Method of synthesysing nucleoside-5'-triphosphates, labelled with radioactive isotopes of phosphorous in alpha-position

FIELD: biology.

SUBSTANCE: present invention relates to biotechnology, more specifically to obtaining nucleoside-5'-triphosphates, labelled with phosphorous-32 (phosphorous-33) in the alpha-position, and can be used for analysis in molecular biology, genetics and medical biochemistry. The method is realised through treatment of labelled nucleosidephosphate in a buffer solution with a mixture of deoxyribonucleoside monophosphate kinase of bacteriophage T5 and pyruvate kinase with subsequent chromatographic purification of the target product.

EFFECT: simple method of obtaining nucleoside-5'-triphosphates and stable output of the target product.

4 ex

 

The invention relates to the field of biotechnology, particularly to a receiving nucleoside 5'-triphosphates labeled with phosphorus-32 (phosphorus-33) in the alpha-position, and can be used for research in the field of molecular biology, genetics and medical biochemistry.

Precursors for the biosynthesis of nucleic acids labeled with radioactive isotopes of phosphorus, [α32(33)P]NTP (nucleoside-5 triphosphate labeled with phosphorus 32 or 33) and [α-32(33)P]dNTP (2'-deoxyribonucleoside-5 triphosphate labeled with phosphorus 32 or 33), are widely used in fundamental and applied research on physico-chemical biology and biotechnology. Almost all major advances in molecular genetics, genetic engineering, including the development of techniques decode the primary structure of nucleic acids based on using nucleotides labeled with radioactive isotopes of phosphorus.

The known method of synthesis of nucleoside 5'-triphosphates labeled with radioactive isotopes of phosphorus in the alpha position, comprising in sequential phosphorylation labeled nucleoside-5'-monophosphate to the target compound using the enzyme preparation nucleotidebinding. The method consists in the incubation of labeled nucleoside-5 monophosphate with the enzyme preparation in the presence of buffer components, ATP (adenosine-5 triphosphate, system and phosphorylation of nucleotidebinding/regeneration of ATP, consisting of pyruvate kinase (EC 2.7.1.40) and phosphoenolpyruvate. The entire synthesis lasts for 30-40 min at 37°C, followed by chromatographic purification of the target compounds (R.Hurlbert, N. Furlong "Methods Enzymol." Part A, 12, 193, 1967). The main disadvantage of this method is that the enzyme preparation used nucleotidebinding is a mixture of proteins obtained alpacamania fractionation of the lysate of E. coli cells. Impurities of various enzymes and other substances in the cell lysate, does not allow to achieve a high yield of the target product, as well as reduce its molar (specific) activity and make it difficult for the final cleanup.

A method of obtaining a nucleoside 5'-triphosphates labeled with radioactive isotopes of phosphorus in the alpha position with high molar activity, in which instead of the total drug nucleotidebinding E. coli using individual enzymes: adenylatecyclase (EC 2.7.4.3), guanidines (EC 2.7.4.8), cicadellinae (EU 2.7.4.14), timetracking (EC 2.7.4.9) and urodilatin (EU 2.7.4.22), isolated from E. coli cells (Ch.K. Bierbricher "Anal. Biochem.", 95, 419, 1979). The main disadvantage of this method is the necessity of preliminary groundwork five pure enzymes for synthesis, which significantly increases with aImost process.

Known closest to the claimed method, in which the enzymatic scheme receiving nucleoside-5'-[α-32R] triphosphates used for phosphorylation commercial preparations coding adenylate kinase, guanidines and nucleotidesequence manufactured by Boehringer-Mannheim". The last of these enzymes (nucleotidesequence) is not an individual protein, and a mixture of enzymes obtained by the fractionation of liver homogenate of bovine (T.F.Walseth, P.S.T.Yuen, M.C.Moos "Methods Enzymol.", 195, 29, 1991).

Technologically this method is similar to the previous one as the composition of the reaction mixture, and equipment, and the differences in the use of other enzymes for the first phosphorylation. Easy, fast and affordable commercial enzyme preparations did this method attractive for laboratory syntheses, and for industrial production.

The main disadvantage of this method is that the enzyme preparation nucleotidesequence is poorly purified mixture cicadellinae, timetracking and urodilatin mixed with ballast proteins and non-specific dephosphorylates enzymes. The relative content cicadellinae, timetracking and urodilatin varies in different batches of the drug 10 or more times. This complicates carrying out the synthesis of the in and prevents the achievement of the sustainable yield of the target product.

The invention solves the problem easier way to obtain [α32(33)P]NTP and

32(33)P]dNTP and achieving a stable yield of the target product.

The problem is solved by a method of synthesis of nucleoside 5'-triphosphates labeled with radioactive isotopes of phosphorus in the alpha position, such as the processing of labeled nucleosidase in a buffer solution with a mixture nucleotidesequence and pyruvate kinase, followed by chromatographic purification of the target product, as nucleotidesequence use dezoksinukleozidtrifosfaty bacteriophage T5.

The enzyme dezoksinukleozidtrifosfaty bacteriophage T5 (EC 2.7.4.13) synthesized in E. coli cells after infection (infection) by bacteriophage T5 with its subsequent allocation (Galina V. Mikoulinskaia, Tony I.Gubanov, Andrei A.Zimin, Igor V. Kolesnikov, Tony A. Feofanov, and Anatolii I. Miroshnikov. Protein Expression and purification, 2003, 27, 195-201).

Studies have shown that the enzyme dezoksinukleozidtrifosfaty bacteriophage T5 able to fosforilirovanii not only [5'-32(33)R]dNMP (2'-deoxynucleoside-5'-monophosphate labeled with phosphorus 32 or 33)and [5'-32(33)R]NMP (nucleoside-5'-monophosphate labeled with phosphorus 32 or 33). For the synthesis of labelled phosphorus compounds of this enzyme gives good results for 30 min incubation at 37°C. the Last stage - phosphorylation is sootvetstvujushij diphosphate derivatives to trifosfatnogo - carried out using the pyruvate kinase.

Thus, the technical result of the claimed method is to simplify the process of obtaining a nucleoside 5'-triphosphates labeled with radioactive isotopes of phosphorus in the alpha position, due to the use instead of the three enzymes in the known method (adenosinemonophosphate, guanozinmonofosfata and nucleotidesequence) - one enzyme in the claimed method dezoksinukleozidtrifosfaty bacteriophage T5, also in stabilizing the yield of the target product. In the known method the yield of the target products are sharply different: for deoxyadenosines output reaches 60%, for deoxyguanosine-5'-triphosphate output reaches 40%, and for timeinterest ranges from 10 to 40% depending on the quality nucleotidesequence, which is used for synthesis. In the present method due to the use of dezoksinukleozidtrifosfaty bacteriophage T5 yield of the target product rises to 75-90%.

The method is as follows.

In the reaction mixture containing Tris-HCl buffer (pH 8.0); magnesium chloride; potassium chloride; DTT (dithiothreitol); ATP and phosphoenolpyruvate add [5'-32P]dCMP (2' deoxycytidine-5'-monophosphate labeled with phosphorus 32) or [5'-33P]dAMP (2 deoxyadenosine-5'-monophosphate, labeled phosphorus 33), T5-kinsui the piruwatkinaza. The mixture is incubated at 37°C for 30 minutes and Then the mixture analyze TLC (thin layer chromatography) plate PAYS (polyethylenimine)pulp in 0.5 M potassium phosphate buffer (pH 4.0) to determine the yield of the reaction. After chromatography the plate is dried and visualize using phosphoimager or autoradiographically. The yield of the reaction is 90% of the radioactivity. The target product from the reaction mixture allocate using reversed-phase HPLC (high performance liquid chromatography), ion-pair mode on a column of C-18 in a gradient of ethanol.

The invention is illustrated by examples

Example 1. Synthesis of [α-33P]dCTP (2 deoxycytidine-5'[α-32P]-triphosphate).

In the reaction mixture by volume of 100 µl composition:

50 mm Tris-HCl buffer pH 8.0

5 mm magnesium chloride

0.2 M potassium chloride

5 mm DTT

0.5 mm ATP

5 mm phosphoenolpyruvate

add 10 MCI [5'-32P]dCMP (about 2.5-3 nmol substances), 5 units of activity dezoksinukleozidtrifosfaty bacteriophage T5 (T5-kinase) and 5 units of activity of pyruvate kinase. The mixture is incubated at 37°C for 30 minutes and Then an aliquot (volume of 0.2-0.3 µl) was analyzed on TLC plate PAYS pulp in 0.5 M potassium phosphate buffer pH 4.0 to determine the yield of the reaction. After chromatography the plate is dried and visualize using phosphoimager or autoradiographically. The output is eacli is 90% of the radioactivity. The target product from the reaction mixture allocate using reversed-phase HPLC ion-pair mode on a column of C-18 in a gradient of ethanol. The final product yield [α32P]dCTP - 7.5 MCI (75%) of the original connection.

Example 2. Synthesis of [α-32P]ATP (adenosine-5'-[α-32P-triphosphate).

In the reaction mixture by volume of 100 µl composition:

50 mm Tris-HCl buffer pH 8.0

5 mm magnesium chloride

0.2 M potassium chloride

5 mm DTT

0.005 mm ATP

5 mm phosphoenolpyruvate

add 5 MCI [5'-32P]AMP (adenosine-5'-monophosphate labeled with phosphorus-32, about 1.5-2 nmol substances), 5 units of activity T5-kinase and 5 units of activity of pyruvate kinase. The mixture is incubated at 37°C for 30 minutes and Then an aliquot (volume of 0.2-0.3 µl) was analyzed on TLC plate PAYS pulp in 0.5 M potassium phosphate buffer pH 4.0 to determine the yield of the reaction. After chromatography the plate is dried and visualize using phosphoimager or autoradiographically. The yield of the reaction is 90% of the radioactivity. The target product from the reaction mixture allocate using reversed-phase HPLC ion-pair mode on a column of C-18 in a gradient of ethanol. The final product yield [α32P]ATP - 4.5 MCI (90%) of the original connection.

Example 3. Synthesis of [α-33P]dATP (2'-deoxyadenosine-5'-[α-33P]-triphosphate).

In the reaction mixture by volume of 100 µl composition:

50 mm Tris-HCl b is fer pH 8.0

5 mm magnesium chloride

0.2 M potassium chloride

5 mm DTT

0.5 mm ATP

5 mm phosphoenolpyruvate

add 10 MCI [5'-33P]dAMP (about 2.5-3 nmol substances), 5 units of activity T5-kinase and 5 units of activity of pyruvate kinase. The mixture is incubated at 37°C for 30 minutes and Then an aliquot (volume of 0.2-0.3 µl) was analyzed on TLC plate PAYS pulp in 0.5 M potassium phosphate buffer pH 4.0 to determine the yield of the reaction. After chromatography the plate is dried and visualize using phosphoimager or autoradiographically. The yield of the reaction is 90% of the radioactivity. The target product from the reaction mixture allocate using reversed-phase HPLC ion-pair mode on a column of C-18 in a gradient of ethanol. The final product yield [α33P]dATP - 8.5 MCI (85%) of the original connection.

Example 4. Synthesis of [(a-32P] CTP (citizen-5'-[α-32R]-triphosphate).

In the reaction mixture by volume of 100 µl composition:

50 mm Tris-HCl buffer pH 8.0

5 mm magnesium chloride

0.2 M potassium chloride

5 mm DTT

0.5 mm ATP

5 mm phosphoenolpyruvate

add 6 MCI [5'-32P]CMP (piridin-5'-monophosphate labeled with phosphorus-32, about 2-3 nmol substances), 10 units of activity T5-kinase and 5 units of activity of pyruvate kinase. The mixture is incubated at 37°C for 30 minutes and Then an aliquot (volume of 0.2-0.3 µl) was analyzed on TLC plate PAYS pulp in 0.5 M potassium phosphate buffer is pH 4 to determine the yield of the reaction. After chromatography plate Vessiot and visualize using phosphoimager or autoradiographically. The yield of the reaction is 90% of the radioactivity. The target product from the reaction mixture allocate using reversed-phase HPLC ion-pair mode on a column of C-18 in a gradient of ethanol. The final product yield [α32P]CTP 5.0 MCI (80%) of the original connection.

The method of synthesis deoxyribonucleoside-5'-triphosphates labeled with radioactive isotopes of phosphorus in the alpha position, such as the processing of labeled nucleosidase in a buffer solution with a mixture nucleotidesequence and pyruvate kinase, followed by chromatographic purification of the target product, characterized in that as nucleotidesequence use dezoksinukleozidtrifosfaty of bacteriophage T5.



 

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