JPS59500081A - Method for producing a given polyribonucleotide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background and prior art of manufacturing method for certain polyribonucleotides There are generally three steps involved in producing proteins using genetic engineering techniques. be. The first step is to experiment with the desired gene that contains the code for a specific protein. It can be produced indoors or isolated and purified. The second step is this gene recombination with a suitable transfer vector such as a plasmid. Third The step is to transfer this recombinant vector to a specific microorganism, and this microorganism By inducing the production of a specific gene product.

The present invention is directed to a method of implementing said first step. Currently, in vitro The method for producing genes by sequentially adding cleotides is a chemical method (I. ra + K, and R1gg31 A, + 5science 109: 14 01 (1980) + (Khorana, H, G, + 5science 203: 614 (1979), 1m elementary procedure (S, Gillam + P, Jahnke + C, Astell TS, Ph1llips + C, A. Hutchinson + M.

Sm1th, Nucelic Ac1ds Res, 6.2973 (19 79) and the production of the desired DNA or RNA molecule using solid phase technology. Ru. The chemical procedure for DNA synthesis is difficult because the reactions involved are nonspecific. After the cropping stage, the desired product is rolled over to ensure sufficient purification. did Therefore, chemical methods of DNA and RNA synthesis are clearly less desirable than enzymatic methods. Not good.

The reason is that more starting material is required to obtain the same yield, resulting in higher costs. The process of purification is time consuming, and at each stage a portion of the product is wasted by us. and many steps involved in protecting and deprotecting reactive sites. In addition, there is a risk of contamination with degrading enzymes that destroy synthetic materials and an opportunity for error. I'm happy with many things.

postreotide There have been many reports on the method of synthetically synthesizing RNA0 using enzymes. this polynucleotide The phosphorylase method is limited to the production of oligodeoxynucleotides.

Under regulated conditions, polynucleotide phosphorylase primarily processes single nucleotides. The otide is added to a short oligodeoxynucleotide, and this adduct is then chromatinized. Isolated by topography. The phase method involves attaching a nucleotide chain to a solid support or material. This is done by stepwise addition of nucleotides using the chemical methods described above. be called.

゛The present invention synthesizes RNA of a predetermined sequence using Tosaku T4 RNA'J gauze. It teaches compositions of compounds useful in the process and methods for making them. Synthetic RN A is transcribed into DNA, the synthetic gene is inserted into the plan, mid, and inserted into the microorganism. Methods for introducing and enabling expression are known in the art.

Enzyme T4RNAIJ isolated from E. coli infected with bacteriophage T4 The existence, purification, and mechanism of the enzyme have been reported (5ilber, R., Malathi.

v, g, and Hurwitz + J, Proc, Natl, Acad, Sci.

(1972) 69: 3009). The catalytic action of this enzyme allows donor nuclei to The phosphate group at the 5' end of leotide and the 3' end of the acceptor oligonucleotide The following phosphodiester bond is formed between the hydroxyl group and the hydroxyl group.

R Japanese patent application 1980-19003 (published on 2nd and 9th 1980) is T4R Using NA ligase, the 3' end of a nucleotide sequence that does not have a phosphate group at the end polynucleotides by adding one mononucleotide diphosphate (pNp) to the end. It teaches extending nucleotides. This method uses triglycerides as the starting substrate. nucleotides, which can be obtained commercially or by chemical means. Adenosine triphosphate (ATP) is used as the energy source for the reaction. ) Request all. Moreover, this invention only teaches adding one base. can produce oligos or polynucleotides that require sequential addition. Powerful.

HOApApAOH+pCp+ATP ----->H□ApApApCp+A MP+PPi Abbreviations used herein for convenience are shown in Table 1.

N, X, Y, Z Arbitrary glue Bonucleotide DNA Deoxyribonucleic acid RNA ribonucleic acid BAP Bacterial alkaline phosphatase VPD Snake venom phosphogesterase AppNp　A”　p”　pN”　p　p　(For detailed explanation, see the main text) ) p2'-2' d-type phosphodiester bond TEAB triethyl hydrogen carbonate ammonium liquid DTT D L-dithiothreitol (Flerand's reagent) PNK polynucleotide kinase nbztO-nitrobenzyl group pnbztO-! unitrobenzyl phosphate group UV ultraviolet rays MOPS 3-(N-morpholino)propanesulfonic acid HEPES　’　　　　-2-Hydroxethylpiveracin-N’-2-ethanes sulfonic acid The present invention teaches the synthesis of biologically active RNA and DNA molecules. . All nucleotide sequences are indicated by the respective abbreviations according to Table 1.

Unless otherwise specified, all phosphodiester bonds between dinucleotides are 3/-5/ Demeru.

Summary of the invention The invention disclosed herein is directed to mono-, di-8 oligo- or polynucleotides ( A ribonucleotide sequence (donor) having two or more bases (acceptor) Using the enzyme T4RNAIJ gauze that specifically adds to the 57-37 linkage at the 3' end of This book teaches a method for total synthesis of double-stranded RNA molecules consisting of a predetermined base sequence. be. T4RNA in the presence of catalyst! The shortest receptor that is affected by J gauze is the 5' position. It is a dinucleotide that has been oxidized at the nucleotide position.The receptor is a trinucleotide or If longer, the 5' end may be phosphorylated or hydroxylated, allowing the donor to become an acceptor. can be added to the 3' end of one base as a donor at the end of the acceptor Regarding the end-addition method, the starting materials for the receptor are commercially available, or a dinucleotide synthesized by a chemical method known in the art; , the first step is to phosphorylate the 5' free end. This step is carried out by the enzyme polynuclease. Mixing dinucleotides and ATP in the presence of creatate kinase (PNK) (Richardson C, C, Proc.

Natl, Aeod, Sci, U, S, A, 54.158 (1965)) , the above-mentioned kinase specifically phosphorylates the 5' end of the dinucleotide shown in Formula 1. do. If the receptor is trinucleotide or longer, this phosphorylation step is unnecessary. is necessary.

PNK (1) 0HXpYOH+ATP----)pXpYOH-rADP second stage is the receptor dinucleotide, (AppNp) (where N is adenine, cytosine , Gua=n, r)? a ribonucleotide selected from the group consisting of syl and isosine; The compound pi me adenosine p2- (3' nucleotide-phosphorus) acid)-5' pyrophosphoric acid. This compound APpNI) and its The synthesis method is disclosed in the specification of the co-pending application (). RNA IJga By the reaction of known receptor ribonucleotides with AppNp in the presence of enzyme, formula 2 is obtained. The indicated nucleotides are produced. This reaction requires ATP as an energy source do not.

RNA ligase (2)　PXpYOH+AppNp-----)pXpYpNpshipA book The invention also provides that in the presence of ATP and RNA ligase, the 5' phosphate of the donor is ligated to the acceptor. It binds to the 3' hydroxyl group of and elongates or generates a bonucleotate (Formula 3) As such, a donor RNA molecule with two or more bases is attached to an acceptor ribonucle. This paper discloses a method for adding ocide to ocide. The link at the 3' end of the donor molecule Due to the acid group, its terminus is not reactive to RNA ligase, resulting in Further addition of donor molecules is prevented. In this method as well, the receptor For leotide, phosphorylation of the 5' end is required, but for trinucleotides or Longer than that, phosphorylation is unnecessary (Equation 4).

RNA ligase (4) pXpYOH+Appnbzl−−−−−−−’> pXpYpnb To phosphorylate the 3' end of the zl donor molecule, in the presence of RNA ligase The 5' phosphorylated dinucleotide is reacted with the compound Appnbzl (Formula 4). n The bz1 part is removed by exposing the generated nucleotide to strong light. (Formula 5).

The generated extended ribonucleotides are then processed by bacterial alkaline phosphatase (BAP). ) to cleave off the terminal phosphate group and act as a receptor. or phosphorylate the 5′ end of bodinucleotide kinase (PNK). ) to convert it into a donor (26).

Each of the reactions described above are just a few of the many ways nucleotides can be produced. These people The method can be applied not only to the dinucleotides described herein, but also to longer oligonucleotides. It can also be used effectively for codes.

thus synthesizing a complete RNA molecule with the code for a specific protein be able to.

For each reaction described below, it is necessary to identify and confirm the reaction products. be. Throughout these examples the identification of the reaction products is 250, 260 and 280n. This is done by determining the absorption ratio at m and comparing this ratio with a known standard. be exposed. Identification and purity were determined in ammonium citrate buffer at pH 5.0 and formate. Filter paper electrophoresis in ammonium buffer, pH 2.8, 800 volts, 1 hour. It is also verified by law.

DESCRIPTION OF SPECIFIC EMBODIMENTS The present invention uses T4RNA! for the production of nucleotides having a predetermined base sequence. j gauze It teaches the use of Two methods are disclosed in the outline outlined above. has been done. One method is to use receptor ribonucleons with at least two bases. It is necessary to add a donor mononucleotide to the compound. Other methods are 2 or more This involves adding a donor nucleotide with more than 100 bases. these two people The decision as to which method to use depends on the composition and origin of the nucleotide sequence of interest. depends on the availability of commercially prepared nucleotides used as starting materials. Ru.

Monoribonucleotide addition A trinucleotide with a predetermined base sequence from a known dinucleotide and an AI)I)Np compound. The team that synthesizes nucleotides, the first thing they need is 57 Hakusan of dinucleotides. This is phosphorylation at the wood end. This can be done using enzyme polynucleases under procedures known in the art. This is accomplished using otidokinase.

Following the phosphorylation step, all that is required is to add the phospho-labeled dinucleotide to the reaction mixture. It is often isolated from other components. This fractionates the reaction mixture and The cleotide-containing fractions were collected and mixed into this phosphorylated dinucleotide assembly. This is achieved by treatment with ATPase, which cleaves pyrophosphate from ATP. It will be done. Therefore, we believe that it is possible to isolate phosphorylated dinucleotides by fractionation. Medo The ATPase used in Example 1 is T4 RNA IJ gauze ffIg. Although it is obtained as a living organism and has been purified, any ATPase should be effective. be.

The phosphorylated dinucleotide or other type of acceptor nucleotide is then AI)P Incubated with NPNp compound.

At that time, the specific AI used) l) NP compound B, N is the 3' end of the dinucleotide Add a compound that will become the nucleotide to be added to the end. Preferred reaction conditions are described below. This is shown in the example below.

Finally, the generated nucleotides are treated with phosphatase to add The terminal phosphoric acid can be removed, leaving a hydroxyl group for the reaction. phosphata The conditions for the phosphatase reaction differ depending on the phosphatase used, but Well known in the art. The nucleotide after phosphatase treatment is VC is prepared for sequential addition as described below.

Addition of two or more nucleotides A donor ribonucleotide with two or more bases is combined with an acceptor ribonucleotide. The addition to the 3' end of the donor phosphorylates the 5' and 3' ends of the donor. , this phosphorylated donor together with the acceptor in the presence of RNA ligase and ATP. This can be done by culturing in Donor ribonuc for this reaction Reotide is a 5' phosphorylated dinucleotide, or a phosphorylated 5' end 1 or I wish I could use an oligonucleotide with a 5' free end.

This ribonucleotide is used to phosphorylate the 3' end of the donor ribonucleotide. pnbzl was incubated with ApPnbzl in the presence of RNA ligase, resulting in pn The bz1 portion is attached to the 3' end. The reaction mixture is boiled and the RNA ligase After becoming inactive, it is mixed with ATP in the presence of Bo/IJ nucleo+tidokinase. , the 5' end of the donor molecule is phosphorylated.

After the donor is separated, it is exposed to strong light such as a 300 watt mercury lamp to remove the donor. The nbz1 portion can be removed leaving the 5' and 3' phosphorylated ends. child The donor can be added to the acceptor with the nbz1 moiety intact, i.e. without removal. I can do it.

After adding the donor ribonucleotide to the acceptor, the resulting extended nucleotide is attached to the host. the donor by removing all phosphate groups by treatment with sphatase. can be converted into a form that acts as a receptor. Or an elongated nucleotide Treating the donor with PNK to phosphorylate the 5' end and make it available for use as a donor. Can be done.

The preferred method described for the D″f2AEf2AE safety step is All selection stages are performed using high-pressure liquid chromatography, thin layer chromatography, etc. Matography, gel and paper electrophoresis, and 3 and other known separation techniques may also be accomplished without departing from the scope of the invention. I can do it.

Adenylyl (3/-s/) guanosine (A pG) was first mixed with ATP to generate pApG. 15mM AG (SI GMA) 70ut, 1M Tris-HCt (pH 8,1) 60ut, 2-method Captoethanol 10uL, 100mM MgC2z 100ut, 2 0mM ATP 150uL, H2O 50uL and PNK (1500 units/m L) After incubating the reaction mixture containing 100 ut at 37° for 1 hour, a linear concentration gradient DEAE Sepha using TEAB (p)I 7.6) of O82 ~ 0.8M Fractionation was carried out by Dex-25 column. identified by UV absorption The fourth peak containing ATP and pApG was collected and combined with methanol under reduced pressure. Dry and then lyophilize.

To separate ATP from pApG, the collected fractions were treated with ATP 7-ase. , fractionation was performed using a column.

pApG/ATP mixture (A 260 = 492) 200ut, 100m MDTT 100uA, IM MOPS (pH7,9) 100ut, 100mM MgCt2150ut, azo 800uA and T4 ATP 250 ut of Aase (5000 units/mA) was mixed and cultured at 37°C for 1 hour.

The obtained product was treated with 1 degree linear gradient of 0.2-0.8M TEAB (pH 7.6). Fractionation was carried out using a DEAE Sephadex-25 column. 26 04 The pApG fraction of the second major peak at n'm was collected.

Next, pApGpAp was synthesized using the pApG obtained in the above reaction. Td RN A ligase was obtained using the techniques of Silber et al. pApG (0,87mM ) 112ut, AppAp (3,4mM) 58ut, 1M HEPE S (pH 7,5) 15ut and RNA ligase (1500 units/mt) 50 ut was mixed and incubated for 1 hour at room temperature. The resulting mixture was added to DEAE-Sephadec. pApGl)AI) was fractionated with 50mM sodium citrate buffer. It was identified by electrophoresis at pH 5.0 in the middle of the night. The collected pApGpAp fraction Dry in vacuo in the presence of methanol and lyophilize.

This sample was then treated with bacterial alkaline phosphatase (BAP). pA pGpAp 40ul (approximately 1.0 unit at 260nm), LM) Lith-HCA ( pH8,1) 5ut, BAPc (200 units/ml, Worthin gton (Biochemical) 5 ut and H2O 50 ut at 65℃ After culturing for 30 minutes, a linear concentration gradient of 0.1-1.0M TEAB (pH 7,6) was added. It was fractionated using a DEAg Sephadex-25 column. ApGpA I got a good fraction.

Implementation row 2 Synthesis of pGUp from GAU Trinucleotide GAU (Co11aborative Re5search) The purity was determined by electrophoresis. 26mMAppnbz1.225uA , 3.3 mM GpApU 300ut, IM MOP S (pH 7, 9) 75uL, 0.1M MgCA2300ut, 100mM DTT15 0ut, H2O 120ut, RNA ligase 180ut and 100% dimethylene The reaction mixture containing 150 ut of Rusulfoxide DMSO was incubated at room temperature for 20 hours. Ta. Next, after boiling this reaction mixture 7 for 2 minutes, the boiled mixture was added to 20mM ATP. 300ut, 100mM DTT 200ut and PNK (1500 units/ mL) was mixed with 100 uA and incubated at 37°C for 90 minutes.

This reaction mixture was then mixed with a linear concentration gradient O1IM to 1.0M TEAB (pI (7 , 6) using a DEAB Sephadex-25 column. PGpA The fourth peak containing pUpnbzl was collected and then placed in a cooling chamber with a water jacket. It was exposed to a 300 watt mercury lamp for 15 minutes from a distance of 5 cm.

Example 3 Trivalent AAA (Boe?inger) 34uA, 1.0mM pGI ) Apnbzl (synthesized by the method of Example 2) 70uL, 1.0mM HEPE s (pH 7,83) 40ut, 1.0M MgCA25ut, 20mM ATP (SIGMA, >25ut, bovine serum albumin (B3A) (x my/mg 2uL, RNA ligase (1500 units/mL) 40uL and 20 uL of 100% dimethyl sulfoxide were incubated overnight at room temperature. fed. This reaction mixture was fractionated using a DEAE Sephadex-25 column. . The absorption ratio was 250/26o-1, 17 and 28°/2450". Next, this The nbzl group was removed by exposing the oligonucleotide to a mercury vapor lamp as described in Example 2. I left.

APCpCPU+PCpCpGpAp----)ApCpCpUpCpCpGp Ap (1,2mM) ACCU (can be synthesized using Example 1) 75uL, 1.0mM, pCCGAp (Example 1) 90uL, 1 ．． 0M MOPS (pH 7,9) 30ut, 100mM.

MgCl2, 120uL, 100mM DTT 70u! , DMSO ( 100%) 60uA, 20mM, ATP 60ut, BSA (1 0mS'/mL) 15uA, RNN soligase 1500 units/mA) 60ut and The reaction mixture containing 100 ut of H2O was incubated at room temperature (approximately 24°C) for 16 hours. Ta. This mixture was treated with a linear concentration gradient of 0, i to 2.0 M TEAB (pH 7,5). It was fractionated using a DEAE Sephadex A-24 column, and the 6th peak was Identified as polynucleotide ApCpCpUpCp, CpGPAP by It was done.

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Claims (1)

[Claims] 1. Method for synthesizing RNA with a predetermined base sequence using Fumoto T4RNAIJ gauze in particular for the hydroxylated 3' end of the acceptor with at least two bases. Do not add donors differently, in which case there are two or more said donors. of nucleotide bases, adenosylene is used as the energy source for addition. A method characterized by providing phosphoric acid. 2. One predetermined glue on a donor ribonucleotide having at least two bases In the method of adding bonucleotides: Apl) The ribonuclein which the PNP moiety is phosphorylated at the 3' end from the NP-type compound The receptor ribonucleotide is converted to T4 under conditions convenient for transfer to leotide. When mixed with APPNpm compound in the presence of RNA ligase; N in pNp is a group consisting of adenine, guanine, cytosine, uracil and inosine ) A method characterized by: 3. At least two predetermined bond nucleotides having at least two bases In a method of adding to an acceptor ribonucleotide having a base of: specifically phosphorylates the 3' end of cleotide; Phosphorylate the 5' end of the donor ribonucleotide; each acceptor and donor ribonucleotide Receptor ribonucleotides are transferred to T4 RNA under conditions that allow covalent bonding between leotides. incubating with phosphorylated donor ribonucleotides in the presence of gauze; A method characterized by consisting of stages. 4. The receptor ribonucleotide is a 5' phosphorylated 3' hydroxylated diribonucleotide. nucleotide, oligonucleotide phosphorylated at the 5' end and 5' and 3' selected from the group consisting of each hydroxylated oligonucleotide The method according to claim 2, characterized in that: 5. The receptor ribonucleotide is a 5' phosphorylated 3' hydroxylated diriponuclease. leotide, 5' end/oxidized oligonucleotide and 5' and 3' end selected from the group consisting of end-hydroxylated oligonucleotides. 4. The method according to claim 3, characterized in that: 6.Hydroxylated at the 3' and 5' free ends, Reacting syntrilin [(ATP) in the presence of polynucleotide kinase] a step of synthesizing said 5' phosphorylated 3' hydroxylated ribonucleotide by 5. The method of claim 4, further comprising a step. 7.Hydroxylated or bonucleotides and ATP at the 3' and 5' free ends is reacted with polynucleotide kinase in the presence of polynucleotide kinase to produce phosphorylated ribonucleotides. generate; fractionating the reaction mixture by exchange column chromatography; Treating fractionated reaction mixture portions containing phosphorylated ribonucleotides with ATPase death; fractionating the ATPase-treated reaction mixture to isolate phosphorylated ribonucleotides; 7. The method of claim 6, further comprising each step. 8. Reacting the ribonucleotide and ATP in the presence of polynucleotide kinase. The 5' end of the receptor ribonucleotide is specifically oxidized by 6. The method of claim 5, further comprising the step of: 9. The step of phosphorylating the 5' end of the donor ribonucleotide From the step of reacting otide and ATP in the presence of polynucleotide kinase. 4. The method according to claim 3, characterized in that: 10. The step of phosphorylating the 37-terminal end of the donor ribonucleotide comprises at least two T4 RNA In the presence of ligase, the pnbz1 group is attached to the 3' end of the ribonucleotide. Claim 3, characterized in that the process comprises a step of reacting under conditions favorable to Method described. 11. In the method of synthesizing RNA with a predetermined base sequence, 2a) at least two Phosphorylate a postnucleotide with a known base sequence at the 5' end; b) The above RNA and APpNp are reacted at the lower right of T4 RNA ligase to create App. attaching the PN2 portion of NP to the 3' end of the ribonucleotide; C) treating the generated elongated RNA with an enzyme that cleaves the truncated phosphate group; d.) Repeat steps b and C until ribosugic acid of the predetermined sequence is produced; (Here, N is a group consisting of adenine, guanine, cytosine, uracil, and inosine. (selected from or postretide) A method characterized by consisting of stages. 12. 1) Conditions favorable for transfer of the Np moiety are such that the receptor ribonucleotide along with the APPNP compound in a small amount of HFJPES buffer, RNA! Jga Claims characterized in that the method comprises culturing at 37°C for 1 hour in the presence of enzyme. The method described in box 2.