JPH08224086A - Compound for cutting rna at specific site - Google Patents

Abstract

PURPOSE: To provide a compound having an RNA derivative oligomer bonded to a terminal of a DNA oligomer, capable of easily and site-selectively cutting an RNA independent of the length and base sequence of the molecular chain and enabling the gene manipulation at an RNA level. CONSTITUTION: This new mixed oligomer contains a DNA oligomer for cutting a specific site of an RNA and an RNA derivative oligomer bonded to the DNA oligomer. It has an RNA derivative oligomer having a substituent such as methyl group on at least one of the 2'-site hydroxyl groups of the sugar part bonded to one or both terminals of a DNA oligomer through a phosphoric acid diester bond between the 5'-hydroxyl group and the 3'-hydroxyl group of ribose or deoxyribose part. The oligomer is capable of easily and site-specifically cutting an RNA molecule independent of the length and the base sequence of the molecular chain and useful for the mass-production of valuable proteins, improvement of the function, clarification of the relationship between the structure and the function, intoxication and utilization of harmful RNA virus and the establishment of effective treating method and enables the gene manipulation at an RNA level.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to mass production of useful proteins, improvement of functions, or elucidation of the relationship between structure and function, and on the other hand, detoxification of harmful RNA viruses and their use or establishment of effective therapeutic methods. R is useful for molecular biology research in the process of achieving these
The present invention relates to an oligomer which can selectively cleave a specific position of RNA upon manipulation at the NA level, which is a powerful means for creating a deletion mutant.

[0002]

2. Description of the Related Art Regioselective cleavage of RNA molecules is a powerful tool for structural elucidation and functional studies of many functional RNA molecules. For example, studies on the structure and stability of the translation rate of m-RNA encoding useful proteins and enzyme proteins, studies on the structure and function of r-RNA,
It can be used for research related to the structure and function at the molecular level of RNA virus genes harmful to animals and plants

Conventionally, as a method of specifically cleaving an RNA molecule, a method of using a base-specific RNA degrading enzyme obtained from nature, such as RNase T ₁ or U _2, etc., or a sequence complementary to the RNA molecule is used. RNase H (liponuclease H) by making a double strand with the DNA that it has
To cleave the RNA at the double-stranded site by the action of H. (H. Donis-Keller, Nucl
eic Acids Res. , 7 , 179, 197
9) etc. are known. The former is widely used as a method for determining the nucleotide sequence of RNA molecules because it is difficult to cleave only at a specific position, and cleavage occurs at a large number of locations, rather, it cleaves in a base-specific manner (H. Do
nis-Keller et al, Nucleic
Acids Res. , 4 , 2527, 1977). Regarding the latter, if the DNA chain is lengthened, cleavage occurs at various positions, and conversely, if the length of the DNA is shortened to 4 bases or 6 bases, it is possible to selectively cause cleavage, but if the sequence is short, various types of cleavage occur. Since it is not possible to improve the regioselectivity due to the fact that a double strand can be formed at this position, it is impossible to regioselectively cleave the RNA molecule by a conventionally known method.

[0004]

[Problems to be solved by the invention] Mass production of useful proteins,
In order to improve its function or to elucidate the relationship between structure and function, or to detoxify harmful RNA viruses and their use, or to establish an effective therapeutic method against them, elucidation of the structure and function of functional RNA It is important to develop a technique, which is important for research, that can easily and selectively cleave an RNA molecule regardless of the length or base sequence of its molecular chain or the base sequence.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have reported that the 2'-O-methylated RNA previously reported has a complementary sequence R.
Finding that a stable double chain is formed with the NA molecule (H. Ino
ue et al, Nucleic Acids Sy
mposium Series. , No. 16, 16
5,1985) (1), the stable double-stranded RNA chain and the 2'-O-methylated RNA chain are
A new finding was obtained that NaseH did not cleave together.

[0006] That is, the present inventors have found that a mixed oligomer in which DNA is directly linked to 2'-O-methylated RNA can be conveniently produced, and that the mixed oligomer and an RNA molecule having a complementary sequence are used. Make a double chain,
By treating with RNaseH, the DNA oligomer portion in the mixed oligomer is 2'-O- at both ends or one end.
DNA even though methylated RNA is linked
Distinguished from oligomers, that is, only at that site RN
Based on this finding, the present invention was completed based on the finding that it can selectively cleave an RNA site complementary to the DNA oligomer, which serves as a recognition cleavage site for aseH.

That is, the present application is a mixed oligomer in which an RNA derivative oligomer is bound to one or both ends of a DNA oligomer. The number of bases of the DNA oligomer in the mixed oligomer of the present invention is 3 to 6, preferably 4
~ 5. Furthermore, the bond between the DNA oligomer and the RNA derivative oligomer is usually via a phosphodiester bond between the 5′-hydroxyl group and the 3′-hydroxyl group of the ribose or deoxyribose moiety, and the bond between the 2 ′ of the sugar moiety of the RNA derivative oligomer is 2 ′. It is preferable that at least one of the -position hydroxyl groups is an RNA derivative oligomer having a substituent.
That is, the compound for cleaving RNA at a specific position (mixed oligomer) of the present invention comprises a complementary DNA (-strand) capable of site-selectively cleaving RNA (+ strand).
A part of the chain) is replaced with an RNA derivative, and when the enzyme having ribonuclease H activity is acted on, the DNA
RNA selectively at the position corresponding to the unsubstituted portion of the (-strand)
It is characterized in that the (+ strand) can be cleaved. The compound for cleaving specific position of RNA of the present invention (mixed oligomer) is complementary DNA (-strand) forming a double strand with RNA (+ strand) for cleavage, and such a compound is It is not necessarily limited to those conventionally known, and includes those that can be prepared using conventional techniques.
In addition, it is not necessary to form a double-stranded portion with DNA (-strand) in the entire RNA (+ strand), and it may be a part. Further, the double-stranded portion may be formed not only at one location but at a plurality of locations.

When a part of the DNA (-strand) is replaced with an RNA derivative oligomer, the part of the DNA (-strand) may be the part of the-strand DNA molecule constituting the double-stranded portion, or When the double-stranded portion is formed at a plurality of locations, it may be the entire DNA (-strand) molecule present at at least one location.

The RNA derivative oligomer has, for example, a substituent described below on at least one of the 2'-hydroxy groups of the sugar moiety.

It is convenient to employ a mixed oligomer described below as the oligomer in which a part of the DNA (-strand) is substituted.

The mixed oligomer is obtained by directly binding the RNA derivative oligomer and the DNA oligomer between the 5'-hydroxyl group and the 3'-hydroxyl group of the ribose or deoxyribose moiety via a phosphodiester bond. can get.

BEST MODE FOR CARRYING OUT THE INVENTION

In the mixed oligomer of the present invention, for example, 2'-
In order to produce the O-methylated oligomer portion, two methods can be roughly classified. One of them is a method of manufacturing according to the method described in the above-mentioned document (1) of Inoue et al. That, 2'-O-methyl -N ⁴ - benzo Irushi cytidine (Compound I), 2'-O- methyl -N ⁶
- benzoyl adenosine (Compound II), 2'-O- methyl -N ² - isobutyryl guanosine (compound III) and 2'-O- to produce methyl uridine (Compound IV), further dimethoxytrityl group the respective compound (-DMT
r) is used to protect the 5'-position hydroxyl group, and the compound (Ia, IIa,
IIIa, IVa) is produced. The 3'-position hydroxyl groups of the compounds (Ia to IVa) are described in the literature (Nucleic Acids).
Res. , 8 , 5461, 1980) to introduce an orthochlorophenyl phosphate group to produce a compound (Ib ₁ , IIb ₁ , III b ₁ , IVb ₁ ) respectively (Method A).

Separately, the compounds (Ia, IIa, IIIa, IV
The 3'-hydroxyl group in a) is described in the literature (Tetrahedron).
Lett. , 24 , 245 (1983) or Nuc.
leic Acids Res. , 11 , 4539 (1
According to the method described in 984)), using a diisopropylamino methoxy phosphine or diisopropylamino cyanoethoxyphosphine, 3'-phosphoramidites compound _{(Ib 2, IIb 2, III} b 2, IVb 2, Ib
₃ , IIb ₃ , IIIb ₃ , IVb ₃ ) (see Examples 11 and 12 described later) (method B).

From the compounds (Ia to IVa), the literature (Nucleic Acids Res., 8 , 550) is used.
7,1980) according to the method described, it can be attached via a spacer to 1% cross-linked polystyrene resin, to produce the respective compounds _{(Ic 1, IIc 1, III} c 1, IVc 1).

On the other hand, the compounds (Ia, IIa, IIIa, IV
From a), the reference (K. Myoshiet al. Nu.
cleic Acids Res. , 8 , 5491 (1
980)) according to the method described in 3'-succinyl compound (I
e, IIe, IIIe, IVe) to the active ester form (If, II
f, III f, IV f), which is a long chain alkylamino control pore glass (Long chain alk).
ylamino controlled pore g
and the compound (Ic
₂ , IIc ₂ , IIIc ₂ , IVc ₂ ) can be produced (see Example 13 below).

On the other hand, regarding the DNA oligomer portion in the mixed oligomer, reference is made to the literature (M. Ikehara e.
t al, Proc. Natl. Acad. Sci. U
SA. , 81 , 5956, 1984) (2) (method A) and the compound (Id ₁ , IId ₁ , III)
It can be produced by sequentially condensing d ₁ , IVd ₁ (method A).

Alternatively, the literature (Science 230 , 2
81 (1985)).
_{d 2, IId 2, III d} 2, IVd 2) according to the method of Table 2, or the compound _{(Id 3, IId 3, III} d 3, IVd
_It can be produced by sequentially coupling ₃ ) (method B).

[0018]

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[0019]

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[0020]

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[0021] Thus, sequentially Compound III b ₁ Compound IVc ₁ as a starting _{material, IId 1, IId 1, IVd} 1, III d 1 I
By condensing II b ₁ , IIb ₁ , and Ib ₁ under the reaction conditions shown in Table 1 (see Example 1 described later), a mixed oligomer (compound V) having the following sequence could be produced. 3'UmGmAATGGmAmCm5 '(V) (units with m indicate O-methylated RNA, others indicate DNA oligomers)

Similarly, the following oligomers (compound VI to
VIII) was produced.

3'UmGmAmATGGmAmCm5 '(VI) 3'UmGmAATGGAmCm 5' (VII) 3'UmGmAmAmTGGACm 5 '(VIII)

A DNA oligomer (compound IX) having the same sequence as these mixed oligomers is prepared according to the above-mentioned reference (2) by Ikehara et al.
The small RNA oligomer (X) having a sequence complementary to X) is described in the literature (S. Iwai et al, Che.
m. Pharm, Bull. , 33, 4618, 198
5) It was produced according to the method described in (3).

3'd (TGAATGGAC) 5 '(IX) 5'ACUUACCUG3' (X)

Mixed oligomers having different sequences (XI, XI
I) and RNA oligomers (XIII, XIV) having complementary sequences thereto were also produced by the methods described in the above-mentioned documents (2) and (3).

3′CmAmTTCAUmAmGm5 ′ (XI) 3′GmUmCCAACmCmAm5 ′ (XII) 5′GUAAGUUAUC3 ′ (XIII) 5′CAGGUUGGU3 ′ (XIV)

On the other hand, high molecular weight RNA WS-S (+) (90
mer, compound XV) is planned to be prepared by utilizing an In Vitro transcription reaction using SP6-RNA polymerase, in which case WS-S (+) RNA is accurately prepared.
The double-stranded DNA (XVI) in which the WS-S (+) sequence was directly linked to the SP6-promoter sequence was chemically and enzymatically synthesized according to the scheme of FIG. did.

First, DNA oligomers (1 to 9) were chemically synthesized according to the method described in the literature (Science 230 281 (1985)), divided into two steps, and double-stranded DNA (XVI) was obtained by a ligase reaction. Obtained compound (XV
I) is SphI and Sma of M13.mp19 vector
Subcloning into E. coli JM1
03 is transformed into a transcription vector M
13AJ-1 was manufactured.

The synthetic double-stranded DNA sequence portion of the obtained transcription vector M13AJ-1 is described in the literature (Proc. Nat.
l. Acad. Sci. USA. , 74 , 5463 (1
977)) and the correct nucleotide sequence was confirmed using the M13 dideoxy method.

Next, M13AJ-1 was digested with the restriction enzyme SmaI.
After linearization with SP6-polymerase,
References (Nucleic Acids Res., 12 ,
7035 (1984)). Vi
tro. By performing RNA transcription reaction, RNAWS-S (+) (XV) transcribed from the 5'end was accurately produced as expected.

The high molecular weight RNA WS-S obtained here
In order to study the cleavage reaction of (+) (XV), it was examined to chemically synthesize a mixed oligomer using method B, which is simpler and more widely used than method A. That is, using the compound IVc ₂ as a starting material, the reaction conditions shown in Table 2 which are used for ordinary DNA automatic synthesis (see Example 1 described later).
4)), the compounds III b ₂ , III b ₂ , II
_{I b 2, Ib 2, Ib} 2, IIIb 2, IIb 2, IVb 2,
_{Ib 2, IVb 2, IId 2} , III d 2, III d 2, Id 2
Was condensed and purified by a conventional method, a mixed oligomer (XVIII) could be produced almost in the same manner as in DNA synthesis.

3′-UmGmGmGmCmCmGmAmUmCmUmAGGC-5 ′ (XVIII)

Similarly, the following mixed oligomer (XVII,
XIX, XXII to XXIV) were produced. 3'-UmGmGmGmCmCmGmAmUmCmTAGGC-5 '(XVII) 3'-UmGmGmGmCmCmGmAmUmCmUmAmGGC-5' (XIX) 3'-AGGCCmCmAmCmAmCmAm-5 '(XXII) 3'-GGCCmCmAmCmAmCmAm-5' (XXIII) 3'-UmGmAAAGCUmCm (XXIV)

In addition, the compound IVd ₄ was used as a starting material.
IId ₃ , III d ₃ , III b ₃ , Ib ₃ , Ib ₃ , IIb using the reaction conditions shown in (see Example 15 described later).
It was possible to produce a mixed oligomer (XX) by condensing ₃ , Ib ₃ , IIb ₃ , Ib ₃ , and IIb ₃ . 3'TAGGCmCmCmAmCmAmCmAm5 '(XX)

A mixed oligomer (XXI) 3'TAGGCCmCmAmCmAmCmAm5 '(XXI) was prepared in the same manner.

The mixed oligomer obtained is WS-S (+)
Compounds (XVII) which have complementarity with RNA (XV)
~ XIX) has only 3'-end replaced with O-methyl RNA,
Compounds (XX to XXIII) have only the 5'end substituted with O-methyl RNA, and compounds (XXIV) are compounds (V to XIII) described above.
And XI and XII), both ends are O-methyl RNA-substituted mixed oligomers.

The mixed oligomers (XVII to XXIII) are WS-
Compound (XXIV) is a compound designed to cut within the stem between the 19th and 24th positions from the 5'side of S (+) RNA, and mixed oligomer (XXIV) cuts within the loop of the 36th to 46th sequences. It is a compound designed to.

FIG. 2 shows mixed oligomers (XVII to XXIV) and W.
Relationship between complementary sequence with S-S (+) RNA (XV), W
The secondary structure of S-S (+) RNA is shown.

In the derivatives of compounds I to IV used for the preparation of the above mixed oligomer, X is a dimethoxytrityl group (-DMTr) or a monomethoxytrityl group (-M).
Normal DNA or RN such as trityl group instead of MTr)
It can be replaced with a protecting group used in A synthesis. Further, as the protecting group for phosphoric acid, in the method A, instead of the ortho-chlorophenyl group in the structural formula, a usual protecting group for DNA or RNA synthesis such as para-chlorophenyl group, cyanoethyl group, trichloroethyl group is replaced. be able to.
Further, in the method B, in the structural formulas R ₂ and R ₃ , a protecting group such as a dimethylamino group or a morpholino group can be used instead of the diisopropylamino group. Further, as a method related to the method B, it is possible to change to H-phosphonate instead of R ₂ and R ₃ , acylate with pivaloyl chloride, and activate with tetrazole. Furthermore, R
Instead of ₁ , R ₂ and R ₃ , methylphosphonic imidazolide can be used, and a methylphosphonate type oligomer having 5′-hydroxyl group and 3′-hydroxyl group can be changed.

Regarding Z, in addition to a methoxy group, a hydroxyl group or an alkoxy group (a hydroxyl group having a substituent such as an ethyl, propyl or butyl group which cannot be removed by a chemical treatment in ordinary nucleic acid chemistry) can be substituted, and OQ (However, Q means a protecting group usually used for RNA synthesis such as t-butyldimethylsilyl group and tetrahydropyranyl group.).

The cleavage reaction of low molecular weight RNA (X, XIII, XIV) and the confirmation of the cleavage position were carried out as follows.

RNA molecules (X, XIII, XIV) are each added to T
The 5'-hydroxyl group was labeled with 4-polynucleotide kinase and [τ ^-32 P] ATP, mixed with complementary DNA oligomer (IX) and complementary mixed oligomers (V to VIII and XI, XII), respectively, and RNaseH was used. The cleavage reaction was performed, and the reaction solution was examined by homochromatography for the location of cleavage and the degree of cleavage. The reaction conditions used were 40 mM
Tris-HCl (pH 7.7), 4 mM MgC
l ₂ , 1 mM DTT, 4% glycerol, 0.003
% Bovine serum albumin, ³² P-labeled RNA oligomer (X, XIII, XIV) (30 to 40,000 cpm), unlabeled RNA oligomer (X, XIII, XIV) (1 μM to 5 μM) and compound (X) Complementary DNA oligomer (IX), (10 μ
M), or complementary mixed oligomers (V-VIII and X)
I, XII) (10 μM to 25 μM) and mixed with E. co
RNase H derived from li HB101 (Takara Shuzo)
(5U to 10U), and at 20 ° C or 30 ° C,
React for 2-48 hours.

The cleavage position can be confirmed by labeling RNA molecules (X,
XIII, XIV) are respectively SnakeVenom Phos
It was carried out by homochromatography at the same time for those subjected to limited decomposition by phod i'esterase.

The cutting points and the degree of cutting are summarized as follows.

[0046] (Arrows represent cleavage points. The number of arrows qualitatively represents the degree of cleavage. The solid line-represents the O-methyl oligomer portion.)

Actually, a spot for homochromatography was cut out, and a liquid scintillation counter (PAC
Radioactivity was quantified by Thierenkov measurement using KARD TRI-CARB640C).

From the above results, when the RNA molecule (X) is cleaved, in the case of the duplex with the complementary DNA oligomer (IX), the 5th, 6th and 7th right side from the 5'side is cleaved,
There is no selectivity, while double-stranded, like double-stranded, 4
When the complementary mixed oligomers (V, VII) containing DNA oligomers of 5 bases and 5 bases were used, the sixth right side from the 5'side could be selectively cleaved. It was also found that the 8th right side from the 5'side was selectively cleaved when the complementary mixed oligomer (VIII) was used. RNA with different base sequences
In the case of the double strand using the molecules (XIII, XIV) and their complementary mixed oligomers (XI, XII), the same result as the double strand was obtained regardless of the nucleotide sequence.
It was found that the sixth right side from the side was selectively cut.

From the above results, in the case of the low molecular weight RNA (9 mer) used as a model, basically, when a complementary mixed oligomer is produced, the number of DNA oligomers contained is 3 to 6, preferably 4 to 4. Assuming that 5 bases are appropriate and assuming a specific position of the RNA molecule to be cleaved, shift from that position to 4 bases to the 5'side of the RNA molecule, or 1 base from that position to the 3'side. It was found that a complementary mixed oligomer containing a DNA oligomer complementary to the sequence of 5 bases from 5 to 5 ′ side can be produced and treated with RNaseH, which enabled the design of the production of the complementary mixed oligomer. Further, in order to enhance the selectivity, the reaction conditions may be examined by changing the reaction temperature and the amount of enzyme in consideration of usual biochemical knowledge.

Next, in order to examine whether the RNA cleavage reaction method obtained here can be applied to the cleavage of functional RNA having a higher-order structure (secondary structure, tertiary structure) more universally, Polymer RNA WS-S obtained as described above
The RNA cleavage reaction using (+) (XV) was examined. The cleavage reaction of high molecular weight RNA WS-S (+) (XV) and the confirmation of the cleavage position were performed as follows. As in the case of the small RNA (X), the RNA needs to be labeled with radioactive phosphorus, but the WS-S (+) RNA (XV) can be labeled with In Vitr.
Since it was prepared by transcription, it has triphosphate at the 5'end. Therefore, the 3'end is referred to the literature (Nature 275
According to the method described in 560, 1978), 32 pCp was used to bind with RNA ligase and labeled. The reaction was electrophoresed on an 8% polyacrylamide gel containing 7M urea and analyzed by autoradiography to give the products as one mixture of different chain lengths (90mer and 91mer). The cause of this is unknown, but the mixture was separated by preparative gel electrophoresis of the same species and extracted from the gel according to a conventional method to obtain 3'end-labeled WS-S (+) RNA. 0.02 pmol of this
It was dissolved in water to a concentration of / μl and used in the following experiments.

First, 3'end-labeled WS-S (+) RNA
3.3 nM, 40 mM Tris HCl (pH 7.
7) 4 mM MgCl ₂ , 0.003% BSA, 1 m
MDTT, 4% glycerol, mixed oligomers (83
nM to 8.3 nM), RNaseH (0.17 to 0.8)
The reaction was carried out for several hours at 3 Units / μl). However, before adding RNase H, heat at 65 ° C. for 2 minutes,
Annealed. The reaction is carried out at 30 ° C and the loadin
g sol. (10 M urea, 0.02% xylenicanol, 0.02% bromophenol blue) was added to stop the reaction, and electrophoresis was performed on an 8% polyacrylamide gel containing 7 M urea, and the band was determined by autoradiography depending on the position. The cutting position was analyzed. As a marker, 3'end-labeled WS-S (+) RNA was alkali-decomposed and RNaseT
A sample decomposed with ₁ , U ₂ PhyM was used.

The results of the cleavage reaction obtained are shown in FIG. 3 (the cleavage position is indicated by an arrow).

The mixed oligomer having a DNA portion on the 5'side had a relatively higher cleavage activity than that having a DNA portion on the 3'side. In the former case as the cause of this, A ⁷
~ While there is complementarity in the region extending to Strand on both sides of the Stem structure of U ²⁴ , the latter has C ¹⁹ ~ in Stem in the latter.
It is considered that this reaction was influenced by the formation of a stable complementary double strand, which is important because Strand of U ²⁴ has complementarity only. Therefore, although it is not possible to know the difference depending on which end the DNA portion exists, R
The influence of the secondary structure that NA itself can have is extremely important. For example, in the mixed oligomer (XXIV) having a complementarity on the loop, cleavage occurred more rapidly than the other cases.

On the other hand, XVII having a DNA portion on the 5'side,
In the comparison between XVIII and XIX, the complementary regions are the same and the lengths of the DNA parts are different, but the cleavage sites are slightly different. In all of the conditions conducted this time, XVIII had the cleavage site only at G ²¹ -G ²² . DN from this XVIII
In XIX, where the A part is one shorter, in addition to G ²¹ -G ²² ,
Cleavage was also performed at a position closer to one 5'side (on the mixed oligomer), and conversely, in XVII where the DNA portion in the mixed oligomer was lengthened to the 3'side, the cleavage site was shifted by one in that direction. There is. Therefore, 2'-O-methyl RNA and 5'on the 3'side
When a mixed oligomer consisting of DNA is used on the side DNA
It can be said that it preferentially disconnects between the 4th and 5th from the 3'side. This result shows that small RNA (X, XIII,
The results obtained in the case of the mixed oligomer having O-methyl RNA at one end which was not used in the cleavage reaction of (XIV), are consistent with the oligomer design obtained as a result of the small RNA (X) cleavage reaction. ing.

Conversely, 2'-O-methyl RN on the 5'side
Mixed oligomers (XX, XXI) consisting of DNA on the A and 3'sides
In the case of using, the cleavage activity is low due to the influence of the secondary structure of WS-S (+) (XV), but it is 1 in XXI.
There was a disconnection at some point. Small RNA in the case of mixed oligomer (XXIV) having O-methyl RNA at both ends
As compared with the case of (X, XIII, XIV), cleavage occurred at the 3'side of the single-base RNA molecule, which was shifted by one base from the result of the low-molecular RNA (X, XIII, XIV) cleavage reaction.

The confirmation of the above cutting point will be described later (Example 2).
As described in 3), the 5'end of the cleavage site is labeled with ³² P,
It was obtained by digestion with nuclease P ₁ into 5'-nucleotide units and analysis by filter paper electrophoresis. When XXI was used, it was estimated from the migration position of the cleaved oligomer by the electrophoretic analysis of FIG.

Summarizing the above results, the above-described design of oligomer synthesis can be applied when a small RNA does not have a secondary structure when cleaved, but a high molecular RNA has a higher structure. When the RNA is cleaved, whether the cleavage position is within the loop or within the stem, and the mixed oligomer used in consideration of the secondary structure has O-methyl RNA on the 3'side or 5'side. Although it is necessary to design a mixed oligomer in consideration of which one is suitable, basically, the above-mentioned oligomer synthesis design can be applied to the cleavage of a high molecular weight RNA and can be carried out sufficiently selectively. .

On the other hand, the number of DNA oligomers in the mixed oligomer is 3 to 6, preferably 4 to 5, which is appropriate.

In order to enhance the selectivity, the reaction conditions may be examined by changing the reaction temperature and the amount of enzyme in consideration of usual biochemical knowledge.

In this way, the specific cleavage method for RNA molecules in general was established by the present invention.

The present invention will be specifically described below with reference to examples.

[0062]

【Example】

Example 1 Production of Mixed Oligonucleotides (Production Example of 5′CmAmGmGTAAGmUm3 ′ (V) m represents an O-methyl nucleotide unit and the other represents a deoxynucleotide unit) 5′-O-dimethoxytrityl-2 ′ 50 mg of 1% cross-linked polystyrene resin bound with 6 μmol of —O-methyl-uridine was placed in a reaction container equipped with a glass filter, and washed 3 times with 2 ml of a mixed solution of dichloromethane-methanol (volume ratio 7: 3). Next, 2 ml of a 2% solution of benzenesulfonic acid in dichloromethane-methanol (volume ratio 7: 3) was added, and the mixture was shaken for 1 minute. After the reaction, the reaction solution was filtered, and the resin was washed with 2 ml of a mixed solution of dichloromethane-methanol (volume ratio 7: 3). Further, the reaction was carried out for 1 minute with 2 ml of the above-mentioned benzenesulfonic acid solution, followed by washing with 2 ml of the former mixed solution twice and further with 2 ml of pyridine three times. Next, 0.3 ml of pyridine was added, and the resin was dried by distilling pyridine off under reduced pressure. Then 5'-dimethoxytrityl -2'-O-methylguanine-3'-o-chlorophenyl phosphate (III b ₁₎ of pyridine solution (20 mg / 0.3 ml) was added and concentration in vaccuo. Next, a pyridine solution of mesitylenesulfonyl-3-nitrotriazole (20 mg / 0.3 ml) was added, and the mixture was heated to 40 ° C and shaken for 20 minutes. The reaction solution was removed from the resin by filtration, and the resin was washed twice with 2 ml of pyridine.

Next, 1.8 ml of a 0.1 M dimethylaminopyridine solution and 0.2 ml of acetic anhydride were added, and the mixture was shaken for 3 minutes to cap the unreacted 5'hydroxyl group with an acetyl group. After the reaction solution was filtered off, the resin was washed 3 times with 2 ml of pyridine.

The above series of operations are repeated, and then IId
₁ , IId ₁ , IVd ₁ , III d ₁ , III b ₁ , IIb ₁ , I
Nucleotide units were condensed in the order of b ₁ to extend the chain length. The yield of each condensation reaction was (47% to 105%). Dimethoxytritanol produced by dedimethoxytritylation was developed in perchloric acid-ethanol to give a color of 5%.
It was calculated from the absorbance at 00 nm. The total yield is 25%
Met.

After the reaction, the resin was washed with dioxane, and 0.5 ml of a 1M dioxane solution of 2-pyridinialdoxime tetramethylguanidine was added to dioxane.
After adding 4 ml and 0.1 ml of water and shaking at 30 ° C. overnight, the solution was filtered, and the resin was washed 3 times with 2 ml of 50% pyridine water. After the filtrate and the washing solution were combined and the solvent was distilled off under reduced pressure, the residue was dissolved in 1 ml of pyridine and placed in a sealed tube.
Add 10 ml of 8% ammonia water, stopper tightly, and 5 at 60 ° C.
Reacted for hours. The reaction solution was concentrated under reduced pressure to dryness, and then reversed-phase silica gel (manufactured by Waters, μ Bondapack C1
(8, particle size 35-100 μ) was applied to a column having a diameter of 0.7 ml and a length of 12 cm, and 5% was used as a mobile phase.
To a linear concentration gradient of 35% acetonitrile from 50 to 50
Column chromatography was performed using a mM acetic acid-triethylamine buffer solution, and quantification was performed by absorbance at 254 nm to separate 5'-dimethoxytrityl mixed oligonucleotides. After the solvent was distilled off under reduced pressure, 1 ml of 80% acetic acid aqueous solution was added, and the mixture was allowed to stand at room temperature for 20 minutes. The reaction solution was distilled off under reduced pressure and then azeotropically distilled with water to remove acetic acid.

The residue was dissolved in water and washed with ethyl acetate,
It was distilled off under reduced pressure and purified by high performance liquid chromatography. That is, with Ncleosil C18, a linear concentration gradient of 13% to 21% was applied as a mobile phase to 0.1.
A 1 M acetic acid-triethylamine buffer solution (pH 7.0) was used at a flow rate of 1 ml / min, and the eluted main peak was collected to obtain 0.07 μmole (yield 1.17%) of the mixed oligonucleotide.

The obtained mixed oligonucleotide was analyzed by high performance liquid chromatography using an anion exchange column (DEAE2SW) to give a single peak,
Confirmed that it is pure.

Table 1 shows one cycle of the chain extension reaction.

[0069]

[Table 1]

The nucleotide sequence was confirmed using a mixed oligonucleotide 0.05 OD unit and the 5'terminal hydroxyl group was ³² P.
Phosphorylation was carried out by labeling with the literature (Proc. Natl. Aca
d. Sci. , USA. , 70 1209-1213
1973). The base sequence was confirmed to be correct by the autoradiography.

Other mixed oligonucleotides (VI-VIII,
And XI and XII) were similarly manufactured by the above-mentioned method.

Example 2 Cleavage Reaction of Double Strands Labeled RNA oligomer (X, 20,000 epm) and unlabeled R
NA oligomer (X, 1 μM) and complementary DNA oligomer (IX, 10 μM) prepared according to the method described in Ikehara et al. (2) were added to 40 mM Tris-HCl.
(PH 7.7), 4 mM MgCl ₂ , 1 mM DT
T, 4% glycerol, 0.003% BSA (Coop
er Biochemical Inc. , Worth
IntonBiochemicals, Nuclea
RNase H (5 U) was added to the solution (total volume 20 μl), and the mixture was reacted at 20 ° C. 4 μl of each was sampled over time and analyzed by homochromatography (FIG. 4).

Next, each spot was cut out, the radioactivity was measured with a liquid scintillation counter, and the cleavage rate was calculated (FIG. 5). The cutting rate according to the cutting point is shown in the graph (Fig. 6).

The above results are qualitatively expressed by using arrows to indicate the cleavage site and the degree of cleavage, and the results are as follows. From the 5'side to the 5'side of RNA oligomer (X), the 6th and 7th positions are shown. In between, the 7th and 8th intervals were cut.

(Result) (Arrows represent cleavage points. The number of arrows qualitatively represents the degree of cleavage. The solid line-represents the O-methyl oligomer portion.)

[Example 3] Cleavage reaction of double strand and labeled RNA oligomer (X, 40,000 cpm) and unlabeled R
NA oligomer (X, 5 μM) and complementary mixed oligomer (VI or VII, 25 μM each) were separately added to the same buffer as in Example 2 with RNaseH (5 U) (total volume 10 μl), and kept at 20 ° C. for 48 hours ( 7), or 30 ° C
The reaction was carried out for 16 hours (FIG. 8) and analyzed in the same manner as in Example 2.

(Result) (Arrows represent cleavage points. Number of arrows qualitatively represents the degree of cleavage. Solid line-represents O-methyl oligomer moiety) (Results)

As described above, when the mixed oligomer (VII) is reacted at 30 ° C. for 16 hours, the RNA oligomer (X) can be selectively cleaved between the 6th and 7th positions from the 5 ′ side. It was

Example 4 Regarding double strand cleavage reaction, labeled RNA oligomer (X, 30,000 cpm) and unlabeled R were used.
NA oligomer (X, 1 μM) and complementary mixed oligomer (V, 10 μM) were mixed with RNa in the same buffer as in Example 2.
Add seH (10 U) (total volume 20 μl), 20 ℃
Alternatively, the mixture was reacted at 30 ° C. for 17 hours and analyzed in the same manner as in Example 2 (FIG. 9).

(Result) (Arrows indicate cleavage points, and solid line-indicates O-methyl oligomer portion).

As described above, by using the mixed oligomer (V), the RNA oligomer (X) could be selectively cleaved between the 6'th and 7th positions from the 5'side.

Example 5 Double Strand Cleavage Reaction Labeled RNA oligomer (X, 30,000 cpm) and unlabeled R
NA oligomer (X, 1 μM) and complementary mixed oligomer (VIII, 10 μM) were added to RN in the same buffer as in Example 2.
aseH (10 U) was added (total volume 20 μl), 20 ° C.
Alternatively, the mixture was reacted at 30 ° C. for 17 hours and analyzed in the same manner as in Example 2 (FIG. 10).

(Result) (Arrows represent cleavage points. The number of arrows qualitatively represents the degree of cleavage. The solid line-represents the O-methyl oligomer portion.)

As described above, by using the mixed oligomer (VIII), the RNA oligomer (X) could be selectively cleaved between the 8th and 9th positions from the 5'side.

[Example 6] Regarding the double strand cleavage reaction, a labeled RNA oligomer (XIII, 30,000 cpm), an unlabeled RNA oligomer (XII, 1 µM) and a complementary mixed oligomer (XI, 10 µM) were used in Example 2. In a buffer similar to
Add NaseH (0, 2, 5, 10U),
(Total volume was 20 μl), reacted at 30 ° C. for 1 hour, and analyzed in the same manner as in Example 2 (FIG. 11).

(Result) (Arrows indicate cleavage points, and solid line-indicates O-methyl oligomer portion).

As described above, the RNA oligomer (XIII) was selectively cleaved between the 6th and 7th positions from the 5'side.

Example 7 Regarding cleavage reaction of double strand, labeled RNA (XIV, 30,000 cpm) and unlabeled RNA oligomer (XIV, 1 μM) and complementary mixed oligomer (XII)
10 μM) in the same buffer as in Example 2
eH (5 U) was added (total volume 20 μl), 20 ° C., 30 m
in, 12 hr, 30 ° C., 30 min, 12 hr. The analysis was performed in the same manner as in Example 2 (FIG. 12).

(Result) (Arrows indicate cleavage points, and solid line-indicates O-methyl oligomer portion).

As described above, the RNA oligomer (XIV) could be selectively cleaved between the 6th and 7th positions from the 5'side.

Example 8 RNA oligomer (X, XI
II, XIV) Method for labeling phosphate of each 5'hydroxyl group with ³² P RN produced by the method described in Iwai et al. (3)
A oligomer (X) of 20 pmde and 30 μCi [r
^-32 P] ATP (3000 Ci / mmol, NEN) 50 mM Tris-HCl (pH 8.0), 10 mM
MgCl ₂ , 1 mM EDTA, 6 mM DTT,
Dissolve in 0.1 mg / ml Gelatin (total amount 25μ
l) T4-polynucleotide Kinas
e (1 U, Takara Shuzo Co., Ltd.) was added, and the mixture was reacted at 37 ° C. for 1 hour for 5 ′ phosphorylation. The reaction solution was extracted with phenol-chloroform and applied to a Sephadex G-75 column (total volume 10 ml). As a mobile phase, 10 mM triethylamine-carbonate buffer (pH 7.0) was used, and the fractions containing the first radioactivity were collected and used as labeled RNA oligomer.

Labeled RNA oligomers were similarly prepared for RNA oligomers (XIII, XIV).

[Example 9] Conditions for homochromatography An appropriate amount (tens of thousands of cpm) of an enzyme reaction solution was added to POLYGRAM.
CEL 300 DEAE / HR-2 / 15 (Mec
Herey-Nagel) and spot Homom
After developing with ix IV at 60 ° C., it was exposed to an X-ray film (Kodak X-Homat RP film Eastman-Kodac) overnight at −80 ° C. Hom
For the preparation of omix, see the literature (E. Jay et.a.
l. Nucleic Acids Res. 1 331
1974).

Example 10 Venom phosp
Limited Degradation by H Odiesterase (Preparation of VPD Degradation Product) 5′-end-labeled RNA oligomer (X, XIII, XIV,
50,000 to 70,000 cpm), carrier RNA 0.2 to 0.3O
D (Tolura yeast RNA Type VI
SIGMA), 0.25M Tris-HCl (p
H8.0), 50 mM MgCl ₂ , VPDase 0.
2 to 0.3 μg (Boehringer) 10 in total
μl and react at 37 ° C for 2, 5, 10, 20, 3
Every 0 minutes, 2 μl of each sampled 5 mM
Place in an Eppendorf tube containing 5 μl of EDTA,
The reaction was stopped at 100 ° C. for 2 minutes. The respective reaction solutions were mixed and used as a marker for homochromatography (indicated as VPD degradation product in FIGS. 4, 7, 8, 9, 10, 11, 12).

Example 11 2′-O-Methylnucleoside-3′-phosphoramidites (Ib ₂ , II
_{b 2, III b 2, IVb} 2 Synthesis N ⁶⁾ of - benzoyl-5'-O-dimethoxytrityl -
2'-methyladenosine (compound IIa) 344 mg
(0.5 mmol) was dissolved in 2 ml of anhydrous THF, and 362 μl (2 mmol) of diisopropylethylamine (DIPEA) was stirred at room temperature in an argon stream, and then 300 μl (1.5 mmol) of N, N diisopropylaminomethoxychlorophosphine was added. Added in 1 minute. After 5 minutes, a white precipitate of DIPEA hydrochloride was formed. After stirring at room temperature for 45 minutes, the reaction was allowed to proceed with silica gel 60T.
LC (mobile phase, n-hexane: acetone = 1: 1/5%
Triethylamine). The raw material spot (R _f value 0.52) disappeared, and a new IIb ₂ spot became R _f.
After confirming that a value of 0.76 appeared, ethyl acetate 2 was added under ice cooling.
0 ml was added. Then, 15 ml of an ice-cooled saturated sodium hydrogen carbonate aqueous solution was added twice, and further ice-cooled saturated saline solution of 15 m was used.
After washing once with 1, the organic phase was dried over anhydrous sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated to dryness. The obtained oily residue was dried under reduced pressure, dissolved in 2 ml of dichloromethane, and added dropwise to 200 ml of n-hexane cooled to -50 ° C with stirring to purify a white precipitate. The white precipitate of IIb ₂ was collected by filtration, immediately placed in a desiccator, and dried under reduced pressure at room temperature overnight to obtain 407 mg of IIb ₂ in a yield of 95%.

By the same operation, N ² -isobutyryl-5'-O-monomethoxytrityl-2'-O-methylguanosine-3'-phosphoramidite (IIIb ₂ ), N
⁴ -benzoyl-5'-O-dimethoxytrityl-2 '
-O-methyl cytidine-3'-phosphoramidite (I
b ₂₎ and 5'-O- dimethoxytrityl -2'-O-
Methyluridine-3'-O-phosphoramidite (IVb
₂ ) was synthesized in almost the same yield.

The ³¹ P-NMR spectrum and FAB-
The data of Mass spectrum and UV absorption spectrum are shown. ³¹ P-NMR spectrum Measurement solvent: CDCl ₃ , external standard: trimethyl phosphate (PPM) Ib ₂ 148.5470, 148.3509 IIb ₂ 149.4520, 148.5998 IIIb ₂ 149.5726, 148.9618 IVb ₂ 148. 8788, 148.3132 (these gave two signals as isomer).

[0098] Example 12 2'-O- methyl nucleoside-3'-beta-cyanoethyl -N, N-diisopropylamino phosphine _{(Ib 3, IIb 3, III} b 3, IVb
₃ ) Preparation of N ⁶ -benzoyl-5′-O-dimethoxytrityl-
2'-O-methyladenosine 344 mg (0.5 mmo
l) was dissolved in 2 ml of THF (distillation in the presence of sodium metal), and 362 μl (2 mmo) of diisopropylethylamine (DIPEA) was stirred under an argon stream at room temperature.
l), and then 1.5 mmol of N, N-diisopropylamine-β-cyanoethylchlorophosphine. 5
After a minute, a white precipitate of DIPEA hydrochloride was formed, but the mixture was further stirred at room temperature, and after 30 minutes, the reaction progressed with silica gel 60TLC.
(Mobile phase n-hexane: acetone = 1: 1/5% triethylamine). After confirming that the spot of the raw material (compound IIa) had disappeared and the spot of the amidite (compound IIb ₃ ) had appeared, 20 ml of ethyl acetate was added under ice cooling. Then 2 with 15 ml of saturated aqueous sodium hydrogen carbonate solution.
After washing once with 15 ml of an ice-cooled saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated to dryness, and the obtained foamy residue was dissolved in 2 ml of ethyl acetate containing 0.2% triethylamine and applied to a column packed with 2.5 g of silica gel 60. Eluted with ethyl acetate containing 0.2% triethylamine, the pure fractions of compound IIb ₃ were collected and concentrated to dryness to give 407 mg (0.485 mmo
l), obtained in a yield of 97%.

By the same operation, N ² -isobutyryl-5'-O-monomethoxytrityl-2'-O-methylguanosine-3'-phosphoramidite (Compound III
b _3), N ⁴ - Benzoyl-5'-O-dimethoxytrityl -2'-O-methylcytidine-3'-phosphoramidite (compound Ib ₃₎ and 5'-O- dimethoxytrityl -2'-O- methyluridine-3'-phosphoramidite (compound IVb ₃₎ was obtained in substantially the same yield.

Below, ³¹ P-NMR spectrum and FAB-
The data of MASS spectrum and UV absorption spectrum are shown. ³¹ P-NMR spectrum Measuring medium CDCl ₃ , external standard trimethyl phosphate (8 ppm) Compound Ib ₃ 148,8127,148,3224 Compound IIb ₃ 149,2125,148,4582 Compound IIIb ₃ 148,4582,148,2545 Compound IVb ₃ 148,8203,148,3601 FAB-MASS spectrum ^{M-H + (m / z} ) compound Ib ₃ 864 compound IIb ₃ 888 compound IIIb ₃ 840 compound IVb ₃ 762 UV absorption spectrum measurement solvent: ethanol (nm) compound Ib _3: λ _max . 305, 260, 236 λ _min . 289,250,223 Compound IIb ₃ λ _max . 279, 234 λ _min . 257,223 Compound IIIb ₃ λ _max . 280, 254, 235 λ _min . 272, 244, 227 Compound IVb ₃ λ _max . 264, 233 λ _min . 253,226

[0102] Example 13 2'-O- methyl linkage solid support _{(Ic 2, IIc 2, III} c 2, IVc
₂ ) Synthesis of N ⁶ -benzoyl-5′-O-dimethoxytrityl-
2'-O-methyladenosine (IIa), 344 mg
(0.5 mmol) and 4-N, N dimethylaminopyridine (DMAP) 92 mg (0.75 mmol) were dissolved in anhydrous pyridine 2 ml, and succinic anhydride 750 mg (0.75 mmol) was added with stirring at 30 ° C. After stirring overnight, the reaction mixture was concentrated to dryness, the obtained oily residue was dissolved in 20 ml of chloroform, and 0.1 M triethylamine-carbonate buffer (TEAB buffer) pH 7.5, 20 was added.
Washed 3 times with ml. The chloroform layer was dried over anhydrous sodium sulfate and then concentrated to dryness. The resulting residue (IIe
And a mixture of DMAP) were dissolved in 2.7 ml of DMF, and 200 mg of pentachlorophenol (0.75 mm)
ol) and dicyclohexylcarbodiimide (DCC)
154 mg (0.75 mmol) was added and it stirred at 30 degreeC overnight. Next, the precipitated insoluble material was filtered off, the filtrate was concentrated to dryness, and the resulting residue was dissolved in 3 ml of chloroform, and the column of silica gel 60 equilibrated with chloroform (20
g, diameter 4 cm x length 2.5 cm). Chloroform was used as a mobile phase, and the eluted pure fraction of IIf (silica gel 60TLC, R _f value 0.81 at chloroform: methanol = 20: 1) was collected and concentrated to dryness to obtain IIf of about 90%. Got at a rate.

By the same operation, If, III f, IV
f was obtained in almost the same yield. R _f value on silica gel 60 TLC R _f value Mobile phase If 0.76 Chloroform: methanol = 15: 1 IIIf 0.68 Chloroform: methanol = 10: 1 IVf 0.74 Chloroform: methanol = 15: 1

Next, a controlled pore glass support (CPG / long chainalkylamine) was used.
e, Pore Dia. 500Å, Particle
size 122-177 μ, NH ₂ —: 30 μmol
/ G, Pierce Chemical Co), 68
0 mg (NH ₂ −: 20 μmol) was placed in a reaction container equipped with a glass filter, washed with 1 ml of DMF, and dried in an argon gas stream for 1 minute. Next, the active ester form (If, IIf, IIIf, IVf) (0.45 mmol) was dissolved in 4.5 ml of DMF, and 114 μl (0.82 mmol) of triethylamine was added. This solution was added to the CPG prepared above and sealed up tightly, and the mixture was shaken at 30 ° C. overnight, and then the reaction solution was filtered off by argon gas pressure. Solid phase carrier is 5m
It was washed 3 times with 1 L of DMF, then 3 times with 5 ml of THF, and dried under an argon gas stream for 1 minute. Then DMAP
273 mg, 2,6 lutidine 940 mg, acetic anhydride 0.
Add a mixed solution of 44 ml and 4 ml of THF, and add 30 ml at 30 ° C.
It was shaken for a minute to acetylate unreacted amino groups. The reaction solution was filtered off, washed 3 times with 5 ml of THF, then 3 times with 5 ml of diethyl ether, dried in an argon stream, and further dried in vacuum. The resulting nucleotide-support (I
_{c 2, IIc 2, III c} 2, IVc 2) a small amount weighed, 3
After the dimethoxytrityl (or monomethoxytrityl group) was cleaved with% trichloroacetic acid, the released tritanol was subjected to colorimetric determination with a perchloric acid-ethanol solution. As a result, the amount of nucleoside bound was 29 to 30 μmol / g.

Example 14 Preparation of mixed oligonucleotide (from methoxy form) (5′CGGAUmCmUmAmGmCmCmGmGm
Production example of GmUm3 '(XVIII), m represents an O-methyl nucleotide unit, and the others represent a deoxy nucleotide unit. )

8 mg of 5'-dimethoxytrityl 2'-O-methyluridine and 0.2 μmol of controlled pore glass (compound IVc ₂ ) bound to a cartridge were packed in a cartridge, and a DNA synthesizer model 380A (Appl) was used.
Fluoroamidites were added to III b ₂ and III by the procedure shown in Table 2 after setting
b ₂ , III b ₂ , Ib ₂ , Ib ₂ , III b ₂ , IIb ₂ ,
IVb ₂ , Ib ₂ , IVb ₂ , IId ₂ , III d ₂ , III
A protected mixed oligomer was synthesized by repeating 14 cycles using d ₂ and Id ₂ in this order. Next, according to the procedure shown in Table 3, an ammonia solution was cut out from a solid phase carrier (CPG) into a 3 ml vial, sealed, and heated at 50 ° C. for 12 hours to remove an acyl group at the base. After cooling, ammonia was removed, the mixture was washed twice with 1 ml of ethyl acetate, and the aqueous layer was concentrated to dryness. The resulting residue is 0.1 containing 10% acetonitrile.
M acetic acid-triethylamine buffer solution pH 7.0 (TEA
A) Reversed phase silica gel (Waters) dissolved in 200 μl
Prep PAK-500 / C-18) manufactured by the company was applied to a column having a diameter of 1 cm and a length of 10 cm, and 0.1 M TEAA (pH 7.0) was applied with a linear concentration gradient of 10% to 35% acetonitrile as a mobile phase. Column chromatography was carried out to quantify the absorbance at 254 nm, and 2.2 OD units of 5'-dimethoxytrityl mixed oligonucleotide were separated. After distilling off the solvent under reduced pressure, 1 ml of 80% acetic acid aqueous solution was added, and the mixture was allowed to stand at room temperature for 10 minutes. The reaction solution was distilled off under reduced pressure and then azeotropically distilled with water to remove acetic acid. The residue was dissolved in water, washed with ethyl acetate, evaporated under reduced pressure and purified by high performance liquid chromatography. Ie YMC pack ODS
(Yamamura Kagaku Co.) 5% to 25% as mobile phase by column
0.1M T with linear gradient of% acetonitrile
Using EAA (pH 7.0) at a flow rate of 12 ml / min, the eluted main peak was collected to obtain 0.91 OD unit (about 6 nmol, yield 3.0%) of mixed oligonucleotide XVIII.

By the same operation, 5'CGGATCmUmAmGmCmCmGmGmGm
Um3 ′ (XVII) with a yield of 2.5%, 5′CGGAm
UmCmUmAmGmCmCmGmGmGmUm3 '
(XIX) with a yield of 3.0%, 5'AmCmAmCmAm
CmCmCGGA3 '(XXII) at a yield of 6.8%,
5'AmCmAmCmAmCmCmCGG 3 '(XXIII)
With 8.6% yield of 5'CmUmCGAAAGmUm
The 3 '(XXIV) was isolated in 3.2% yield, respectively.

One cycle of the chain extension reaction operation (0.
2 μM CGPresin used) and CGPresin release operation are shown in Tables 2 and 3, respectively.

[0108]

[Table 2]

[0109]

[Table 3]

Example 15 Production of mixed oligonucleotide (using β-cyanoethyl form) (5′AmCmA
Production Example 1 of mCmAmCmCmCmGGAT3 ′ (XX). m represents an O-methyl nucleotide unit. Others represent deoxynucleotide units. ) Controlled pore glass cartridge (App 2) to which 0.2 µmol of 5'-dimethoxytritylthymidine is bound.
Lied Biosystems) and the phosphoramidite was treated with IId ₃ , III by the procedure shown in Table 2.
d ₃ , III d ₃ , Ib ₃ , Ib ₃ , Ib ₃ , IIb ₃ , I
Using b ₃ , IIb ₃ , Ib ₃ and IIb ₃ in this order, 11 cycles were repeated to synthesize a protected mixed oligomer. Next, according to the operation shown in Table 3, the ammonia solution was put into a 3 ml vial, which was separated from the solid phase carrier (CPG), sealed, and heated at 50 ° C. for 9 hours to remove the β-cyanoethyl group and the base acyl group. did. After cooling, the ammonia was removed, the mixture was washed twice with 1 ml of ethyl acetate, and the aqueous layer was concentrated to dryness. The remaining residue is 0.1% containing 10% acetonitrile.
1M acetic acid-triethylamine buffer (TEAA p
H7.0) dissolved in 200 μl and reversed-phase silica gel (Wa
ters, Prep. PAKC-500 / C-18)
Was applied to a column having a diameter of 1 cm and a length of 10 cm and 0.1 M TEAA (pH 7.0) with a linear concentration gradient of 10% to 35% acetonitrile applied as a mobile phase.
Column chromatography using 254 nm
9.5 OD units of 5'-dimethoxytrityl mixed oligomer were separated from the absorbance at. After the solvent was distilled off under reduced pressure, 1 ml of 80% acetic acid aqueous solution was added, and the mixture was left at room temperature for 10 minutes. The reaction solution was evaporated under reduced pressure and then azeotroped with water under reduced pressure to remove acetic acid. The residue was dissolved in water, washed with ethyl acetate and evaporated under reduced pressure, and purified by high performance liquid chromatography. That is, with a YMC pack (manufactured by Yamamura Scientific Co., Ltd.) column, 0.1 M TEAA (pH) was applied with a linear concentration gradient of 5% to 25% acetonitrile as a mobile phase.
7.0) at a flow rate of 1.2 ml / min, the main peak eluted was collected, and 1.9 OD units (about 16 n) were collected.
Mixed oligonucleotide (XX) with a molar yield of 8.1%)
I got

By the same operation, the mixed oligomer 5 '
AmCmAmCmAmCmCmCGGAT3 '(Compound (XXI)) was obtained in a yield of 9.3%.

Example 16 Synthesis of Double-Stranded DNA (XVI) Commercially available compounds (Id _{4 to} IVd ₄ ) and (Id _{2 to} IVd)
₂ ) DNA using an automatic DNA synthesizer using (ABI)
Oligomers (1-9, chain length 17-47) were synthesized and purified by a conventional method. 1 nmo of each DNA oligomer
0.5 M Tris-HCl (pH 7.
5) 4 μl, 0.1 M MgCl ₂ · 4 μl, 0.2 m
M ATP 10 μl, 0.1 M DTT · 4 μl, H ₂
It was dissolved in 14 μl of O, 2 μl (5 U) of T4-polynucleotide kinase (Takara Shuzo) was added, and the mixture was reacted at 37 ° C. for 40 minutes, and subsequently the same amount of kinase was added and reacted for 40 minutes. 5'-phosphorylated compound (1,2,5,6) (A
(Liquid) and (3, 4, 7, 8, 9) (Liquid B) were separately mixed to obtain A liquid and B liquid. Approximately 3 in a 80 ° C water bath
It cooled to 20 degreeC over time. 2 mM ATP4 in solution A
Add 0 μl, 0.1 M DTT 40 μl, T4-DNA ligase (Takara Shuzo) 1 μl (175 U) (Total)
100 μl) Reaction was carried out at 15 ° C. for 20 hours. For solution B, 25 μl of 2 mM ATP, 25 μl of 0.1 M DTT,
0.5 μl (87 U) of ligase was added and the same reaction was performed. After the reaction liquids were treated with phenol, gel filtration column (Superose ^TE 6H10 / 30) mobile phase: 0.05M Tris HCl 1mM EDTA
(PH 8.0), fractionated, duplex A: 590 pm
ole (1.07 OD), B: 311 pmole (0.
62 OD) was obtained.

Next, A chain 347 pmole and B chain 311 p
Mole was mixed, and 140 μl of reaction solution (50 mM T
ris HCl (pH 7.5), 10 mM MgC
₁₂ in 0.2 mM ATP, 10 mM DTT), 3
After left at 7 ° C for 1 hour, annealed at room temperature to give T4-D.
Add NA ligase 0.5 μl (87 U),
The reaction was carried out for 1.5 hours. After phenol-chloroform treatment, ethanol precipitation treatment was performed and the mixture was dried under reduced pressure.

6 mM Tris HCl (pH 7.
5), 6 mM MgCl ₂ , 125 mM NaCl, 7 m
Add 95 μl of M DTT solution to dissolve and add 5 μl of SphI
1 (20 U) was added, and the mixture was kept at 37 ° C for 30 minutes, and then continuously at room temperature for 2
Reacted for days. Phenol-chloroform treatment followed by ethanol precipitation treatment was applied to the gel filtration column (Superose ^TH 6H10 / 30) described above, and fractionated.
After ethanol precipitation, 68 pmole (7.6 μg) of double-stranded DNA (XVI) was obtained.

Example 17 Transcription vector M13-
Preparation of AJ-1 M13mp19 2 μl (Takara Shuzo, 0.1 μg / 1 μ
l) 6 mM TrisHCl (pH 7.5), 6 mM
In 20 μl of MgCl ₂ , 125 mM NaCl, 7 mM DTT solution, add SphI (2U Takara Shuzo), and add 37
The reaction was carried out at 30 ° C for 30 minutes. Phenol-chloroform treatment and ethanol precipitation were carried out by a conventional method, and then 10 mM
Tris HCl (pH 8.0), 10 mM MgCl
₂ , 20 mM KCl, 7 mM DTT solution 20 μl
Inside, SmaI (5 U, Takara Shuzo) was added and reacted at 30 ° C. for 30 minutes. After ferrule-chloroform treatment and ethanol precipitation by a conventional method, the mixture was dried under reduced pressure. Next, 50 mM Tris HCl (pH 7.5), 10 mM
MgCl ₂ , 0.2 mM ATP, 10 mM DTT
Double-stranded DNA obtained in Example 16 dissolved in 20 μl of solution
(XVI) 0.04 μg was added, and T4-DNA ligase 2U
Was added, and the reaction was continued at 16 ° C for 3 hours and then at 4 ° C overnight. This solution was used for the subsequent transformation in 1 μl, 2 μl and 4 μl each.

Literature (Method in Enzymo)
E. LOGY 101 20-78 1983). coli JM 103 competent C
ell was transformed and white plaques were screened. Each of the phages was taken from about 10 white plaques, and E. coli JM 103 solution 1.
Add to 5 ml (bacteria were cultured overnight at 37 ° C and diluted 100-fold), incubate at 37 ° C for 6 hours, and 12000r
After centrifugation for 5 minutes at pm, a supernatant was obtained. According to the conventional method, single-stranded D
Each NA was obtained and the synthetic double-stranded portion inserted by the dideoxy method was sequenced to obtain the target clone.

[0117] To 1 l of a large amount of JM 103 solution (0.3 OD / 570 nm) that had been mass-cultured, 100 µl of the above supernatant was added, and the mixture was incubated at 37 ° C for 7 hours, according to a conventional method.
445 μg of the target vector M13AJ-1 was obtained.

Example 18 Preparation of WS-S (+) RNA 50 μg of M13-AJ-1 was SmaI, 10 mM Tris HCl (pH 8.0) containing 70 units, 1
0 mM MgCl ₂ , 20 mM KCl, 7 mM DT
After reacting in 100 μl of T solution at 30 ° C. for 2 hours, it was confirmed that 1% agarose formed a linear chain. Phenol-
Chloroform treatment, ethanol precipitation, dissolution in 1 ml of water, and UV measurement at 260 nm (0.84O
D / ml: 42 μg). It was again dried under reduced pressure, dissolved in 84 μl of water, and used for the next transcription reaction. The method is the literature (Nuc
leic Acid Res. 12 7035 (198
According to 4)), add 10m to a 1.5ml Eppendorf tube.
5 μl of M ATP, GTP, UTP and CTP respectively
100 μl of [5- ³ H] -UTP (100 μl each)
ci, 2.2 × 10 ⁸ dpm / 7.3 nmole, manufactured by Amersham) were added, and the mixture was dried under reduced pressure.

Next, 5 times the transfer buffer (200 mM
Tris / HCl (pH 7.5), 30 mM MgC
l ₂ , 10 mM spermidine) 20 μl, 100 mM
DTT 10 μl, RNasin 5 μl (150 U), the above SmaI cut DNA solution 30 μl (15 μg),
10 μl (150 U) of SP6-RNA polymerase and 25 μl of water treated with diethylpyrocarbonate were added to make 100 μl in total, and the mixture was reacted at 40 ° C. for 1 hour. Furthermore,
3 μl (45 U) of SP6-polymerase was added and reacted for 2 hours. Add 15 μl of RQIDNase (15
U) and RNasin 4 μl (120 U) are added, and the temperature is 37 ° C.
It was reacted for 15 minutes. The extract was extracted once with 100 μl of phenol-chloroform, 100 μl of chloroform and 100 μl of ethyl ether, and then dried under reduced pressure. (All reagents used above were kits manufactured by Promega and Biotech, except [ ³ H] UTP (manufactured by Amersham Japan).)

Next, it was dissolved in about 200 μl of water and NENS
Treated with ORB20 (manufactured by Japon), 50% eluent about 1
ml to dryness under reduced pressure, and 10 mM Tris HCl (pH
7.5), dissolved in 500 μl of 0.25 mM EDTA.

Sampling 5 μl of the resulting solution,
Liquid scintillation counter (Packard, TR
I-CARB4040), and calculated WS-S
It was confirmed to contain 3 μg of (+) RNA. Also, 26
It was estimated to contain about 9 μg of DNA by UV measurement at 0 nm.

The WS-S (+) RNA solution obtained here was used for the experiment of the following Example 19.

[Example 19] 3'- ³² pCp labeling of WS-S (+) RNA WS-S (+) RNA 336 ng (11.2 pmol) 0.56 μM [5'- ³² P] pCp (Amersham Japan 3000 Ci / mmol 0.825 μM ATP 6 μM HEPES (pH 8.3) 50 mM MgCl ₂ 10 mM DTT 3.3 mM DMSO 10% (v / v) BSA 10 μg / ml glycerol 15% (v / v) RNA ligase (PL Pharmacia) 2U ──────────────────────────────────── ↓ Total volume 20 μl 4.5 ° C, 16 hours

The reaction was carried out under the above conditions. However, RNA
65 ° C. before adding ligase and [5′- ³² P] cPc,
After heating for 5 minutes, it was immediately cooled with ice. After the reaction, 20 μl of loading solution (10% urea, 0.
02% xylene cyanol, 0.02% bromphenol blue) was added, and the mixture was applied to 8% polyacrylamide gel (length 50 cm, thickness 0.5 mm) containing 7 M urea, and electrophoresed at 2000 V for 2 hours. After the electrophoresis, the gel was subjected to autoradiography, the portions having different chain lengths were separately cut out, and extracted from the gel according to the method of Maxam-Gilbert. The extract is NENSORB ^™ 20
(1) and 3'- ³² pCp-labeled WS-S (+) RNA desalted by (Du Pont) and different in chain length.
0.725 pmol of each RNA having a short chain length (total 1.45 pmol, yield 13%) was obtained. The quantification was performed by β-dose measurement by the Cherenkov method. Then 0.02
H ₂ O was added and dissolved to a concentration of pmol / μl, and stored at −80 ° C. ³² pC for the next cleavage reaction
p-labeled WS-S (+) RNA was used.

Example 20 RNA Strand Cleavage Reaction A general method is shown below. 3′- ³² pCp-labeled WS-S (+) RNA 3.3 nM mixed oligomer 83 nM to 8.3 μM Tris-HCl (pH 7.7) 40 mM MgCl ₂ 4 mM BSA 0.003% DTT 1 mM glycerol 4% RNaseH (Manufactured by Takara Shuzo) 0.17 to 0.83 units / μl ───────────────────────────────────── Total volume 6 μl

Prior to the addition of RNase H, the mixture was 6
Annealing was performed by heating at 5 ° C. for 2 minutes. Reaction is 3
Perform at 6 ℃ in loading solution at 0 ℃
was stopped by adding on, and 2 μl was electrophoresed on an 8% polyacrylamide gel containing 7 mol urea. (Thickness 0.2 mm, length 35 cm, 2000 V, 2 hours)

Mixed oligomers 8.3 μM (2,500 times as much as RNA), RNaseH 0.17 units /
FIG. 13 shows the result of autoradiography by 8% PAGE after the reaction with μl for 2 hours.

In lane8, a cleavage occurs between the 43rd G and the 44th A on the loop. lane10 has occurred is selectively cleaved between G ²¹ and G ²² in the stem. The lane 11 is cut at two places. lan
The DNA44mer of e2 could not be selectively cleaved, and
It is not cleaved at all by the complementary DNA 6mer of ne3.

FIG. 14 shows that the amount of enzyme was 0.83 units / μ.
It is the result of performing the same reaction with l. Due to the increased amount of enzyme, lanes 6 and 7 are cleaved.

FIG. 13 and FIG. 14 show the samples that were heated during the migration (FIG. 14) and those that were not heated (FIG. 13).
In 9 and 10 (11), a change in the migration position reflecting the structural change of RNA due to the influence of the mixed oligomers is observed.

[Example 21] Under the same conditions as in Example 20, the amounts of the mixed oligomers were changed to 1/10 and 1/100.
(25 times to 2500 times RNA), RN
FIG. 15 shows the result of an experiment using aseH of 0.42 units / μl.

Lanes 5, 6, 7 and 11, 12, 13
From the results, it can be seen that when the mixed oligomers (XXIV and XVIII) were used, even if the mixture was reduced to 25 times, the cleavage occurred and the raw materials disappeared. When the mixed oligomer (XIX) was used, cleavage occurred, and the cleavage occurred between G ²¹ and G ²² and between G ²² and G ²³ . When the mixed oligomer (XXI) was used, a slight cleavage occurred at 2500 times and a cleavage occurred between G ²¹ and G ²² .

[Example 22] Preparation of RNA chain length marker 30,000 to 60,000 cpm of 3'- ³² pCp-labeled WS-S
Expanded RNA S using (+) RNA
The reaction was performed according to the instruction manual (using RNase T ₁ manufactured by Sankyo Co., Ltd.) using an Equating Kit (manufactured by Pharmacia), and 2 μl of the reaction solution was used for electrophoresis.
That is, 1 μl of 3'terminal ³² P-labeled WS-S was used for alkaline decomposition.
(+) RNA (about 50,000 cpm) and carrier t
RNA 2 μg / μl 1 μl, CO ₃ / HCO ₃ solution 1
μl was added, and the mixture was heated at 90 ° C. for 6 minutes and cooled with ice, and 3 μl of urea-dye solution (5 mM Tris, 5 mM boric acid, 1 mM
EDTA, 0.01% xylesianol FF, 0.01
% Bromphenol blue, 10 M urea) was added, and 2 μl thereof was applied to electrophoresis.

RNase T ₁ digestion was performed with 1 μl of the 3'end ³²
P-labeled WS-S (+) RNA (about 50,000 cpm) and buffer (33 mM sodium citrate pH 5.0,
1.7 mM EDTA, 0.04% xylene cyanol FF, 0.08% bromphenol blue, 1 mg / m
l carrier tRNA, 7M urea) 3 μl, distilled water 1
μl, RNaseT ₁ (manufactured by Sankyosha) 0.01 unit /
After adding 1 μl of μl and reacting at 55 ° C. for 12 minutes, the mixture was cooled with ice, and 2 μl of the mixture was applied to electrophoresis.

RNase U ₂ digestion was performed with 1 μl of 3'end ³²
P-labeled WS-S (+) RNA (about 50,000 cpm) and buffer (33 mM sodium citrate pH 3.5,
1.7 mM EDTA, 0.04% xylene cyanol FF, 0.08% bromphenol blue, 1 mg / m
l carrier tRNA, 7M urea) 3 μl, distilled water 1
1 μl of RNase U ₂ (Pharmacia) 2 unit / μl was added, and the mixture was reacted at 55 ° C. for 12 minutes. After the reaction, the mixture was ice-cooled, and 2 μl thereof was applied to electrophoresis.

Example 23 5 ′ of RNase cleavage product
Terminal base analysis (analysis example of cleavage product when 5'CmUmCGAAAGmUm3 '(XXIV) is used)

WS-S labeled with ³² pCp at the 3'end
(+) RNA 0.02 pmol (about 200 cpm: measured by Cherenkov method) of 5'CmUmCGAAAGmUm
Solution A was added to 6 µl of the cleavage reaction solution with 3'and RNaseH.
(0.1 M Tris-HCl, 10 mM triethylamine, 1
200 μl of mM EDTA, pH 7.7) was added for dilution, and the NENSORB ^™ 20 nucleic acid purification cartridge (D
u PONT), washed with 3 ml of solution A and then with 1 ml of distilled water, and then eluted with 500 μl of 50% methanol water. The eluate was concentrated under reduced pressure to dryness, and
e Journalof Biological Ch
chemistry, 252 , 3176-3184 (197).
7))) 5'-terminal phosphate with high specific activity ³²
Exchanged for P. That is, 0.5 M imidazole-hydrochloric acid buffer solution 1 μl, 0.1 M magnesium chloride 1 μl, 1 mM spermidine 1 μl, 1 mM EDTA was added to the concentrated dry solid.
1 μl, 50 mM dithiothreitol 1 μl, distilled water 2
μl, 1 μl of 3 mM ADP were added and dissolved, and the mixture was heated at 65 ° C. for 2 minutes and immediately ice-cooled.

Next, this mixed solution was mixed with [γ- ³² P] ATP,
20 pmol and 100 μCi (manufactured by Amersham Japan) were added to a container which had been concentrated and dried in advance, and 1 unit of 7 units / μl of T4 polynucleotide kinase (manufactured by Takara Shuzo) was added and reacted at 37 ° C. for 30 minutes. 10M urea, 0.02% xylene cyanol, 0.
10 μl of a solution containing 02% bromphenol blue was added, and the whole amount was electrophoresed by 8% polyacrylamide gel (thickness 0.2 mm, length 35 cm) containing 7 M urea (2
000 V, 2 hours) and separated. After electrophoresis, autoradiography is performed to cut out a band near the chain length of about 48, cut into fine pieces, and 0.5 M ammonium acetate, 0.1 m
300 μl of M EDTA was added and left at room temperature for 1 day. The gel was removed by centrifugation and the eluate was concentrated to dryness under reduced pressure. The resulting 48-chain-length cleavage product was solution A (0.1 M tris-hydrochloric acid, 10 mM triethylamine, 1 mM EDT
A, pH 7.7) 200 μl was added for dilution and carrier t
4 μg of RNA was added, and the mixture was applied to a NENSORB ^™ 20 nucleic acid purification cartridge (manufactured by Du PONT), and 3
500μ after washing with ml of solution A and then 1 ml of purified water
It was eluted with 1 of 50% methanol water. Eluate (800c
(measured by pm Cherenkov method) is concentrated under reduced pressure to dryness, then distilled water 8 μl, 0.4 M ammonium acetate pH 5.0 1
μl, and nuclease P1 (Yamasa Shoyu Co., Ltd.) 0.1
μg / 1 μl was added and dissolved to react at 37 ° C. for 30 minutes. The reaction solution was Toyo Roshi No. 51 (40 cm), and filter paper electrophoresis (900 V, 3.5 mA, 90 minutes) in 0.2 M morpholine acetate buffer (pH 3.5).
Standard 5'CMP, 5'AMP, 5'GMP, 5'UM
It was carried out with the P and 3'end ³² pCp-labeled yeast 5S RNA digested under the same conditions with nuclease P1. As a result of radioautography after electrophoresis, 5'AMP was detected as shown in FIG. 16-A. From the above, it was confirmed that the cleavage site was between the 43rd G and the 44th A.

As for the cleavage product obtained by using XVII and XVIII, WS-S (+) whose 3'end was labeled with ³² pCp was used.
Approximately 0.02 pmol (approximately 10
00 cpm: measured by Cherenkov method), and 5'-end analysis was performed in the same manner. That is, the 5'-terminal phosphate was treated with 3300 cpm as the cleavage component exchanged with high specific activity ³² P.
(Cleaved product of XVII) 4800 cpm (Cleaved product of XVIII)
Each was isolated, completely decomposed with nuclease P1, subjected to acidic filter paper electrophoresis, and the result of its radioautography is shown in FIG. 16-B. Both have G at the 5'end
, And the combined consider a comparison with the chain length marker (FIG. 14), the cutting position XVII is C ²⁰ _G ^21, XVIII is G ²¹ _G ²²
Was confirmed.

[0140]

As described above, according to the present invention,
RNA molecules can be selectively cleaved at specific positions regardless of the length of their molecular chains, and thus various functional R
The structure elucidation and function research of NA will be facilitated, and it is expected to be used for mass production of useful proteins.

[Brief description of drawings]

FIG. 1 is a double-stranded DNA in which WS-S (+) sequences are linked.
(XVI) shows a chemical and enzymatic synthesis method.

Figure 2: Mixed oligomers (XVII-XXIV) and WS-S
Relationship between complementary sequence of (+) RNA (XV) and WS-S
(+) Shows the secondary structure of RNA.

[Fig. 3] Breakage points when various mixed oligomers are used

FIG. 4 is a homochromatography showing a time course of cleavage of a compound consisting of a labeled RNA oligomer, an unlabeled RNA oligomer and a complementary DNA oligomer according to the present invention.

FIG. 5 is a graph showing the cutting rate at the cutting point and the time in FIG.

FIG. 6 is a graph showing the cutting rate at the cutting point and the time in FIG.

FIG. 7 is a homochromatographic diagram showing a time course of cleavage of a compound consisting of a labeled RNA oligomer, an unlabeled RNA oligomer and a complementary mixed oligomer.

FIG. 8 is a homochromatographic diagram showing the time course of cleavage of a compound consisting of a labeled RNA oligomer, an unlabeled RNA oligomer and a complementary mixed oligomer.

FIG. 9 is a homochromatographic diagram showing a time course of cleavage of a compound consisting of a labeled RNA oligomer, an unlabeled RNA oligomer and a complementary mixed oligomer.

FIG. 10 is a homochromatographic diagram showing the time course of cleavage of a compound consisting of a labeled RNA oligomer, an unlabeled RNA oligomer and a complementary mixed oligomer.

FIG. 11 is a homochromatographic process showing the time course of cleavage of a compound consisting of a labeled RNA oligomer, an unlabeled RNA oligomer and a complementary mixed oligomer.

FIG. 12 is a homochromatographic diagram showing the time course of cleavage of a compound consisting of a labeled RNA oligomer, an unlabeled RNA oligomer and a complementary mixed oligomer.

FIG. 13 is an autoradiography showing a cleavage process of 3′- ³² pCp-labeled WS-S (+) RNA and a mixed oligomer.

FIG. 14 is an autoradiography showing a cleavage process of 3′- ³² pCp-labeled WS-S (+) RNA and a mixed oligomer.

FIG. 15 is an autoradiography showing a cleavage process of 3′- ³² pCp-labeled WS-S (+) RNA and a mixed oligomer.

FIG. 16 is an autoradiography showing an example of 5′-terminal base analysis of the RNase cleavage product in FIGS.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Susumu Shibahara 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Central Research Institute of Ajinomoto Co., Inc. (72) Sachiko Mukai 1-Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa 1 Central Research Laboratory, Ajinomoto Co., Inc. (72) Inventor Toru Nishihara 1-37 Sachimachi, Kurashiki City, Okayama Prefecture

Claims (5)

[Claims] 1. A mixed oligomer in which an RNA derivative oligomer is bound to one or both ends of a DNA oligomer. 2. The mixed oligomer according to claim 1, wherein the bond between the DNA oligomer and the RNA derivative oligomer is via a phosphodiester bond between the 5′-hydroxyl group and the 3′-hydroxyl group of the ribose or deoxyribose moiety. 3. The mixed oligomer according to claim 1, wherein the number of bases of the DNA oligomer is 3 to 6. 4. The RNA derivative oligomer, wherein at least one 2′-hydroxyl group of the sugar moiety has a substituent RN.
The mixed oligomer according to claim 1 or 2, which is an A derivative oligomer. 5. The RNA derivative oligomer, wherein R has at least one 2′-position hydroxyl group of the sugar moiety has a methyl group.
The mixed oligomer according to claim 1 or 2, which is NA.