or cytosine monodeoxyribonucleotide, respectively, and the shaded X represents any one of adenine monodeoxyribonucleotide, guanine monodeoxyribonucleotide, cytosine monodeoxyribonucleotide, and thymine monodeoxyribonucleotide. Represents a monodeoxyribonucleotide of the formula
Is a thiophosphate bond [-OP (= S) (OH)
-O- or -OP (= O) (SH) -O-],
m and n each represent an integer of 1 or 2 which may be different, and * represents a base pair between complementary nucleotides. ) 2. The method according to claim 1, wherein the active group and the 5′-hydroxyl group of the nucleobase are protected by a protecting group, and the 3′-hydroxyl group is converted from the monodeoxyribonucleoside (solid phase) bound to the carrier via a cross-linking structure. After removing the protecting group for the 5'-hydroxyl group, the solid phase is washed, and (b) the active group of the nucleobase and 2'- and 5 '
A ribonucleoside-3 ′ in which a hydroxyl group is protected by a protecting group;
-O- (β-cyanoethyl) phosphoramidite or deoxyribonucleoside-3′-O- (β-cyanoethyl) phosphoramidite in which the active group of the nucleobase and the 5′-hydroxyl group are protected with a protecting group are reacted, (C) acetylating the unreacted 5′-hydroxyl group and then washing the solid phase, and oxidizing with an oxidizing agent and washing the solid phase in any order. Less than,
(A) to (c) are repeated, (e) the solid phase is treated with alkali to break the bond between the 3′-hydroxyl group and the carrier,
(F) removing another protecting group.
A method for producing a stabilized ribozyme. 3. The method according to claim 2, wherein the oxidizing agent contains iodine and / or tetraethylthiuram disulfide. 4. A method for cleaving or inactivating a target RNA, comprising reacting the target RNA with the stabilized ribozyme according to claim 1. 5. A pharmaceutical, veterinary, pesticide or reagent comprising the stabilized ribozyme according to claim 1 as an active ingredient.