JPH0371109B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing oligoribonucleotides. If oligoribonucleotides with specific nucleotide sequences can be obtained, it is possible to prepare specific genes or
It can be used to adjust enzymes that use A as a substrate, analyze reaction mechanisms, and study interactions between proteins and nucleotides. Such oligoribonucleotides can be prepared by organic chemical methods, but they can also be prepared relatively easily and with good yield by methods using enzymes. Currently, as a method using an enzyme, there is a method in which ribonucleotide trimers or more are prepared by an organic chemical method or an enzymatic method, and the materials are linked together using RNE ligase. However, the specificity and reaction mechanism of RN eligase for the phosphorus donor and phosphorus acceptor base sequences are not yet clear, and conventional methods may not be able to obtain oligoribonucleotides with the desired base sequences. Ta. Therefore, the present inventors conducted studies to develop a method for preparing oligoribonucleotides efficiently and accurately using a certain material using a certain simple method, and as a result, the present invention was achieved.

The gist of the present invention is that when producing an oligoribonucleotide by linking two types of oligoribonucleotides using RN eligase, (A) the 5'-terminal hydroxyl group is A group whose hydrogen atom is represented by the following general formula () [In the formula, R ¹ represents a phenyl group, R ² represents a hydrogen atom, or R ¹ and R ² represent a group forming a morpholine ring together with N] It becomes a phosphate acceptor substituted with The oligoribonucleotide primer and (B) the hydrogen atom of the 2′-terminal hydroxyl group have the following general formula () [In the formula, R ³ represents a hydrogen atom or an alkyl group]
A method for producing an oligoribonucleotide, characterized in that it uses an oligoribonucleotide that is a phosphorus donor and is substituted with a group represented by the following.

To explain the present invention in detail, RN Eligase (hereinafter referred to as RNA ligase) used as a catalyst is Biochimica et Biofisica Acta Vol. 562.
Following the method described on pages 149-161 (1979),
It is used that has been purified to the extent that almost no ribonuclease is present.

Specifically, T4 RNA ligase can be obtained by disrupting E. coli infected with T4 phage and separating and purifying the disrupted solution by ammonium sulfate precipitation, column chromatography, or the like.

Phosphorus receptors for RNA ligase include oligoribonucleotides in which the hydrogen atom of the 5'-terminal hydroxyl group is substituted with a group represented by the above general formula (). As such oligoribonucleotides, trimers or more are used. Examples of trimers include those represented by the following general formula ().

In the above formula, R ¹ represents a phenyl group, R ² represents a hydrogen atom, or R ¹ and R ² represent a group that forms a morpholine ring together with N. B ¹ , B ² and B ³
each independently represents an adenyl group, a guanyl group, a cytosyl group or a uracil group. Although the above examples are trimers, corresponding tetramers to decamers can be mentioned as well.

On the other hand, examples of the phosphorus donor include oligoribonucleotides in which the hydrogen atom of the hydroxyl group at the 2' end is substituted with a group represented by the above general formula (). Examples of such oligoribonucleotides include trimers of the following general formula ().

In the above formula, B ⁴ , B ⁵ , and B ⁶ are adenyl groups,
Represents a guanyl group, cytosyl group or uracil group. R ³ represents hydrogen or an alkyl group. Of course, it may be a corresponding tetramer or more.

Therefore, such a phosphorus acceptor and a phosphorus donor can be easily produced by the following reactions (a), (b), and (c).

(a) Production of dimer by reverse reaction of ribonurease (hereinafter abbreviated as RNase) Journal of Biochemistry, Vol. 81, 1237-1246
It is produced according to the following reaction formula () according to the method described in Page (1977).

XpN＞p+NRNase ――――――→ XpNpN … () In the above reaction formula, where N>p is the ribonureoside
Represents cyclic p in which the hydroxyl groups at the 2' and 3' positions together form an ester with one phosphoric acid (the same meaning is shown below).

(b) Production of trimer Using the dimer obtained by the reaction in (a) as a primer, polynucleotide phosphorylase (hereinafter abbreviated as PNPase) is used to convert the hydrogen atom of the hydroxyl group at the 2-position into an orthonitrobenzyl group. A trimer is produced by extending the substituted ribonucleoside diphosphate by one residue. The reaction formula is as follows.

XpNpN＋PPNφ PNPase ――――――→ XpNpNpNφ …() In the above reaction formula, φ is the 2′- of the ribonucleotide
Indicates an orthonitrobenzyl group substituted with the hydrogen of OH.

(c) Production of phosphorus donor and phosphorus acceptor The trimer obtained by the reaction in (b) is treated with (A) acid to deprotect the protecting group -N(R ¹ R ² ) of 5' phosphate. (B) to remove the orthonitrobenzyl group at the 2' position by irradiation with light,
Phosphorus receptor. The reaction formula is as follows.

XpNpNpNφH+ ---→ pNpNpNφ (phosphorus donor) ...() , can be produced by a similar method.

In the method of the present invention, oligoribonucleotides are prepared using RNA ligase using the phosphorus acceptor and phosphorus donor as raw materials.

The reaction involves a phosphorus donor and a phosphorus acceptor,
RNA ligase, adenosine triphosphate, Mgcl ₂ ,
This is done by adding to a buffer solution containing dithiothreitol and optionally bovine serum albumin, and keeping it warm at about 4°C for one day or more.

After the reaction, oligoribonucleotides of the reaction product can be easily obtained by adding the reaction product to an ion exchanger column such as DEAE-Sephadex (trademark) and performing column chromatography. The generated oligoribonucleotide has substituents at the 5' and 2' ends, so using this as a material, a phosphorus donor and a phosphorus acceptor are obtained in the same manner as in the above method, and then ligated with RNA ligase to further lengthen the oligoribonucleotide. A chain of oligoribonucleotides can be produced.

According to the method of the present invention as described above, oligoribonucleotides can be easily produced by the following process.

() Attach pNp as a ligand to a suitable solid support using the phosphoamidate method, and convert the 3'-terminal phosphate into a cyclic phosphate.

() The support is immersed in RNase and high-concentration ribonucleoside to react, and after the reaction, the support is removed by centrifugation.

() Next, it is immersed in PNPase and ppNφ to react, and only the reactant support is taken out.

Since nucleosides cannot be attached in the RNase reaction and the ligand remains monomeric, it cannot serve as a primer for this reaction.

() This support is irradiated with light to remove the terminal φ group, and then placed in a solution of RNA ligase and pNpNpNφ to react.

The support-containing ligands obtained by centrifuging the reaction product in () are trimers and hexamers in which pNp and the final base are protected by φ. If the same reaction is repeated again, more nonamers will be included. After the required number of connections, the support is immersed in a weak acid solution and these synthetic materials are recovered in solution. The product is monomer+3(n+1)mer(n
is the number of RNA ligase reactions), separation and purification is extremely easy.

According to the method of the present invention as described above, synthetic materials are prepared for various base sequences, and oligoribonucleotides with desired base sequences can be easily prepared using a uniform reaction system as desired. There are advantages.

Hereinafter, the method of the present invention will be explained in more detail with reference to Examples.

In the following examples, C, A and U all represent ribonucleosides, C represents cytosine as a base, A represents adenine as a base, and U represents uracil as a base. ani indicates the aniline residue when forming phosphoramidate. p is phophate;
>p represents cyclic P forming an ester with the 2' and 3' hydroxyl groups of the ribonucleoside.

φ represents an orthonitrobenzyl group substituted for the 2'-OH hydrogen of the ribonucleotide.

PP stands for diphosphate.

For example, anipApUpCφ is R ¹ in equation ()
is a phenyl group, R ² is a hydrogen atom, B ¹ is an adenyl group,
It means a compound in which B ² is a uracil group and B ³ is a cytosyl group.

Example 1 (1) ani pApUpCφ, pApUpCφ and ani
Preparation of pApUpC (a) Preparation of PPCφ Add pocl ₃ 0.2ml to Cφ300mg (0.8mmol)
(2.18 mmol) and 2.5 ml of po(OCH ₃ ) ₃ were added and reacted at 4° C. for 2 hours to obtain pcφ. Add triethylamine to pcφ to make a salt, add 8 ml of t-butanol, 8 ml of H ₂ O, 0.4 ml of morpholin and 0.8 g of dicyclohexylcarbodiimide/16 ml of t-butanol, react under reflux for 1 hour, and then add 0.4 ml of morpholin and 0.8 ml of dicyclohexylcarbodiimide. g
was added and reacted to obtain pcφ morpholidate.

Separately, phosphoric acid 160μ and (n-c ₄ H ₉ ) ₃ N600μ
was added to 2 ml of viridine and dried, 2 ml of viridine and pcφ morpholidate were added, and the mixture was reacted overnight at 37°C to obtain ppcφ. The yield was 30%.

(B) Preparation of anipA>p pAp Ba salt, Dowex 50×2 (100 to 200 mesh) aniline salt, and 50 ml of water were mixed and allowed to stand at room temperature overnight to obtain pAp aniline salt.

0.89 mmol of pAp aniline salt, 10 ml of water, 25 ml of t-butanol, 1 ml of aniline, and 2.06 g of dicyclohexylcarbodiimide were mixed and reacted under reflux for 3 hours to obtain anipA>. The yield was 29%.

(c) Preparation of ani pApU Journal of Biochemistry Vol. 81 1237
~1246 (1977), R.
Prepared using Nase U ₂ .

A 0.1 M Na acetate acetate buffer pH 4.5 containing 1500 units of R Nase U ₂ and 800 μmol of uridine was added at 0°C to
Ani pApu was obtained by standing overnight at 1°C. Yield 41
%.

(d) Preparation of ani pApUpCφ According to the method described in Biochimica Biofijica Acta Vol. 565 (1979) pp. 192-198,
ppCφ11.2mM, ani pApU2.5mM, tolyl hydrochloride
0.1M of pH 8.5, 2mM of Mncl ₂ and 50 units of PNPase derived from Micrococcus rhizodeicticus (manufactured by Sigma) were placed in a colored bottle and left overnight at 37°C.

Preparative separation using high performance liquid chromatography [8 mm diameter, μBONDPAKC ₁₃ (sold by Water Co., Ltd.) was used as the packing material. Flow rate 4ml/min.

Developed with 0.05M NH ₄ HCO ₃ containing 20% CH ₃ OH].
ani pApUpCφ was obtained with a yield of 23.2%. Product identification was determined by digestion with R Nase T ₂ and ani pAp and Up.
It was confirmed that Cp was produced in a ratio of 1:1:1 by using a separately synthesized sample.

(e) Preparation of ani pApUpC 50 ml of ani pApUpC and 50 ml of water were placed in a donut-shaped light irradiation flask and irradiated with light (>290 nm) using a 100 W ultra-high pressure mercury lamp. Irradiation was performed for 12 hours under cooling with tap water.

In addition to a diethylaminoethyl cellulose (DEAEA25) column (0.7 x 10 cm),
NH ₄ HCO ₃ OM → 1M gradient, and ani
pApUpC was obtained. Yield 56%.

(f) Preparation of pApUpCφ ani pApUpCφ containing 12% methanol 0.05M
Add 100μ of NH ₄ HCO ₃ and 10μ of 1NHC
It was kept at 37°C for 24 hours at pH 1.5. Preparative separation using high-speed liquid chromatography [8 mm diameter, μBONDPAKC ₁₃ (sold by Water Co., Ltd.) was used as the packing material.
Contains 25% methanol at a flow rate of 1ml/min
Developed with 0.05MNH ₄ HCO ₃ ] with a yield of 73%.
pApUpCφ was obtained.

(2) Reaction using T4RNA ligase ani pApUpC0.1mM (5nmol),
pApUpCφ0.2mM (10nmol), Tris-HCl pH8.0
50mM, MgCl ₂ 10mM, dithiothreitol
5 units of T4 RNA ligase was added to 45 μl of a reaction solution containing 10 mM adenosine triphosphate and 0.1 mM adenosine triphosphate, and the mixture was allowed to react at 4° C. for 3 days.

The enzyme was removed from the reaction solution using an ultrafilter with a molecular weight of 40,000 (Ultrafree, manufactured by Millipore), and the enzyme was removed using high-performance liquid chromatography [4 mm diameter, packing material: Hanui Pharmaceutical Co., Ltd.]
Using COSMOSIL C18, 20% methanol 0.05M
[developed with NH ₄ HCO ₃ ], and the yield was 75%.
pApUpCpApUpCφ was obtained.

The obtained hexamer was identified by decomposing it with RNase T ₂ and using separately synthesized preparations to confirm that the decomposition products contained ani pAp, Up, Cp, and Cφ in a ratio of 1:2:1:1:1, respectively. This was done by checking.

Claims (1)

[Claims] 1. When producing an oligoribonucleotide by linking two types of oligoribonucleotides using R.N. Eligase, as the two types of oligoribonucleotides, (A) a 5'-terminal hydroxyl group; hydrogen atom is a group represented by the following general formula () [In the formula, R ¹ represents a phenyl group, R ² represents a hydrogen atom, or R ¹ and R ² represent a group forming a morpholine ring together with N] It becomes a phosphate acceptor substituted with The oligoribonucleotide primer and (B) the hydrogen atom of the 2′-terminal hydroxyl group have the following general formula () [In the formula, R ³ represents a hydrogen atom or an alkyl group]
1. A method for producing an oligoribonucleotide, which comprises using an oligoribonucleotide as a phosphate donor substituted with a group represented by the following.