CS224618B2 - Method for carrying out a synthese of polydesoxynykleotide - Google Patents

Description

The invention relates to a process for the preparation of polydesoxynucleotides from 2'-desoxynucleotide-5'-triphosphates by a DNA polymerase enzyme having more than one free mercapto group not belonging to the active center in the presence of a divalent cation and optionally an exemplary primary complex in an aqueous buffered pH medium 6.5 to 9.5, corresponding to the enzyme used, at a temperature of 15 to 42 ° C.

An exemplary primary complex is an exemplary molecule that is identical to the desired polydesoxynucleotides produced.

It is known that enzymatic synthesis of polydesoxynucleotides is carried out in a homogeneous phase manner. Such a method is described, for example, in the following publications: J. F. Eurd and S. D. Wells,

J. Mol. Blol. 53, pp. 435-459 (1970); Bollum, F.J., Procedures in Nucleie Aeid Research, p. 591, Cantoni, G.L., and Davies, D, R, New York, Harper 8 Row (1966); J. Sagi, A. Szabolcs, A. Szemzo and L. Otvos, Nucleic Acid Research, 4, pp. 2,767-277 (1977).

In these methods, the buffered reaction mixture comprises a substrate, an exemplary primary complex, an enzyme, and cations that provide optimal enzyme function. The reaction mixture is incubated, after completion of the reaction, the enzyme is denatured (for example by heating the reaction mixture), then the secreted enzymatic protein is removed from the mixture. From the polydesoxynucleotide mixture obtained, high molecular weight polymers are obtained by dialysis purification.

The polydesoxynucleotides thus prepared are used as DNA models for the physical, chemical, biochemical and biological studies of nucleic acids.

A commonly used DNA model is, for example, a 2'-desoxysdenosine-5'-monophosphate (dAMP) and thymine-5'-monophosphate (dTMP) copolymer with a strictly alternating sequence and a double helix.

...- dAMP-dTMP-dAKF-dTMP -..., which has the abbreviation: poles d (A-T).

In the case of DNA-derived DNA polymerases, the nucleotide sequence of a new DNA strand is determined by the nucleotide sequence of the sample DNA introduced into the mixture. Thus, for the synthesis of the d (AT) poles, such d (AT) poles of the exemplary primary complex are required, and by carrying the corresponding exemplary primary complex, various polydesoxynucleotides can be produced, such as poly (dA) poly (dT), d (GC) poles. the poles d (IC), poly (dG) poly (dC), poly (d1) poly (C), wherein A is adenine, thymine, G guenine, inosine and C cytosine.

Individual DNA-poly, meres enzymes, on the other hand, are also able without the externally introduced exemplary primary complex of the so-called d-A-T / day synthesis. Such an enzyme is, for example, the enzyme S. coli DNA polymerase I.

A disadvantage of the known methods for the enzymatic preparation of polydesoxynucleotides is that the enzyme can be used only once and that the removal of the enzyme is associated with its denaturalization in the processing of the reaction mixture.

It is therefore necessary to develop a method of preparation that does not have this deficiency.

It is the basis of the invention that the above object is achievable when the synthesis of polydesoxynucleotides is carried out using an enzyme bound to a solid support. Thus, the enzyme can be removed from the reaction mixture by simple filtration and can be reused.

It is a further object of the invention to recognize that DNA polymerase enzymes having more than one mercapto group that do not form part of the active center of the enzyme can bind to the solid support and so use for this purpose. Indeed, these groups can bind the enzyme to a suitable carrier by substituting the proton, without causing the loss of activity or decrease in activity.

Accordingly, the invention relates to a process for the preparation of polydesoxynucleotides from 2'-desoxynucleoside-5'-triphosphates with a DNA polymerase enzyme having more than one non-active mercapto group in the presence of a divalent cation and optionally an exemplary primary complex in the an aqueous buffer medium having a pH of 6.5 to 9.5, corresponding to the enzyme used, at a temperature of 15 to 42 ° C. According to the invention, an enzyme with mercapto groups capable of binding to a solid support by a covalent bond is used as the DNA polymerase enzyme.

In carrying out the process of the invention, an immobilized reagent having thiol groups is used as the solid carrier.

Solid carriers that are capable of binding a mercapto group are suitable for enzyme binding. Such carriers include agarose-modified mobilized thiol reagents such as Thiopropyl-Sepharose 6B (Pharmacia Information Booklet, 977) or activated Thiol-Sepharose 4B (Pharmacia Information Booklet, 1974).

The structure of Thiopropyl 1-Sepharose 6B is

wherein the matrix is modified agarose. Thiopropyl-Sepharose 6B contains 20 µmol of 2-thiopyridyl groups per ml of swollen gel; 1 g of lyophilized gel occupies a swollen volume of approximately 3 ml. The 2-thiopyridyl group masked and activated immobilized thiol group can be released in various ways, but can also be reacted directly with the mercapto group to form a disulfide bond. According to the information material from Pharmacia is the molar extinction coefficient while cleaved 2-thiopyridone at 343 nm 8.08 16¾ xx ^-1 cm ^- '.

The selected solid support was swelled in potassium phosphate buffer (pH 7.4), packed into the column, and baked dissolved in the buffer.

According to the invention, enzymes are used which contain free mercapto groups capable of reaction. These include the isolated DNA polymerase type I enzyme (Kornberg polymerase) from Escherichia coli. It is a spherical molecule without a subunit of molecular weight 109,000 g / mol containing 975 amino acids. Its active center also contains a lysine and ariginine molecule. There is one single sulfide bond and one sulfhydryl group in the molecule. The sulfhydryl group can be reacted with iodoacetate and mercury salts without reducing activity.

Similarly to the enzyme from E. coli, isolated DNA-polymerized type I enzymes are inhibited from other bacteria (Bacillus subtilis, Micrococcus lisodeicticus and cycobacterium tuberculosis) by non-sulfhydryl agents, i.e. similar to the immobilization of the enzyme from E. coli. At the same time, however, the four types of bacteria-isolated DNA polymerase enzymes type II and III are inhibited by sulfhydryl agents so that they cannot bind to the solid support in the above manner. Of the mammalian organisms isolated so far, DNA polymerases (EC.2; 7.7.7.) Also inhibit alpha-polymerases and gamma-polymerases, while beta-polymerase with sulfhydryl agents does not.

The enzyme loaded on the column, dissolved in the buffer, is flushed with the next enzyme into the column, and then the mixture is pumped through the peristaltic pump 10 times for as perfect a bond as possible. The reaction proceeds according to the reaction scheme.

R-S-S ~ <O> + H-SH-> R-S-S-E + S = / C? S where means

E Type I DNA polymerase enzyme a

R Sepharose-O-CH ₂ -CH-CH ₂ -.

OH

The amount of bound enzyme can be directly determined by measuring the molar absorption of light of the cleaved 2-thiopyridone moiety, indirectly by measuring the amount of DNA that is produced by enzyme-catalyzed synthesis.

After the enzyme binding reaction is complete, the buffer is drained, the gel is washed with additional fresh buffer at first until the run-off buffer spectrum is not the same as the pure buffer, which is about 10 ml. Wash with a further 40 ml at a rate of 1 ml / min. A spectrophotometer is used for the spectrophotometer.

The enzyme thus coupled is then used for the synthesis of polydesoxynucleotides. The reaction mixture used for the synthesis comprises the deoxynucleotides used in the aqueous medium as a substrate and an exemplary primary complex.

The reaction mixture is adjusted to a pH of 6.5 to 9.5 with a buffer whose quantity and quality must ensure optimal functioning of the enzyme used. In addition, a metal salt ensuring optimal enzyme function, preferably a magnesium salt, is added to the reaction mixture at an appropriate concentration.

The column is heated to 37 ° C and the reaction mixture is also applied at 37 ° C; it is then pumped through the corresponding peristaltic pump.

The progress of the reaction can be monitored in various ways, for example by measuring the radioactivity of the acid-insoluble substance, paper chromatography, analysis of the end product or the control reaction.

In the case of measurement of radioactivity, the reaction mixture must also contain a labeled substrate,

For example, dATP may be used for this purpose. The measurement is performed by taking a 25 µL sample with a Hsmilton mlipipette over a period of time from the effluent column and aliquots on GF / G glass filter plates (2.5 cm diameter, Whatmen). Place in a solution (10 ml glass filter solution, 0 to 5 ° C for 10 minutes) of cold 5% trichloroacetic acid at 0 to 5 ° C (TGA) and 1% sodium pyrophosphate, wash twice with 5% trichloroacetic acid solution ε then twice with 96% ethyl alcohol and finally twice with diethyl ether and air dried. Radioactivity in acid-insoluble polymer molecules (a polymer consisting of more than 10 monomer molecules) is measured in admixture with a toluene Packard spectrometer. In this way, the control reactions can also be monitored.

Paper chromatography is suitable in the case of radioactive substrates for quantitative and inactive substrates for qualitative monitoring of the reaction. On a 1 cm wide and 15-18 cm long paper strip of DE 81 (DEAE-cellulose, whetman), 5-25 µl of the reaction mixture is applied. A 0.15 M ammonium bicarbonate solution is used as the composition:

R ^. the nucleotide values are 0.35 to 0.55, the polynucleotides remain at the starting point.

A solution containing 2.5 moles of sodium chloride and 0.038 moles of ethylenediaminetetraacetic acid per liter of solution in a 1: 0.65 by volume ratio is added to the gelled and washed (260 nm controlled) reaction mixture and then held for 10 minutes at temperature 70 ° C. After cooling, shake three times with equal volumes of a 24: 1 mixture of chloroform and isoamyl alcohol. The two phases are separated (by centrifugation or on phase separating paper, Ahatman) and the aqueous phase is dialyzed twice with distilled water under stirring (4 ° C, continuously). The conductivity of the dialyzed solution is measured. When the conductivity after the flow of 0.8 l of double-distilled water reaches the value of double-distilled water (0.2 to 0.6 µS), it is dialyzed with an additional 0.4 to 0.5 liters of double-distilled water. Then the product is lyophilized to dryness and taken up in 1C- ² mol of TRIS.HCl (pH 7.4), and Ifc ^-1 mol of ethylenediaminetetroacetic acid; the solution has a volume of 500 µl. Aliquots of the obtained solution are subjected to determination of concentration, ultraviolet spectrum, thermal hyperchromicity ε of the activity of the sample. The polymer solution was stored at -25 ° C.

Control reaction: To 165 µl of the reaction mixture (the same composition as that used in the reaction with the mobilized enzyme) was added an amount of enzyme corresponding to ε incubated at 37 ° C. The sample is taken over the same time periods as for the controlled reaction and applied to the glass filter (GF) C as described above, processed and measured.

*

Storage and re-use of immobilized enzyme are carried out as follows:

After draining and washing the reaction mixture, the gel is washed with an additional 10 mL of 60 mM Knifux phosphate buffer followed by 20 mL of 0.02% sodium azide solution and stored in the column or after removal at 3-6 ° C. After washing the sodium azide solution, the enzyme can be reused; when reused, its initial activity barely decreases by 0 to 5. The most re-investigated and stabilized immobilized enzyme was E. coli DNA-polymerssa I, Grade II: over two months, its activity did not change over six times repeated use.

The process according to the invention has the following important advantages:

a) The solid support covalently bonded with DM-polymer is involved in the synthesis of the desoxypolynucleotide by reaction with the hetrogen phase, and can be removed from the reaction mixture without denaturalization after completion of the reaction.

b) The bound enzyme may be reused.

e) The bound enzyme can also be used in continuous operation. The process according to the invention is illustrated in more detail by the examples.

Example 1

a) Preparation of Thiopropyl-Sepharose 6B g Lyophilized commercial thiol reagent Thiopropyl-Sepharose 6B was swelled for 15 minutes at room temperature in 60 mK potassium phosphate buffer (pH 7.4), then packed into a jacketed glass column and blended. glass filter (5 * 5 cm) and washed with 200 ml of the above buffer to wash the commercially added stabilizer.

b) ICO units (20 µl) of E. coli MKE 600 DNA polymerase I, step I with a specific activity of 3,850 units / mg protein by Hamilton micropipette are added to approximately 0.5 ml of the buffered, gel-free buffer. (One unit of DNA polymerase enzyme incorporated 10 nmoles of total nucleotide at 37 ° C over 30 minutes into an acid-insoluble polymer in the presence of the corresponding exemplary primary complex in a certain buffer). Slowly drip it into the gel layer (0.1 to 0.2 ml / min) and rinse with a further 10 ml of buffer at room temperature and then pump 10 times with a peristaltic pump.

c) Synthesis of poles d (A-T)

The columns were thermostated at 37 ° C and then 10 ml of a reaction mixture of the following composition: 60 mmol / l of kallumphosphate (pH 7.4), 6 mol / l of magnesium chloride, mmol / l [µP dATP whose specific activity was 10.9 decays / min per mole, 1 mmol / l dTTP,

100 µmol [p] / 1 activated with d / A-T poles of a sample primary complex dissolved in twice distilled water to a volume of 10 ml. The dead space corresponding to 2 to 3 ml of buffer (spectrophotometrically checked) is poured out, then the reaction mixture is pumped through the peristaltic pump at a rate of 0.5 ml / min. The progress of the reaction can be monitored by measuring the radioactivity.

r

After a reaction time of 24 hours, the amount of polymer formed by incorporation of 1 H dAMP into the acid-insoluble substance is 3.04 mg. 45% of the substrate is used.

From the thus obtained poles d (A-T), it is separated by dialysis of a low molecular weight substance. The amount of pure and native high molecular weight d (A-T) poles of 200 COO g / mol is 2.2 mg.

Example 2

By the method described in Example 1, 150 units (100 µl) of the E.coli enzyme MRE DαA polymerase I, stage II with a specific activity of 116 units / mg protein was bound to the immobilized thiol reagent. The synthesis was carried out as described in Example 1. Mmol / l iP dATP and 2 mmol / l dTTP are used as substrates. The amount of polymer synthesized during this 4 hours is 2.37 mg, measured by radioactivity, substrate consumption 18%. The molecular weight of the isolated and purified polymer is low (5 to 200,000 g / mol) and is 1.78 mg.

Example 3

The non-immobilized thiol reagent binds, as described in Example 1, 45 units (50 µl) of the B. coli MRE 600 DNA-polymerase I fragment (enzyme A according to Klenow) with a specific efficiency of 6,940 units per mg protein. Synthesis was also carried out as described in Example 1. 0.5 mmol / l dATP and 0.5 mmol / l dTTP were used as substrates. According to radioactivity measurements, the amount of total polymer synthesized is 22.74 mg over 24 hours,

224613 substrate consumption is 82%. The isolated, purified poles d (A-T) has a high molecular weight (greater than 20,000 g / mol) and is 2.12 mg.

Claims (1)

Process for the synthesis of polydesoxynucleotides from 2'-desoxynucleotide-5'-triphosphates such as 2'-desoxyadenosine-5'-triphosphate and thymidine-5'-triphosphate or 2-desoxyguanosine-5'-triphosphate and 2'-desoxycytidine-5'- triphosphate, a DNA-polymeric enzyme having at least one non-active mercapto group in the presence of a divalent cation and optionally an exemplary primary complex in a buffered aqueous medium having a pH of 6.5 to 9.5 corresponding to the enzyme used at I5 to 42 ° C, characterized in that an enzyme bound by a covalent bond to a solid carrier capable of binding a mercapto group, which is an immobilized agent containing a thiol group, is used as the DNA polymeric-sucking enzyme.