JPS6336756B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing oligonucleotides. More specifically, the present invention relates to a method for enzymatically producing oligonucleotides containing guanylic acid.

In recent years, as the role that nucleic acids play in important biological functions such as transmission of genetic traits and protein synthesis has been elucidated, it has become increasingly important to understand the relationship between their structure and function. ing. One research method is often used to learn the relationship between structure and function by synthesizing oligonucleotides with a desired base sequence, that is, oligonucleotides with known base sequences, and examining the effects on the protein synthesis system. It is. When attempting to produce oligonucleotides with known base sequences as reagents used in such research, it is relatively difficult to produce oligonucleotides containing guanylic acid.

The present inventors conducted studies to develop an enzymatic production method for oligonucleotides containing guanylic acid, and found that
This invention has been achieved. That is, the gist of the present invention is to react an oligonucleotide primer and guanosine diphosphate at 20°C to 55°C in the absence of a base-specific nuclease using polynucleotide phosphorylase obtained from a bacterium belonging to the genus Thermus . The present invention relates to a method for producing an oligonucleotide, which is characterized by obtaining an oligonucleotide containing guanylic acid.

To further explain the present invention in detail, the bacteria belonging to the genus Thermus include Thermus thermophilus ,
Examples include Termus flavus . These strains include Thermus thermophilus HB8 (ATCC27634,
InternationalJ.of Systematic Bacteriol. 24 ,
102-112), Thermus Flavus BS1, AT61, AT62 (these are Agr.Biol,
chem. 36 , 2357-2366). In particular, Thermus Thermofilas is preferred.

Polynucleotide phosphorylase can be obtained from these bacteria by known methods (e.g. Proteins, Nucleic Acids, Enzymes vol. 13, No. 9, 845~, or The Journal of
Biological Chemistry vol.243, No.2, 231~)
It can be obtained by separating and purifying it. For example, Thermus Thermophilus HB8 is mixed with a suitable medium such as 5 g of polypeptone and 4 g of yeast extract.
The cells were cultured in a medium consisting of 1 g of glucose, 1 g of glucose, 2 g of sodium chloride, and 1000 ml of water at 60°C to 85°C with aeration and agitation, and the resulting bacterial cells were ground with alumina and extracted with Tris-HCl buffer containing magnesium ions. , ultracentrifugation, ammonium sulfate fractionation, column chromatography, isoelectric focusing, etc., and can be purified by polyacrylamide gel electrophoresis. And this Thermas Thermofilas
Polynucleotide phosphorylase from HB8 is
Eur. J. Biochem. 77 , 575-583 (1977).

In the present invention, using the polynucleotide phosphorylase obtained as described above, oligonucleotide primers and guanosin diphosphate are reacted at 20°C to 55°C in the absence of a base-specific nuclease to generate oligonucleotides containing guanylic acid. Obtain nucleotides. As an oligonucleotide primer,
Oligonucleotides with arbitrary base sequences can be used depending on the purpose. To give a specific example, A _P A _P A [adenylyl-(3'→5')] ₂ adenosine, A _P
5')] adenosine, [uridylyl-(3'→5')] uridine, commonly known as U _P U _P U, and the like.
These oligonucleotides may be commercially available products, or may be produced by known methods. For example, _{APAPA is} available from Boehringer Mannheim.

The amount of guanosine diphosphoric acid to be used is preferably close to an equivalent molar ratio to the oligonucleotide primer, for example, about 3 to 4 moles. The reaction temperature is selected from 20°C to 55°C, preferably 30°C to 45°C. If the temperature is higher than this range, a large amount of polyguanylic acid will be produced, which is not preferable. On the other hand, if the temperature is lower than this range, the reaction progresses slowly and is not preferable.

The reaction is carried out, for example, with polynucleotide phosphorylase of 0.5 units/ml or more, preferably 1 unit/ml to 13
units/ml, magnesium salt such as magnesium chloride 0.1mM to 3mM, oligonucleotide primer 0.05mM to 2mM, guanosin diphosphate 0.5
mM to 10mM and 50mM Tris-HCl buffer (PH
8.5) is reacted for 5 to 45 hours at the above-mentioned temperature in the absence of a base-specific nuclease.

When the reaction is performed as described above, for example, A _P
From A _P A is A _P A _P AG and A _P A _P A _P G _P G, A _P A
From A _P A _P G _P G, from U _P U _P U, U _P U _P U _P G,
You can get each.

After the reaction is complete, the product can be prepared using conventional methods, e.g.
DEAE (Trademark, Pharmacia Co.
They can be separated by column chromatography using ion-exchange gel (manufactured by the company).

According to the method of the present invention, oligonucleotides containing guanylic acid can be obtained in good yield.

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

Reference example (Culture of Thermus thermophilus HB-8,
Purification of polynucleotide phosphorylase) Thermus Thermophilus HB-8
(ATCC27634) was cultured using a medium containing 5 g of polypeptone, 4 g of yeast extract, 1 g of glucose, and 2 g of NaCl. That is, first, the above bacteria are inoculated into two 20 ml test tubes containing about 3 ml of the above medium, and the tubes are left in an incubator (75°C) for 2 days and nights. Next, this was planted in 8 Sakaguchi Kolben bottles (No. 5) containing the above medium 1, and cultured with shaking at 75°C overnight. Furthermore, this was planted in 4 Jia Farmers 20, and aerated culture was carried out at 75℃ for about 8 hours.
Collect bacteria at the late stage of logarithmic growth (Cretz number approximately 400) (using a shear press type continuous centrifuge), and add approximately 2
Wash gently with The bacterial cells were stored frozen at -20°C. The yield of bacterial cells was about 700 g in wet weight based on 80 g of the culture medium.

190 g of the above bacterial cells were suspended in a buffer containing 0.01M magnesium acetate, and Sigma Chemical CO.Ltd.
After adding 0.5 μg/ml of deoxyribonuclease (manufactured in Japan), the mixture was ground with alumina, centrifuged at 16,000 g for 20 minutes,
The supernatant was then ultracentrifuged at 78,000g for 18 hours.
Collect the supernatant from the bottom 1/3. Sedimentation classification is
Re-extract with Tris-Mg buffer, perform ultracentrifugation again, and collect the bottom 1/3 of the supernatant.
The nucleic acid fraction is then removed with streptomycin and the precipitated fraction containing 35-55% ammonium sulfate is collected. After desalting by dialysis, it is subjected to QAE-Sephadex A-25 (ion exchange resin manufactured by Pharmacia, trademark) column chromatography. After washing with 0.15M NaCl,
When flowing out with a concentration gradient of NaCl 0.15-0.40M,
Polynucleotide phosphorylase activity appears around 0.3M. The active fraction was concentrated on a short column of DEAE cellulose, eluted with 1M Tris (PH 8.1), and transferred to Sephadex G-200 [same as above. ]
(2cm x 72cm) column chromatography,
0.01M Trilith (PH8.2), 1mM MgCl ₂ ,
Elute with 0.05M NaCl, 1mM mercaptoethanol.

This was further adsorbed onto Whatman's Microgranular DEAE-Cellulose DE-51 (ion-exchange cellulose manufactured by Whatman, (trademark)), and a buffer solution of 0.01M Tris-HCl (PH8.2), 1mM MgCl ₂ and 1mM 2-mercaptoethanol was used. NaCl0.1~0.25M
When flowing out with a concentration gradient of , activity appears around 0.15M. The active part was collected and adsorbed onto hydroxyapatite, and 0.01M Tris-HCl (PH7.4) was added to
When ammonium sulfate is flowed out with a concentration gradient of 0.1 to 0.6M in a buffer solution of mM2-mercaptoethanol and 0.1M NaCl, activity occurs around 0.2 to 0.25M. Next, 10 ml of this active fraction was subjected to isoelectric point fractionation using a 110 ml column chromatograph. After applying electricity at 400 V for 40 hours in a Tris-hydrochloric acid buffer containing 1% ampholine [LKB buffer, (trademark)] solution, 1 ml was taken out and 0.01 ml was used to measure polyadenylic acid synthesis activity. The activity classification was obtained. Each active fraction was collected and dialysis was performed to remove ampholine, and the isoelectric point
4.3, an enzyme with a molecular weight of 200000 (Sephadex G-200) was obtained.

The polyadenylic acid synthesis activity test was conducted as follows.

That is, Tris-HCl (PH9.0) 10mM,
_MgCl2 1mM, ADP 1mM ( ^14C -ADP1μCi/
A total volume of 0.2 ml of a reaction solution consisting of 10 μM of µmole), (Ap) ₃ A and the above enzyme is reacted at 70°C for 20 minutes, and after the reaction is cooled on ice. Take that 25μ and Whatman3MM
It was spotted on activated paper chromatography at the origin of Whatman Paper (trademark) (1.5 x 10 cm) and developed with ethanol:1M ammonium acetate (PH5.5) (1:1). After development, the origin was cut out, dried, and measured using a scintillation counter.

Note that 1 unit of this enzyme is defined as the amount that produces 1 μmole of adenosine monophosphate in 1 hour at 70°C.

Example 1 (1) Reaction Magnesium chloride 0.5mM, A _P A _{P A} 0.4mM,
Guanosin diphosphate 2mM, polynucleotide phosphorylase obtained in the above reference example 1.61 units/ml and 50mM Tris-HCl buffer (PH8.5)
A total volume of 1 ml containing the mixture was reacted at 37°C for 20 hours. The reaction mixture was transferred to a 0.5 x 25 cm DEAE-Sephadex tube.
A-25 (ion exchange gel trademark manufactured by Pharmacia Co.) column was added, and a linear concentration gradient elution from 0.25M to 0.65M ammonium bicarbonate (total volume 200ml) was performed at a flow rate of about 25ml/hour at room temperature. 2
g was collected as one fraction, and the absorption at 260 nm of each fraction was measured. Three absorption peaks are obtained, of which the second peak (40th to 56th fraction), which is thought to be a tetramer, is collected and desalted by freeze-drying, and the structure of the product is determined as follows. did.

0.5A ₂₆₀ units of the product obtained as described above, spleen phosphodiesterase (Worthing-
ton Biochemical Cu.) 20μg and 10mM
10μ of the reaction mixture containing Tris/HCl buffer (PH8.5) was reacted at 37°C for 1 hour. The reaction mixture was separated by two-dimensional thin layer chromatography. That is, a cellulose plate (7 x 7
cm ² ), the first direction (dimension) was treated with a 5:3 (volume ratio) mixed solvent of isobutyric acid and 0.5M NH ₄ OH, and the second direction (dimension) was treated with ethanol.
It was developed with a 50:50 (volume ratio) mixed solvent of 1M ammonium acetate (PH5.5). Spots were detected by UV, cut out, and extracted with 0.6 ml of 0.01 MHCl. The absorption of the extract was measured at 260nm and 280nm,
The ratio of each component was determined. Ap and G 3.02:1.00
It was confirmed that this tetramer was (Ap) ₃ G. In this reaction, the yield of (Ap) ₃ G from (Ap) ₂ A was 33%.

Claims (1)

[Claims] 1 Using polynucleotide phosphorylase obtained from a bacterium belonging to the genus Thermus, an oligonucleotide primer and guanosine diphosphate were combined in the absence of a base-specific nuclease for 20
A method for producing an oligonucleotide, the method comprising obtaining an oligonucleotide containing guanylic acid by reacting at a temperature of 55°C to 55°C.