JPH02195883A - Cutting method of specific part of polyribonucleotide by ribozyme - Google Patents

Abstract

PURPOSE:To obtain polyribonucleotide useful for cutting and detoxication of viroid single chain cyclic RNA as a cause of plant dwarfing by constructing ribozyme having special nucleotide sequence and cutting activity at specific part in polyribonucleotide sequence. CONSTITUTION:A polyribonucleotide having nucleotide sequence expressed by formula I. A polyribonucleotide containing nucleotide sequence of C is cut at a part expressed by arrow by using (1) a polyribonucleotide containing nucleotide sequence A and not containing nucleotide sequence B, and a polyribonucleotide containing nucleotide sequence B and not containing nucleotide sequence C, or (2) a polyribonucleotide containing nucleotide sequence A and nucleotide sequence B, and not containing nucleotide sequence C.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a polyribonucleotide constituting a lipozyme that has catalytic activity to cleave a specific site in a polyribonucleotide sequence, and also relates to This invention relates to a method of cleaving specific sites in other polyribonucleotide sequences using nucleotides.

As an example of the industrial application of the present invention, it is expected that it will be possible to cleave and detoxify viroid-main-stranded circular RNA, which is thought to be the cause of the disease of various plants.

(Prior Art) There are several reported examples of RNA or polyribonucleotides having self-cleavage activity. Zaug et al. found that Tetrahymena precursor liposomal RNA forms liposomal RNA by self-cleavage (N
ature, 324.429-433° (I986)
), Hutchins et al. found that the positive and negative RNA transcripts obtained from the complementary DNA of avocado sun blotch viroid each self-cleaved, and that the nucleotide sequences near the cleavage site were highly homologous. RNA transcripts have similar secondary structures near the cleavage site, and we predicted that cleavage would occur at specific sites in specific sequences (Nucleic Ac1
d Res, 14.3627-3640, (I98
6) Uhlenbeck synthesized 24n+er and 19rser polyribonucleotides containing the consensus sequence of this viroid using T7 RNA polymerase and synthetic NA, and found that self-cleavage occurred at a specific site of the 24mer ( Nature,
328.596-600. (See Figure 1 of (I987)).

Epstein et al. found that a transcript of newt satellite DNA with a chain length of about 300 strands self-cleaves at a specific site, and the nucleotide sequence near the cleavage site of this is similar to the nucleotide sequence near the cleavage site of avocado sun blotch viroid. (Cel
l, 48.535-543. (I987)) Previously, the present inventors chemically synthesized two types of 21-mar polyribonucleotides containing the cleavage site of this newt satellite RNA, and when they were formed into double strands, one strand was identified. It was found that the cleavage occurred at the site of (FEB).
S Lett,, 228.228-230, (I9
88), see Figure 1). At the same time, we investigated the sequences essential for self-cleavage and found that base pairs 9-14 A-U and base pairs 10-13 C-G can be exchanged with each other. These RNAs, or RNAs having polyribonucleotide cleaving activity, are collectively called polyribonucleotide cleavage enzymes.

However, all of the bozymes that have been found so far self-cleave, and none that cleave other RNA or polyribonucleotide molecules have been found.

(Problems to be Solved by the Invention) The present inventors have studied polyribonucleotide sequences that do not self-cleave but recognize specific sequences of other polyribonucleotides and cleave at specific sites. As a result, by using two molecules of polyribonucleotides with two different sequences, or polyribonucleotides having the above two types of sequences in the same molecule, which three-dimensionally recognizes a polyribonucleotide with a specific sequence, discovered a method for cleaving polyribonucleotides having the specific sequence, and completed the present invention. That is, the present invention relates to a polyribonucleotide containing a specific sequence constituting a ribozyme that has the function of cleaving other polyribonucleotides at a specific site, and further relates to a polyribonucleotide containing a specific sequence that constitutes a ribozyme that has the function of cleaving other polyribonucleotides at a specific site. The present invention relates to a method of cleaving a specific site of a third polyribonucleotide having a specific sequence by constructing a ribozyme from the polyribonucleotide having the specific sequence.

The present invention is a polyribonucleotide containing the nucleotide sequence represented by A and not containing the nucleotide sequence represented by C of formula Cb); or use a polyribonucleotide that is not
Using a polyribonucleotide containing the nucleotide sequence represented by the nucleotide sequence represented by and the nucleotide sequence represented by B but not containing the nucleotide sequence represented by C, the polyribonucleotide containing the nucleotide sequence represented by C is indicated by an arrow. The present invention relates to a method of cutting at a cutting site. : 3・115・ (In the formula, U is uracil, A is adenine, C is cytosine,
G represents a guanine nucleotide, R represents any of uracil, adenine, or cytosine nucleotide, 8
X3 to X5 corresponding to 3 to S5, Y1 to Y2 corresponding to X□~X! represents any of uracil, adenine, cytosine, or guanine nucleotides that can form hydrogen bonds, and corresponds to S,~S2. , V
Y3-Y4 corresponding to a-v4 and υ□-v2 corresponding to Z1-Z2 are hydrogens with matching integer numbers in at least two group combinations among the three group combinations. Represents any uracil, adenine, cytosine, or guanine nucleotide that can form a bond. ).

In particular, in formula (b), X! corresponding to S, ~S2! ~
Y3 to Y4 and Z corresponding to X2, ■3 to v4.

~Z2 corresponds to ~J2, or ~J2 is any of uracil, adenine, cy[-sine, or guanine nucleotides that can form complementary hydrogen bonds in the combination of these three groups, respectively, with matching integer numbers. This invention relates to a method for cleaving certain polyribonucleotides.

Particularly preferably, in formula (b), R, Sl, Wt
, (I1a, Xs, Y3, Z2 and 2. are adenine'/L/otide, S2, Sl, S,
t, V2, W4. Yt and Y4 are cytosine nucleotides, Vl, V4, X2, X3, X4, Y
2 and Z4 are guanine nucleotides, Ss, V3.
The present invention relates to a polyribonucleotide cleavage method in which W2, us, Xs, Zs and Z3 are uracil nucleotides.

The A chain of formula (b) is a polyribonucleotide having a nucleotide sequence represented by formula (a): "ACCCLIGAAAGCUG" (a
) (wherein U represents uracil, A represents adenine, C represents cytosine, and G represents guanine nucleotide).

A novel polyribonucleotide, particularly preferred 1. (Means to be solved by the invention) A) Synthesis of a polyribonucleotide chain from a polyribonucleotide is carried out after protecting the 2'-hydroxyl group and the 5'-hydroxyl group. This can be carried out by condensation of unit nucleotides using the solid-phase phosphoramidite method (Sequential Biochemistry Experiment Course 1, Gene Research Methods 1, pp.
35-61. 1986, Tokyo Kagaku Doujin). That is, the 2″-hydroxyl group is protected with a tetrahydrapyranyl group, etc., the 5′-hydroxyl group is protected with a dimethoxytrityl group, etc., and the base portion is protected as necessary, and a nucleoside is reacted with a phosphoramidite. By this method, a completely protected unit nucleotide (nucleoside 3'-0-phosphoramidite) can be obtained.The polyribonucleotide chain is elongated by protecting the 5'-hydroxyl group with a dimethoxytrityl group, etc., and converting the 3' This can be carried out by removing the protecting group of the 5'-hydroxyl group of a nucleoside resin containing the desired base, in which the hydroxyl group is bonded to the carrier via a crosslinked structure, and then reacting the desired unit nucleotides in sequence.

B) Group 71. Next, the methyl group attached to the phosphate group is removed, the polyribonucleotide chain is cut out from the resin by alkali treatment,
By removal of the 2'-hydroxyl protecting group, 5'-hydroxyl protecting group, and base moiety protecting group by acid treatment, followed by purification using desalting treatment, reversed phase, ion exchange chromatography, etc. A desired polyribonucleotide chain can be obtained.

C) To confirm the base sequence of a polyribonucleotide chain, label the 5' end of the polyribonucleotide chain, fragment it with snake venom phosphogesterase, and perform, for example, electrophoresis or homochromatography. It is possible to do this by conducting such things as
．． 1986, Tokyo Kagaku Doujin). Chain length can also be determined by performing electrophoresis on labeled polyribonucleotide chains.

D) Cleavage of polyribonucleotide cleavage of other polyribonucleotide strands by the polyribonucleotide strand can be confirmed by the following procedure (Chemistry, Vol. 43, No. 5, p.
p, 348-349, (I988)). The 5'-end of the polyribonucleotide chain used as a substrate is labeled, and a buffer solution containing a polyribonucleotide chain having catalytic activity, magnesium chloride, and sodium chloride is added to this and kept warm.

After a certain period of time, the reaction is stopped by adding EDTA to the reaction solution, and this solution is subjected to polyacrylamide gel electrophoresis. After electrophoresis, the cleavage rate can be calculated by cutting out the gel and quantifying the radioactivity at the cleavage site.

Hereinafter, as a reference example, a method for synthesizing a nucleoside 3'-0-phosphoramidite, and as an example, cleavage of a ribonucleotide chain will be described, but the present invention is not limited thereto.

Reference Example Nucleoside 3″-〇-phosphoramidite was synthesized by the reaction represented by the following formula.

(In the formula, ThP represents a tetrahydropyranyl group, D represents a trityl group, and B' represents any one of benzoyladenine, isobutyrylguanine, benzoylcytosine, and anisoyluracil.) The synthesis in which B' is benzoyl adenine, isobutyrylguanine, benzoylcytosine and anisoyluracil will be described.

1) B' = benzoyladenine benzoyladenosine 2'-tetrahydropyranyl-
52-dimethoxytrityl compound ((I) 0.61 mg,
0.8 tomol) was dehydrated with pyridine, dichloromethane (2 ml) and isopropylethylamine (0.8 tomol)
, 56 ml, 3.2 mmol) and purged with nitrogen to dissolve chlorodiinpropylaminomethylphosphine (
0.19 ml, 0.9611 I+uol) was added dropwise over 2 minutes.

After stirring at room temperature for 40 minutes and confirming that the raw material had disappeared, ethyl acetate (30011) was added, and the mixture was washed with saturated sodium bicarbonate solution and saturated brine. After 1PS (manufactured by Wandman) filtration, the solvent was distilled off and silica gel chromatography (Wakogel C-300, 15 g) was carried out.

(ethyl acetate) to obtain the desired product as a foamy substance.

Collection flO,72: (0,781110O1) 98χ
Hz), 2.0-1.4 (m, 6)1. -CH2-of
Thp), 1.2-0.9(o, 128.・NCI(
(Cqt)t).

IP-NMR (CDCl) δppm:
148.15. 148.68 Compounds (2) in which B' is isobutyrylguanine, benzoylcytosine, and anisoyluracil were also synthesized in a similar manner. The yield and physical properties are shown below.

2) B' = isobutylguanine, yield R: 88%'H
-NMR (CDCl) δppa+: 11.5 (m,
I) I, 8M), 8.7 (n, IFI, NH), 7.
79(s, It(,)14), 7.7-7.2(m, 9
Ar of trityl).

6.8(m+48,2+2',6.6'Hof tr
ityl), 5.9(m, 2JLl' Jl-, 2-
near, 3H2and H-2').

4.8-4.0 (a+, to 3)! -3', 4' ai
d acetall(of Thp), 3.77(s
, 68. -0CH3of trityt).

3.7-2.9 (Il, IOH, H-5', -0CH2
-of Thp, -HClj-, P-QC) It, -C
Of4(CH3)2), '2.0-1,6(+w
, 6, -CHt-of Thp) mountain 4 (m, 12) 1
．． -11cH(Cut)s).

3-blind P-NMR (CDCh)"" δGll+II:
148.21. 148.483) B' = benzoylcytosine, yield: 9e'H-NMR (CDC+3)
δppm: H-NMR (CDCI) δppm:
8.01 (d, ILH-6Js, g=8.3Hz), ?
,9(m,3)1. +1-6. o-Ar)+7.3(m
, IIH, m-Arand trityl) 6.9 (m
, 4H, 2, 2', 6. (i'Hof trityl)
, 6.17 (m, 1 MIH-1'), 5.31 (d, 1
HJI-5, Js, g: 8.4IHz), 5.0 (br
s, IH, acetall (of Thp), 4.6 (
m, 28. I (-2', 3'), 4.3 (m, II, I
(-4')3.86(s, 311.-0CHz of
anisoyl), 3.81(s, 61(, -01Jl
z of trityl).

3.5(I11,5FI, H-5'-NCR-an
d -0CI (2 of Thp), 3.3 (d
d, 3H, P-OCH3, jp-ocylx: I2.9
Example 1゜Poj1Bofre No. 3''G32. S' B [I) 4) B' = anisoyluracil, yield: 96, formula (I
A@ (hereinafter abbreviated as IIA) of I), and formula (II
The B chain (hereinafter abbreviated as 111B) of I) was synthesized.

That is, 2'-hydroxyl group is replaced by tetrahydropyranyl group, 5'-
A nucleoside resin in which the hydroxyl group is protected with a dimethoxytrityl group is treated with an acid, and the nucleoside resin is synthesized by a solid-phase phosphoramidite method in which the chain is extended in the 5' direction. The method for synthesizing IA will be described, but other polyribonucleotide chains were also synthesized in the same manner.

Chain elongation was performed according to the following formula.

A@ (hereinafter abbreviated as IA) and B@ (hereinafter IB) in formula (I)
It is abbreviated as ), c chain (hereinafter abbreviated as IC) (symbols in the formula represent the following.

DMTr: “'A: Benzoyladenine n: Number of condensed nucleotides [F]: Controlled pore glass (control,
(1edpore glass) ThP and B' have the same meanings as described above.

As a model for polyribonucleotide chain synthesis, an Applied Biosystems (ABI) model 380 DNA synthesizer was used. Nucleoside 3'-〇-phosphoramidite ((4) 18 mg, about 20 μmol) is 0.1
A solution prepared to be dissolved in 6 ml of acetonitrile was set in bottle #]-4 of the synthesizer. Bottle #1
-4 was set with 1x dichloro iff/dichloromethane. Other reagents used were from ABI.

First, the method of Kierzek et al. (Biochemist
ry, 25°7.840-7,846 (I986)
), synthesized nucleoside horse chestnut ((3), Iμ
mol) was treated with 1z dichloroacetic acid/dichloromethane for 120 seconds to remove the dimethoxytrityl group.

Next, nucleoside 3'-0-phosphoramidite (
(4), 20 equivalents) and tetrazole (50 equivalents) were used to oxidize phosphorus in Roamiguite to produce phosphoric acid.

By repeating the above reaction once, a polyribonucleotide (5) having the desired base sequence was obtained.

Example 2 Removal of the protecting group in the polyribonucleotide chain and cutting out from the resin were carried out according to the following formula.

(The abbreviations in the formula represent the following.

B: Any one of adenine, guanine, cytosine, or uracil.

DMTr, Thp[F], n and B' have the same meanings as described above. ) That is, a thiophenol-triethylamine/dioxane solution was allowed to act on Compound (5) for 30 minutes to remove the methyl group as a protecting group for the phosphoric acid group. Thereafter, the resin was cut out by treatment with a concentrated ammonium hydroxide solution at room temperature for 1 hour. The ammonium solution containing the excised polyribonucleotide was sealed and heated at 60°C for 5 hours. After that, the solvent was distilled off, and reversed-phase force chromatography (C18, φ0.7 x 14 cm) was performed.
The peak with the color development of dimethoxytrityl cation caused by perchloric acid was examined, and both components were collected and the solvent was distilled off.

Add 0.01 N hydrochloric acid to this, and add 0.01 N hydrochloric acid. The pH was adjusted to 2.0 with IN hydrochloric acid, and the mixture was left to stand at room temperature for 20 hours to remove dimethoxytrityl groups and tetrahydropyranyl groups. 0. I
After neutralizing with N ammonium hydroxide and washing with ethyl acetate,
ephadex G-25 (φ1.8 x 38cm)
The crude compound (6) was obtained by desalting.

Example 3 The crude compound (6) obtained in Example 2 of ``1 polynucleotide'' was purified by reversed phase high performance liquid chromatography and ion exchange high performance liquid chromatography.

Regarding reverse phase high performance liquid chromatography, YMC
PACK C18 column (φ1.0 x 30cm,
The test was carried out using Yamamura Kagaku Co., Ltd.).

Purification was carried out using a 0.1 M triethylammonium acetate buffer by an io -15% acetonitrile concentration gradient method (flow rate 2 ml/win).

The RNA fraction was collected using an absorption wavelength of 254 nm.

The separation pattern of reversed phase high performance liquid chromatography is shown in Figure 1. The horizontal axis shows time, and the vertical axis shows absorption at 254 nm and acetonitrile concentration.

For ion exchange chromatography, TSK gel DEAE-2SW column (φ0.46x 25c
m, manufactured by Toyo Soda Co., Ltd.). Purification was performed by a 0.5-0.9 M ammonium acetate concentration gradient method using 20% acetonitrile (flow rate 1 m
l/win,). A polyribonucleotide fraction was collected based on the absorption wavelength of 254 nm. The separation pattern of ion exchange high performance liquid chromatography is shown in Figure 2. The horizontal axis represents time, and the vertical axis represents absorption at 254 nm and ammonium acetate concentration.

Through the above separation and purification operations, 4.75 A=bo units of IA were obtained.

Example 4゜8 Cutting IA of polyrinucleotide (
0,015A26° unit, 100 pmol), [γ
-”P] ATP (0.5μl), buffer (Iμm,
250mM Tri 5-HCI (pH 7,6
) + 5On+M magnesium chloride, 50mM mercaptoethanol), T4 polynucleotide kinase (
0.5 μm, 1 unit), add sterile water (3 μl),
It was kept warm at ℃ for 1 hour. In order to remove unreacted [γ-”P]ATP, it was spotted on DEAE Cellulocell TLC,
Homo m i x m (Nucleic Ac1
dRe5earch, 1.331-353 (I974
)). The radioactive portion containing polyribonucleotides was scraped off and eluted from the DEAE cellulose with 2M triethylammonium bicarbonate.

Add snake venom phosphogesterase (0.5 μl) to this.

1 mglo, 5 ml) and kept warm at 37°C.
After 30 minutes, the ice was fractionated to obtain a limited ice melt. About this 1
Electrophoresis at pH 3.5 was performed in the second dimension, and homochromatography was performed in the second dimension to confirm the sequence.

The results for IA, IB and IC are shown in Figure 3.
4 and 5. In Figures 3.4 and 5, the horizontal axis shows the direction of electrophoresis, and the vertical axis shows the direction of migration of homochromatography.

Mu po! The 5'' ends of the rice polyribonucleotides DA and mB of summer bonucleotides were labeled, and the chain lengths were confirmed by electrophoresis on a two-circle polyacrylamide gel containing 8M urea.The results are shown in Figure 6. , 1an
e1: IA.

), ane 2: I B, 1 ane 3: I C
, 1ane 4: I[A, 1ane 5: mB, X in the figure
C indicates a xylene cyan tool. ) Example 5゜The IC used as a substrate has [γ-22P]AT at the 5' end.
P, after labeling with T4 polyoliponucleotide kinase, N
Desalting with ENSORB 20 (manufactured by Dupont),
The protein-removed product was used.

The cleavage reaction was carried out as follows. IC at 2.5pm
ol, and I corresponding to the catalytic polyribonucleotide
A and I B (lane 3), I[A and IB (l
ane 4) or IA and IIIB (lane 5
) for 3.75 P each! 1101 with 25 mM magnesium chloride, 40 mM Tri 5-HCI (
pH 7,5), 20118 common salt composition solution (2,3μ
1) was added and kept at 15°C for 15 hours. As a control,
A buffer solution containing 5 mM EDTA was added instead of 25 iaM magnesium chloride in lane 3, and the mixture was kept warm (lane 2).
) was added to stop the reaction and analyzed by 20x polyacrylamide gel electrophoresis containing 8M urea.

As a marker (lane 1), an IC whose 5' end was labeled was cut with RNase U2 and electrophoresed.

The cleavage rate was determined by cutting out the gel and quantifying it using a liquid scintillation counter. The results are shown in Table 1.

Table 1 - 1ane cleavage rate (%) The results of electrophoresis are shown in FIG. 7. In Figure 7, RNase
U2 cleaves the bond between the purine 3' position of the polyribonucleotide chain and the phosphate of the following nucleotide, and is used as a marker for A2, G3, A6, and A7.
, A8 indicates the mobility of fragments cleaved after the second adenine, third guanine, sixth, seventh, or eighth adenine from the 5' side of each IC. This indicates that the other 1ane was cleaved after the 6th adenine.

FIG. 1 shows the separation pattern of synthetic polyribonucleotide crude compound (IA) by reversed phase high performance liquid chromatography.

FIG. 2 shows the separation pattern obtained by subsequent ion-exchange high performance liquid chromatography.

Figure 3 shows IA (7), Figure 4 shows IB (7), and Figure 5 shows IC (7).
) Shows patterns of base sequence analysis by electrophoresis and homochromatography.

Figure 6 shows IA, IB, IC1IIA by electrophoresis.
Comparison patterns of chain lengths of and mB are shown.

Figure 7 shows IA and IB, IIA and IB, or IA and mB.
The electrophoretic pattern of the cut fragment of IC is shown in FIG.

Claims (1)

[Claims] 1. Polyribonucleotide having a nucleotide sequence represented by formula (I): ^5^'ACCCUGAAAGCUG^3^' (a) (
In the formula, U is uracil, A is adenine, C is cytosine, G
represents a guanine nucleotide. ). 2. Formula (b) [1] A polyribonucleotide containing the nucleotide sequence represented by A but not containing the nucleotide sequence represented by C, and a polyribonucleotide containing the nucleotide sequence represented by B and containing the nucleotide sequence represented by C. or using polyribonucleotides that do not contain
Using a polyribonucleotide containing the nucleotide sequence represented by A and the nucleotide sequence represented by B but not containing the nucleotide sequence represented by C, the polyribonucleotide containing the nucleotide sequence represented by C is indicated by an arrow. Method of cutting at the site ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (b) (In the formula, U is uracil, A is adenine, C is cytosine,
G represents a guanine nucleotide, R represents any of uracil, adenine, or cytosine nucleotide, and S
X_3-X_5, V_1-V_ corresponding to _3-S_5
Y_1 to Y_2 corresponding to 2 and Z_3 to Z_5 corresponding to W_3 to W_5 are uracil, adenine, cytosine, and those having matching integer numbers in the combination of these three groups can form complementary hydrogen bonds, respectively. Represents any guanine nucleotide, X_1 to X_2 corresponding to S_1 to S_2, Y corresponding to V_3 to V_4
W_1~ corresponding to _3~Y_4 and Z_1~Z_2
W_2 represents any one of uracil, adenine, cytosine, and guanine nucleotides that have matching integer numbers that can form complementary hydrogen bonds in at least two of the three group combinations. )
. 3. In formula (b), X corresponding to S_1 to S_2
Y_3-Y_ corresponding to _1-X_2, V_3-V_4
4, and W_1 to W_2 corresponding to Z_1 to Z_2 are uracils in which those with matching integer numbers can form complementary hydrogen bonds, respectively, in a combination of these three groups,
The method for cleaving polyribonucleotides according to claim 2, wherein the polyribonucleotide is any one of adenine, cytosine, and guanine nucleotides. 4. In formula (b), R, S_1, W_1, W_3,
X_5, Y_3, Z_2 and Z_5 are adenine nucleotides, S_2, S_3, S_4, V_2, W_4, Y
_1 and Y_4 are cytosine nucleotides, V_1, V
_4, X_2, X_3, X_4, Y_2 and Z_4 are guanine nucleotides, S_5, V_3, W_2, W_
5. The method for cleaving polyribonucleotides according to claim 2 or 3, wherein X_1, Z_1 and Z_3 are uracil nucleotides.