JPH053786A - Sequence specific rna hydrolase - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme, composed of a hybrid ribonuclease H having cysteine residue in which thiol group is linked to oligodeoxyribonucleotide through covalent bond without affecting enzymic activity and having high selectivity and catalytic activity. CONSTITUTION:A gene capable of coding Escherichia coli ribonuclease H is converted into a gene capable of coding variant type ribonuclease H in which amino acid at the 131-, 135-, 138or 142-position located on the molecular surface is substituted with cysteine by enzymic treatment, subjected to treatment with a restriction enzyme, linked to a vector, transduced into Escherichia coli and expressed to prepare a variant type ribonuclease H. The resultant variant type ribonuclease H is subsequently reacted with a compound, expressed by formula II and prepared by reacting an oligodeoxyribonucleotide having amino group expressed by formula I introduced thereinto with N-(epsilon-maleimidocaproyloxy) succinimide to afford the objective hybrid ribonuclease H in which the variant type ribonuclease having the cysteine residue on the molecular surface is linked to the oligodeoxyribonucleotide without affecting the enzymic activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hybrid ribonuclease H in which ribonuclease H and oligodeoxyribonucleotide are covalently bonded. Further, the present invention is
The present invention relates to a mutant ribonuclease H useful for producing the hybrid ribonuclease H, a DNA encoding the mutant ribonuclease H, an expression vector containing the DNA, and a host cell transformed with the expression vector.

[0002]

BACKGROUND ART Cleavage of RNA in vitro in a sequence-specific manner determines the primary structure of RNA,
It is extremely important for cutting and pasting RNA. However, at present, as a means to achieve that purpose, Inoue et al. (Inoue, H.) (Febsters, 215 , 32)
7-330 [1987]) and Shibahara et al.
S. ) (Nucleic Acid Research, 15 , 440
There is only a method developed by 3-4415 [1987]). This method uses ribonuclease H
Utilizing the property of specifically hydrolyzing only the RNA chain of the DNA / RNA hybrid, first, 2'-O-
A hybrid of a chimeric oligonucleotide containing methylribonucleotide and RNA is prepared, and then Escherichia coli ribonuclease H is allowed to act on the hybrid to cleave the RNA at the region where the hybrid is formed. However, in this method, 1.5 equivalents or more of the chimeric oligonucleotide is required to efficiently cleave RNA, and the reagent for synthesizing the chimeric oligonucleotide is very expensive. More cost issues were pointed out. Furthermore, since annealing for hybrid formation must be carried out before the action of ribonuclease H, operability is complicated, and its improvement has been desired.

On the other hand, the antisense oligonucleotide inhibits the expression of a specific gene in animal cells in the region where messenger RNA or its precursor hybridizes with the antisense oligonucleotide, and the endogenous ribonuclease H (RNA -Minshull, J.) and Hunt because it is cleaved by an enzyme that cleaves only the RNA strand of the DNA hybrid.
Since its discovery by (Hunt, T.) (Nucleic Acid Research, 14 , 6433-6451 [198]
6]), the development of a highly selective cleavage method of RNA in vivo has become remarkably important. This is because such a highly selective cleavage method makes it possible to prevent the expression of undesired traits in various cells, and is expected to have an epoch-making application particularly in the fields of medicine and agriculture.

However, attempts to inhibit in vivo protein synthesis using the above-mentioned antisense oligonucleotides themselves have been the target of messenger R.
It was unsuccessful due to the low efficiency of cleaving NA molecules. The reason for the low efficiency of cleavage is mainly because there is not sufficient amount of ribonuclease H in the intracellular compartment where the antisense oligonucleotide and messenger RNA hybridize. It is considered that this is because the sense oligonucleotide is decomposed by the intracellular nuclease. Therefore, in order to improve the intracellular stability of antisense oligonucleotides, a method for synthesizing oligonucleotides having a modified phosphate bond such as phosphotriester, phosphoramidite, phosphorothioate, or methylphosphonate has been proposed. It has been developed so far (Review: Miller, PS Biotechnology, 9 , 358-362 [1991]).
However, although these oligonucleotide derivatives are certainly less susceptible to degradation by intracellular nucleases, RNA hybridized with them is RNaseH
The efficiency as an antisense was not improved so much because it was difficult to be decomposed by.

On the other hand, there have been some methods to solve the problem that the cleavage efficiency is poor because ribonuclease H is not present in sufficient amount in the intracellular compartment where messenger RNA hybridizes with these antisense oligonucleotides. It has been reported. One is Zuckermann (RN) and Schultz,
P. G. ) In the method developed by
National Science of Academy, 86 , 17
66-1770 [1989]), staphylococcal nuclease and oligodeoxyribonucleotide (22me
It utilizes a hybrid nuclease in which r) is linked by a covalent bond. In this hybrid nuclease, the oligonucleotide portion controls the nucleotide sequence recognition, and the nuclease portion controls the catalytic activity. However, although this hybrid nuclease surely cleaves RNA in a sequence-specific manner, it has poor cleavage efficiency.
(Up to 50%), the operation is complicated, the cleavage product is difficult to re-ligate because it forms 3'-phosphate, there is almost no turnover (repeating action) as an enzyme, and further self-digestion, etc. There are many problems and it is highly desirable to solve these problems. Another one
One is the use of ribozymes discovered by Ceck (TR) (Manual Review of Biochemistry, 59 , 543 [1990]). However, the ribozyme RNA molecule is certainly another RNA
Although it cleaves a molecule in a sequence-specific manner, it has a fatal drawback that its catalytic activity is extremely low.

[0006]

[Means for Solving the Problems] As a result of intensive investigations by the present inventors for the purpose of developing a sequence-specific RNA hydrolase having both high selectivity and high catalytic activity, the molecular surface of ribonuclease H Hybrid ribonuclease H obtained by converting any of the amino acid residues that are present in Escherichia coli and not directly involved in the enzyme activity into a cysteine residue and covalently binding to an oligodeoxyribonucleotide via the thiol group of the cysteine residue. It was found that the above can achieve the intended purpose, and the present invention has been completed.

Therefore, a first object of the present invention is a hybrid in which a mutant ribonuclease H modified so as to have one cysteine residue on the surface of a molecule without affecting enzyme activity and an oligodeoxyribonucleotide are bound. A ribonuclease H, wherein the thiol group in the cysteine residue is involved in covalent bond with an oligodeoxyribonucleotide.

In the present specification, the molecular surface of ribonuclease H means a region capable of coming into contact with water molecules in an aqueous solution. As will be described later, the present inventors have found that E. coli ribonuclease H exists on the molecular surface of 1
Although the amino acid at position 31, 135, 138 or 142 was converted to a cysteine residue and this enzyme was linked to oligodeoxyribonucleotide through its thiol group, almost no decrease in enzyme activity was observed in any case. . As is clear from this, the position of the amino acid residue to be converted to a cysteine residue is not limited to the above-mentioned one, but it is an amino acid residue existing on the molecular surface of ribonuclease H, and it is Any amino acid residue that is not directly involved may be used. By the way, the active center of E. coli ribonuclease H is 10
And 70-position aspartic acid, and 48-position glutamic acid, which is located near these residues in the tertiary structure, 124-histidine, 16-position, 44-position,
It is known that asparagine at the 45th and 130th positions, threonine at the 43rd position, and glutamine at the 72nd position are also involved in catalytic activity and substrate binding.

The mutant ribonuclease H is a natural ribonuclease H in which an amino acid residue existing on the surface of the molecule is converted into a cysteine residue, and in addition to this conversion, a cysteine residue existing outside the surface of the molecule. Is converted to another amino acid residue. It is convenient to use Escherichia coli-derived ribonuclease H, but it is highly homologous to Escherichia coli ribonuclease H in amino acid sequence (30% or more) and has a tertiary structure and an active center similar to E. coli ribonuclease H. Ribonuclease H derived from other organisms can be used, irrespective of natural type or mutant type.

The oligodeoxyribonucleotide used in the present invention has the following structure for binding to ribonuclease H:
Its 5'end is chemically modified. The chemical modification is not limited to a particular one, but it is convenient that a spacer is attached to the 5'end and a maleimide group is provided at the tip of the spacer. An example of a spacer having a maleimide group at the end and capable of being bonded to the 5'end of an oligodeoxyribonucleotide is given below.

[Chemical 1] First, this spacer is used for Amino Modifier II (C
LONTECH) was added to the 5'end of the oligodeoxyribonucleotide by a DNA synthesizer, and then EMCS
By reacting with (Dojindo Laboratory), it can be bound to the 5'end of oligodeoxyribonucleotide. As an example of another spacer, a method of introducing a thiol group into the 3'end of an oligodeoxyribonucleotide has been reported by Zuckerman, RN and Schultz, PG (Science, 238 ). , 1401-1403 (1987)). In this case, the resulting chemical formula of (nucleotide)-(spacer)-(thiol group) is

[Chemical 2] And an enzyme having a thiol group (Enz-SH)
By reacting with

[Chemical 3] A moiety replaces the S-Enz moiety, resulting in the oligonucleotide being covalently attached to the enzyme via an SS bond.

The oligodeoxyribonucleotide is a tetramer or more, and a nucleotide sequence complementary to the nucleotide sequence of the cleavage site of RNA to be degraded.
It may be of any length and sequence as long as it contains (30 mers or less). However, it is preferable that the number of nucleotides is around 10. The oligodeoxyribonucleotide may be modified to enhance the specificity of complementary binding to RNA or to increase the intracellular stability. For example, a chimeric nucleotide in which a part of DNA is replaced by 2'-O-methyl RNA, or an oligonucleotide in which a phosphate bond is replaced by a thiophosphate bond, a phosphate triester bond, or the like can be used as the oligodeoxyribonucleotide of the present invention. . In the present specification, those modified in this manner are also simply referred to as oligodeoxyribonucleotides.

The hybrid ribonuclease H according to the present invention is obtained by reacting a mutant ribonuclease produced by the method described below with an oligodeoxyribonucleotide whose 5'end is chemically modified, and the product thereof is subjected to, for example, a cation exchange column. It can be prepared by separation by HPLC used. In the thus-obtained hybrid ribonuclease H, the oligodeoxyribonucleotide portion is responsible for the base sequence recognition, and the mutant ribonuclease H portion is responsible for the catalytic activity, so that the efficient enzymatic activity is exhibited.

Mutant ribonuclease modified so as to have one cysteine residue on the molecular surface is a recombinant DNA.
It can be produced by a conventional method using a technique, for example, the method shown in Examples below. That is, a DNA encoding ribonuclease H in which one amino acid residue on the surface of the molecule has been converted into a cysteine residue is used as a Gene Amp Kit.
(Takara Shuzo) is prepared by the PCR method. PC
The site mutagenesis using R was performed by the method of R. Higuchi (PCR Protocols (Innis, MA et al.
l.eds.), pp177, Academic Press Inc. (199)
0)). The DNA thus obtained is inserted into, for example, an expression vector having both P _L and P _R promoters of bacteriophage λ, this vector is introduced into an E. coli host, and the host is cultured to produce the target mutant ribonuclease. can do. The DNA encoding the mutant ribonuclease H, the expression vector into which the DNA is inserted, the host cell transformed with the expression vector, and the mutant ribonuclease H produced are all novel, and all of the present invention Configure. Therefore, another object of the present invention is the E. coli ribonuclease H 131, 13
Mutant ribonuclease H in which the amino acid residue at position 5, 138, or 142 is substituted with a cysteine residue, and DN encoding the mutant ribonuclease H
A, an expression vector containing the DNA, capable of expressing the DNA, and a transformant transformed with the expression vector.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[0015]Example   (1) pJAL131C, pJAL135C, pJAL
Preparation of 138C and pJAL142C E. coli ribonuclease H (hereinafter referred to as ribonuclease H
As a method of introducing a DNA chain into
It was decided to use the chemical modification by. First, Ribonuk
Active center of Rease H (Asp10, Glu48, Asp70)
Glutamic acid, relatively close to, with residues facing out
131, glutamic acid 135, arginine 138, methyl
Choose onin 142 and convert each to cysteine
I decided. In addition, conversion to other cysteines is necessary for this conversion.
Of natural ribonuclease H in order to avoid biological modification
Free cysteines (13, 63, 133)
Cysteine-free mutant Ribonuk converted to red alanine
Plasmid pC containing the structural gene for Rase H
A600 (Kanaya et al., Biochemical Journal,27
1, 59-66, 1990) as a template.
Was performed using. Figure 1 shows the PCR primers
5'-primer, 3'-primer and mutation
A primer was synthesized. From glutamic acid 135 cis
Conversion to thein (see Figure 2) involves 5'containing the SphI site
-Primer and primer NO. 2, including SalI
3'-primer and primer NO. 1 for each
Commercially available Gene Amp Kit mixed with Smid pCA600
Amplification of DNA was performed according to the instruction of (Takara Shuzo).
As a result, 400 bp, X defined by SphI-XhoI
A 100 bp DNA strand defined by hoI-SalI was obtained.
It was Clamps remain at these ends
Therefore, the SphI-XhoI fragment and the XhoI-SalI fragment were obtained.
Therefore, SphI and X were added to each amplified DNA fragment.
Add hoI, XhoI, and SalI and digest at 37 ° C for 1 hour
did. Each of these digests was applied to a 1.5% agarose gel.
Upon electrophoresis, a 400 bp SphI-XhoI fragment and
A 100 bp XhoI-SalI fragment was cut out and extracted. one
, Plasmid pJLA504 (West Germany: purchased from Medac)
Also, add SphI and SalI at 37 ° C for 1 hour
After digestion for a while, use 0.7% agarose gel electrophoresis.
Scut out and extract the 4.9 kb SphI-SalI fragment.
It was 400 bp SphI-XhoI obtained as described above
0.1 μg, 0.05 μg of 100 bp XhoI-SalI fragment
And 0.1 μg of the 4.9 kb SphI-SalI fragment were mixed, and D
Using NA ligation kit (Takara Shuzo)
The plasmid was circularized exactly as in the manual. This cyclization
Escherichia coli HB101 was transformed with the plasmid
A transformant pJAL135C was obtained. Also, pJAL142C
For pJAL131C, 13
For 8C, use SstI instead of XhoI
Went to. Detailed restriction of the plasmid thus obtained
Enzyme cleavage maps are shown in Figure 3 and Figure 4 (pJAL131
C (Fig. 3), pJAL135C (Fig. 4), pJAL138C
(See FIG. 3) and pJAL142C (FIG. 4), respectively). Transformation
Substitute Escherichia. E. coli HB101 / pJA
L131C, pJAL135C, pJAL138C, pJ
AL142C deposited with Institute of Microbial Science and Technology
Has been done. Escherichia. E. coli HB101 / pJAL1
31C; Consignment No .: Microtechnology Research Institute, No. 12312, Contract date: Heisei 3
June 17, Escherichia. E. coli HB101 / pJAL1
35C; Consignment No .: Microtechnology Research Institute, No. 12313, Contract date: Heisei 3
June 17, Escherichia. E. coli HB101 / pJAL1
38C; Consignment No .: Microtechnology Research Institute, No. 12314, Contract date: Heisei 3
June 17, Escherichia. E. coli HB101 / pJAL1
42C; Consignment No .: Microtechnology Research Institute, No. 12315, Contract date: Heisei 3
June 17,

(2) Cultivation of transformants and preparation of ribonuclease H-containing cells Plasmids pJAL131C, 135C, described in (1),
E. coli K-12 strain H transformed with 138C and 142C
B101 was shake-cultured at 30 ° C. in LB medium containing 80 μg / l ampicillin. At the time when the turbidity of the culture broth grew up to about 100 in terms of the Klett value, the culture temperature was raised to 42 ° C., and the mixture was further shaken for 4 hours and then collected. The obtained bacterial cells were frozen and stored at -20 ° C.

(3) Extraction and purification of ribonuclease H from transformants HB101 collected from 200 ml of the culture broth obtained in (2) above.
/ PJAL131C and pJAL135C, pJAL13
1.0 g of each of 8C and pJAL142C
10 mM Tris-HCl buffer (TE) containing M EDTA (p
After suspending in 30 ml of H7.5), the cells were disrupted by sonication in ice. Centrifuge supernatant (crude extract) obtained by centrifugation at 15,000 rpm for 30 minutes at 4 ° C. was added to TE (pH 7.5).
It was dialyzed against 5 l at 4 ° C. D equilibrated with the same buffer
E-52 column (4 ml) and P-11 column (2 ml) were passed in this order. Under this condition, C131, C135, C
Mutant ribonuclease H of either 138 or C142
Also passes through the DE-52 column and is adsorbed on the P-11 column. TE (pH 7.5) 4ml, then 0.1M NaC
Each mutant ribonuclease H was eluted from the P-11 column by flowing 4 ml of TE (pH 7.5) containing 1 and linearly increasing the NaCl concentration to 0.5M. Each of the P-11 elution fractions containing these mutant ribonucleases H was concentrated to about 2 ml and then further concentrated to 0.1 MNa.
Sephacryl S-300 (Superfine) equilibrated with 10 mM sodium acetate buffer (pH 5.5) containing Cl
Each mutant ribonuclease H was purified by applying it to a column (φ1.6 × 90 cm). Purified sample is 15% S
DS-PAGE gave a single band and reverse phase HPLC also showed a single peak. The purification yield was about 30 mg / l culture. To identify the purified sample, the peptide fragment obtained by digestion with Achromobacter protease I was mapped by reverse-phase HPLC to confirm the elution position of each fragment peak, and 131, 135, 13
Each peptide containing the amino acids at positions 8 and 142 is
After fractionation, it was performed by N-terminal amino acid sequence analysis.

(4) Synthesis of nonadeoxyribonucleotide NEM-d9mer having a maleimide group at the 5'end The mutant ribonuclease H (C13) shown in (3) above.
In order to introduce a DNA chain into 1, C135, C138 or C142), a nonadeoxyribonucleotide derivative having a maleimide group highly reactive with SH group was synthesized.
The reaction formula is shown in “Chemical Formula 4”.

[Chemical 4] First, a nonameric DNA having a sequence of 5'-GTCATCTCC-3 'was prepared using a DNA synthesizer (Applied Biosystems 380).
A) was manually synthesized according to A), and at the final step, amino modifier II (CLONTECH, N-fluorenylmethyloxycarbonyl-O′-dimethoxytrityl-O ² -cyanoethyldiisopropylamino-) was used.
20 μmol of phosphinyl-3-amino-1,2-propanediol) was added to add an amino group necessary for introducing a maleimide group. The reaction is then cleaved from the carrier with aqueous ammonia, left at 50 ° C. for 6 hours for deprotection, then isolated by reverse phase HPLC and treated with 2% 80% acetic acid for 20 minutes to remove the dimethoxytrityl group. It was The product thus completely deprotected was purified by reverse phase HPLC and ion exchange column chromatography. The reaction formula of the following reaction is shown in "Chemical formula 4". To introduce a maleimide group into this reaction product (see (1) in "Chemical formula 4"), EM
CS (Dojndo Laboratories, N- (ε-maleimidocaproyloxy) succinimide) was reacted in DMF containing 0.1 M sodium carbonate, 2 mM EDTA (pH 7.5) for 1 hour. Then, it was purified by subjecting to Sephadex G-50 by gel filtration, and it was confirmed by reverse phase HPLC that this nonadeoxyribonucleotide derivative (see (2) of "Chemical Formula 4") was single. The yield of this compound was 2.4 OD units.

(5) Introduction of DNA chain into ribonuclease H Mutant ribonuclease H having one cysteine residue obtained in (3) and nonadeoxyribonucleotide derivative (NEM-d9) shown in (4), To connect between the thiol group and the maleimide group, 0.1M Tris-HCl buffer (p
Ribonuclease H (5.4 nmol) and N in H7.0)
EM-d9 (27 nmol) (molar ratio 1: 5) is reacted at room temperature for 1 hour. The reaction formula is shown in "Chemical Formula 5".

[Chemical 5] This reaction solution was applied to a cation exchange column ES-502C (Asahi Kasei) and the peak was fractionated. Figure 5 shows the elution pattern on the cation exchange column.
Is NEM-d9, 1 μg, B is mutant ribonuclease H
(C135) 5 μg, C is NEM-d9-, 1 μg and C13
This is a pattern in which 100 μl of a reaction solution containing 5,5 μg was applied to HPLC. The peak obtained in C is 15% S
When analyzed by DS-PAGE, a single band was observed at the position of molecular weight 21000 as shown in lane 4 of FIG. Ribonuclease H into which this nucleic acid derivative has been introduced
(C135) was named d9-C135. A schematic diagram of this enzyme is shown in FIG. 7. A model of substrate recognition and its degradation by d9-C135 is shown in FIG. 8. In d9-C135, the nucleic acid portion controls RNA sequence recognition, and the enzyme portion controls catalytic reaction. Other mutant ribonuclease H (C
131 or C138) in the same manner as d9-C1.
31 and d9-C138 were obtained. d9-C138 is crude.

(6) The comparative activity of the enzyme activity of each mutant ribonuclease H was defined as 1 unit of the amount of enzyme that produces 1 μmol of the acid-soluble fraction in 1 minute at 37 ° C. using M13 DNA / RNA hybrid labeled with tritium as a substrate. did. The amount of protein is based on the assumption that mutant ribonuclease H and natural ribonuclease H have the same extinction coefficient.
It was determined by measuring the absorbance at 280 nm using 0.1% ₂₈₀ = 2. Regarding the mutant ribonuclease H introduced with nonadeoxyribonucleotide,
When the extinction coefficient when the nucleic acid and the protein were bound one-to-one was calculated, the molecular extinction coefficient at 280 nm was 8.7 × 10 ⁴ , so it was used at 280 nm
The results are shown in Table 1 as determined from the absorbance at. For reference, Table 1 also shows the activity of each mutant ribonuclease H when modified with NEM.

[Table 1]           Specific activity (U / mg) Enzyme name   Unqualified (%)                  Decoration                               NEM (%)    NEM-d9 (%) Natural type 35.9 (100) <0.03 − − − C131 23.2 (64) 7.7 (33) 0.1 (0.43) C135 36.5 (100) 16.9 (72.8) 0.78 (2.1) C138 8.6 (24) 2.4 (27.9)-C142 34.2 (95) 12.7 (37.1) 0.11 (0.32)   -Undecided

Each enzyme has about 25 amino acids relative to the natural type.
% To 100% activity was retained, and about 30% to 70% activity was retained for each unmodified enzyme even when NEM modification was performed. When modified with NEM-d9, the activity was reduced to several% as compared with the unmodified form. in this case,
Since the DNA / RNA hybrid was used as a substrate, it was considered that the oligonucleotide portion of the enzyme modified with NEM-d9 inhibited the enzyme activity.

(7) Cleavage mode of complementary RNA chain To investigate the function of the hybrid enzyme in the present invention, a complementary RNA chain, nonaribonucleotide (5'-GGA)
GAUGAC-3 ') was hydrolyzed. The method was in accordance with the method already filed by Kanaya et al. (Japanese Patent Application No. 01-320231). As the substrate, nonadeoxyribonucleotide (5'-GUCATCTCC-
3 ') and the nonanucleotide duplex of the above nonaribonucleotide and nonaribonucleotide were used. Add 30 μl of the enzyme solution to 9 μl each of nonanucleotide duplex and nonaribonucleotide solution
The temperature was kept at 1.5 ° C. for 1.5 hours and the reaction was stopped by treating at 90 ° C. for 2 minutes. Then, 2 μl of the reaction solution was applied to a 20% polyacrylamide gel containing 7 M urea and electrophoresed for about 2 hours. The radioactivity on the gel was analyzed using a Fujix BA100 bioimaging analyzer using an imaging plate. The pattern of electrophoresis is shown in FIG. The enzymes and substrates in lanes 1 to 4 of FIG. 9 are shown in Table 2 below.

[Table 2]   lane   Enzyme (amount)     Substrate     1-d-r9mer     2 C135 (0.030 pmol) d-r9mer     3 d9-C135 (0.019 pmol) d-r9mer   4 d9-C135 (0.019 pmol) r9mer   Ribonuclease H is a DNA / RNA hybrid
It cleaves RNA strands, but d9-C135
Is cut off, and in C135, Nona Nucleo
Cid duplex 5th and 6th and 6th and 7th
Cleaves between the second residue, but it is 5th in d9-C135
Only between the eye and the sixth residue. That is, d9-C1
In 35, the RNA strand can be selectively cleaved at a certain point.
all right. The cutting mode is summarized in FIG. Well
In addition, the base mass in the reaction solution is 10 pmol and the enzyme
The amount is about 0.02 pmol and about 500 times as much as the substrate is cleaved.
Therefore, it can be seen that this enzyme has turned over.
The same applies to other enzymes other than d9-C135.
Results were obtained.

(8) Enzymatic Kinetic Parameters of Hybrid Enzyme As shown in (7) above, it was revealed that the hybrid enzyme according to the present invention cleaves only the RNA strand at a specific site. It was decided to determine the enzyme reaction rate parameter. The method was based on the method already applied for a patent (Japanese Patent Application No. 01-320231). For the unmodified enzyme, the nonanucleotide duplex solution shown in (7) was used, and for the hybrid enzyme, the nonaribonucleotide solution was used as the substrate solution. The results are shown in Table 3.

[Table 3]enzyme     km (μM)  kcat (min ^-1 )    kcat / kmSubstrate                                         (min ^-1 , μM ^-1 )   Natural type 0.44 141 320 d-r9mer   C131 1.30 95 73 d-r9mer   C135 1.43 131 92 d-r9merC142 1.82 125 69 d-r9mer d9-C131 0.43 23.3 54 r9mer d9-C135 0.20 16.3 82 r9merd9-C142 0.56 21.7 39 r9mer For all hybrid enzymes, unmodified ones have km
1/3 to 1/7 times, kcat is 1/4 to 1/8 times
It was That is, these enzymes have reduced catalytic activity
However, the affinity for the substrate is increasing. Generally an enzyme catalyst
Noh is compared in kcat / km, but hybrid enzyme is uncorrected
Holds 60 to 80% of decorative enzyme and has sufficient catalytic ability
It is considered to be a thing. As described above, the hybrid according to the present invention
Ribonuclease H sequence-specifically cleaves RNA,
Moreover, it retains high catalytic activity. Also the reaction product
Suitable for religation because it has a 5'-phosphate group
And that there is a turnover as an enzyme.
It also has advantages such as.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the sequence of an oligonucleotide used for site-directed mutagenesis.

FIG. 2 is a construction diagram of plasmid pJAL135C.

FIG. 3 is a restriction enzyme digestion map of pJAL131C or pJAL138C.

FIG. 4 is a restriction enzyme digestion map of pJAL135C or pJAL142C.

FIG. 5 is a graph showing an elution pattern from an ES502C cation column when subjected to HPLC, where A is N.
EM-d9 (1 μg), B is C135 (5 μg), C is N
Reaction solution 1 of EM-d9 (1 μg) and C135 (5 μg)
The results are obtained by applying 00 μl to each HPLC.

FIG. 6 is a copy of a photograph of a CBB-stained gel after SDS-PAGE. 1 is a marker and 2 is C135 (1 μm).
g), 3 is 10 μl of a reaction solution obtained by reacting 0.2 μg of NEM-d9 with 1 μg of C135, and 4 is a peak portion of FIG. 7C obtained by ES502C.

FIG. 7 is a schematic diagram of the structure of d9-C135.

FIG. 8 is a schematic diagram of substrate recognition of d9-C135 and its degradation. White band is DNA, Black band is RN
A solid line connecting A, Cys ¹³⁵ and DNA indicates a linker portion. Also, the three black residues Asp ¹⁰ , Glu
⁴⁸ , Asp ⁷⁰ is the active center of ribonuclease H.

FIG. 9 shows a degradation product of nonanucleotide duplex or nonaribonucleotide hydrolyzed by C135 and d9-C135 in the presence of Urea in 20% SDS-P.
FIG. 3 is a copy of a photograph of a gel which has been subjected to AGE electrophoresis and whose radioactivity has been detected by an imaging plate.

FIG. 10 is a schematic diagram showing the cleavage patterns of an unmodified type and a modified type (introduced with NEM-d9).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 15/55

Claims (15)

[Claims] 1. A hybrid ribonuclease H in which a mutant ribonuclease H modified to have one cysteine residue on the surface of a molecule without affecting enzyme activity and an oligodeoxyribonucleotide are bound to each other, wherein the cysteine is Hybrid ribonuclease H, characterized in that the thiol group in the residue is involved in covalent bond with oligodeoxyribonucleotide. 2. The mutant ribonuclease H is derived from Escherichia coli and is located on the surface of the molecule 131, 135, 1.
The hybrid ribonuclease H according to claim 1, wherein the amino acid at position 38 or 142 is substituted with cysteine. 3. The hybrid ribonuclease H according to claim 2, wherein the maleimide group introduced into the oligodeoxyribonucleotide via a spacer and the thiol group in the cysteine residue are covalently bound to each other. 4. The spacer has the formula: — (CH ₂ ) ₅ —CO—N.
The hybrid ribonuclease H according to claim 3, which is represented by HCH ₂ CH ₂ —CH (CH ₂ OH) —. 5. A mutant ribonuclease H having one cysteine residue on the surface of the molecule. 6. The ribonuclease H derived from Escherichia coli, wherein the amino acid at position 131, 135, 138 or 142 located on the surface of the molecule is substituted with cysteine, and the mutant ribonuclease H according to claim 5. 7. Escherichia coli ribonuclease H 131, 1
A DNA encoding a mutant ribonuclease H in which the amino acid residues at positions 35, 138 or 142 are replaced with cysteine residues. 8. An expression vector containing the DNA according to claim 7 and capable of expressing the DNA. 9. A replication origin derived from the plasmid pBR322, a gene of the heat-sensitive repressor cI ^ts857 , both the P _L and P _R promoters of bacteriophage lambda, and the P
The expression vector according to claim 8, which contains the mutant ribonuclease H DNA according to claim 7, which is under the control of both _L and P _R promoters. 10. Plasmids pJAL131C and pJAL
The expression vector according to claim 9, which is 135C, pJAL138C, or pJAL142C. 11. The expression vector according to claim 8, which contains an ampicillin resistance gene as a selectable marker. 12. A transformant obtained by transforming an Escherichia coli host with the expression vector according to claim 8. 13. The transformant according to claim 12, wherein the host is Escherichia coli K-12 strain HB101. 14. Escherichia coli HB101 / pJAL131
C, HB101 / pJAL135C, HB101 / pJA
The transformant according to claim 13, which is L138C or HB101 / pJAL142C. 15. In vitro or in vivo,
A method for selectively and specifically cleaving single-stranded RNA using the hybrid ribonuclease H according to claim 1.