CH672791A5 - - Google Patents

Description

DESCRIPTION

The present invention relates to a process for the synthesis of long-chain DNA, according to which a plurality of oligonucleotide blocks are linked together, in a predetermined order, by purely chemical means, by a phosphotriester condensation process, in solid phase. , using as a support an amine controlled porosity glass.

It is known that the synthesis of polypeptides by gene technology using a synthetic gene is possible by the stages of (1) synthesis of a structural gene; (2) recombination of the gene in an appropriate plasmid; (3) transformation of an appropriate host with the chimeric plasmid formed; and (4) preparing the desired polypeptide by culturing the transformed substance.

In recent times, the development of a DNA probe has attracted public attention as a new avenue for gene technology. It is a method of identifying an unknown DNA and RNA which is a product transcribed by a hybridization of a DNA and a single stranded RNA, which are known in the art, using the properties of DNA and RNA to form a duplex by choosing a complementary substance, a relationship which is exactly that of the molding template and the molded object. As very sensitive and rapid identification is possible using the hybridization method, this method can be applied to the precise diagnosis of a disease by finding a particular DNA and RNA at the level of genes in the blood and cells of patients. and in pathogenic bacteria. As a result, DNA is important as a diagnostic agent given its use as a DNA probe.

With regard to the DNA indicated above as a structural gene and its use as a DNA probe, it is known, as regards its nature, that the longer the base sequence, the more the source of information is important and the more wide the field of use as a DNA probe. However, it is also known that the longer the base sequence, the more difficult it is to synthesize it.

Consequently, it was desirable to have a technique for preparing synthetically easily a long chain DNA.

The conventional method of DNA synthesis is as follows. Relatively short DNA fragments comprising 10 to 20 basic radicals are first prepared by chemical synthesis, then they are combined to prepare fragments having a total double stranded DNA structure presenting the information on the desired peptide synthesis, and finally they are combined using an enzyme called DNA ligase in an appropriate aqueous medium.

By this method, however, only relatively short fragments are made comprising 1 base (monomer), 2 bases (dimer) or 3 bases (trimer) before the block condensation, and it is not possible to synthesize a Long DNA with for example 80 radicals.

Furthermore, in this method, it is essential to use an enzyme called DNA ligase. Therefore, when synthetically preparing double-stranded DNA as a gene, it is necessary that all of the base sequences constituting the double-stranded DNA be synthetically prepared at the same time. Consequently, the above method is not as effective in a method of synthesizing double stranded DNA.

Studies have been pursued to overcome the above technical difficulty, and it has succeeded in synthesizing DNA comprising, for example, 46 bases, using a process called "triester process" (among the so-called "solid" processes). , in which 1% polystyrene is used as support, and the compounds of 4 bases (tetramer) or 5 bases (pentamer) are subjected to repeated condensation, compounds constituting the starting unitary fragments before 2o the block polymerization.

Even by this method, however, the number of bases in the DNA obtained is 50 at most, and it remains difficult to prepare by synthesis a DNA having chains comprising up to 80 to 150 radicals.

In view of the above, further studies have been pursued with attention (1) to the synthesis of long chain DNA carrying as much gene information as possible, and (2) to synthesis in even more advantageous conditions, and she finally made the present invention.

The method according to the present invention has the characteristics specified in claim 1.

The present invention will be further illustrated by the following:

Before the condensation, each block can be obtained in the conventional manner, in which each base is subjected to synthesis in the liquid phase.

The amine controlled porosity glass usually designated by the term "controlied porosity glass" amine (see Tetrahedron Letters, 24, 747 - 750,1983) used in the present invention is used as a support in the solid phase process. The amino group of this substance combines deoxythymidine which is converted to 3'-succinate by the usual method. This is used as a support for the nucleoside. Each of the desired blocks 45 is successively extended, on this resin, in the direction of the 5'-terminal element. As the condensing agent, mesitylene sulfonyl-3-nitrotriazolide (MSNT) is used. The DNA thus obtained is single stranded, and the complementary stranded DNA which is necessary for the preparation of the duplicate DNA can be readily obtained in a similar manner. This duplicate DNA can also be very easily obtained using DNA polymerase, using as a template a short fragment (10 base pairs, for example), which is complementary to the 3'-terminal region of 55-strand DNA. unique got. One of the most important merits of the present invention is that DNA polymerase can be used, since the condensation reaction can be accomplished without the aid of DNA ligase which has been widely used in conventional methods.

The duplicate DNA obtained is combined to give a vector plasmid by the known method, then transformed into bacteria such as Escherichia coli, and the strain is cultured to give the desired polypeptide. In the above states, there are a variety of ways to apply 65 proven gene technologies.

It is possible, in accordance with the present invention, to synthesize DNA having up to 80 to 150 radicals, and it is therefore possible to synthesize

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polypeptides having 15 to 30 amino acids by means known in gene technology. For example, the following polypeptides can be synthetically prepared: the hormone release inhibiting factor (somatostatin containing 14 amino acids), the stimulant of gastric secretion (gastrin, containing 17 amino acids), the ulcer remedy duodenal (secretin, containing 27 amino acids), the stimulant of growth hormone secretion, the agent to increase the level of insulin and sugar in the blood (glucagon, containing 29 amino acids), an agent similar to morphine (beta-endorphin, containing 31 amino acids) and the remedy for hypercalcemia (calcitonin, containing 32 amino acids), etc.

Furthermore, the long chain DNA of the present invention applies not only to DNA base sequences of structural parts of genes, but also to the preparation of general DNA comprising regulatory sites and specific sequences. , as well as a long chain DNA probe recognizing their structures. Accordingly, the present invention can be successfully used for the development of diagnostic agents.

According to the present invention, long chain DNA can be synthesized in a simple manner and in large quantities. The long chain DNA of the present invention can be effectively used (1) as a source of gene information regarding the synthesis of polypeptides and (2) as a source for the application of a DNA probe in gene technology. .

DNA production was 0.1 OD (1 OD equals approximately 50 micrograms) for a maximum of one lot. However, in accordance with the present invention, it is now possible to manufacture in quantities of up to 30 to 50 OD per batch. As a result, one can expect an expansion in the field of use of long chain DNA as a gene and as a DNA probe.

EXAMPLES

The present invention will be further illustrated by examples relating to the synthesis of endorphin, the physiological activities of which, such as analgesic action on the central nervous system and the action of endocrine hormone, are known.

(1) Synthesis of each of the blocks constituting basic sequences containing the endorphin gene.

The amino acid sequence of the endorphins is known, and the base sequence of the corresponding DNA can be freely chosen by referring to a table of codon usage. They will be found below together with their relationship to each of the blocks constituting the DNA base sequences used in the present invention. The upper, middle and lower columns correspond respectively to each block (the numbers therein are the block numbers), to the base sequence and to the corresponding amino acid sequence. Incidentally, restriction enzyme sites are given at both ends of the DNA base sequences. These sites are used to insert the plasmid.

© a - Endorphin

'13 12 11 10'

6 'ACCTGCAGCC CGT CGC TAC GGT GGT TTC ATG

Pstl Arg Arg Tyr Gly Gly Phe Met

"9 8 7 - 6 -

ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG

Thr Ser Glu Lys Ser Gin Thr Pro Leu Val

2 1

5 - 4 - 3 "►

ACT TAA TAG GGCTGCAGGT 3 '

Thr STOP STOP Pst I

672 791 4

(D a - [Leu5 3 - Endorphinè

■ l 3 16— 15 -10-

5 'ACCTGCAGCC ATG TAC GGT GGT TTC ÎTG ^ Pst 1 Met Tyr Gly G1 y Phe Leu

8 -7

ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG

Thr Ser Glu Lys Ser Gin Thr Pro Leu Val

2 1

vs

4 3

"ACT TAA TAG GGCTGCAGGT 3 '

Ihr STOP STOP Pst I

(g) r - (Leu5) - Endorphin

■ 13 —16— 15 10-

e 'ACCTGCAGCC ATG TAC GGT GGT TTC TTG' Pst I Met Tyr Gly Gly Phe Le ^

8

ACT TCT GAG AAG TCT .CAA ACT CCA TTG GTG

Thr Ser Glu Lys Ser Gin Thr Pro Leu Val

2 1

5 - 17 - 3 H M.

ACT TTG TAG GGCTGCAGGT i Thr Leu STOP Pst I '

<S) r - Endorphin

-13 12 11 10-

s ACCTGCAGCC CGT CGC TAC GGT GGT TTC ATG Pst I Arg Arg Tyr Gly Gly Phe Met

8

ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG

Thr Ser Glu Lys Ser Gin Thr Pro Leu Val

2 1

5 17 - 3 ■

ACT TTG TAG GGCTGCAGGT Thr Leu STOP Pst I

Among the blocks constituting the above endorphin genes, block 7 was prepared by synthesis by passing through the stages indicated below.

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bzbz (I) d (DUTr) AgAgcE

TEA bzbz (D)

d (DHTr) Ag Ce ce bzbz d (DHTr) AgAgo "(V)

BSA

'bzbz d Ag Cg ce —1 (VI)

bz (I) d (DHTr) TeCbce

(IV) bzbz d (DHTr) Cg Ag ce

"S NT

bzbzbzbz d- (DHTr) AgAgAgCgcE

(K)

TEA

bzbzbzbz d (DMTr) AeAbAeCpo- (XI)

TEA

bz d (DHTr) TgCgo * (VI)

J MS NT

BSA

bzbz dCe Ag ce

—1 on bzbzbz d (D il T r) TgCgCpAgcE “(X) 1 BSA bzbzbz dïg Ce cp Ag es (XI)

MSNT

l (XI)

bzbzbzbz bzbzbz d (DflTr) Ag Ag Ag Cg 7th Cg Ce Ae ce

TEA

(X IV) bzbzbzbz bzbzbz d (DîlTr) Ag Ae Ag Cg Tg Cg Cg Ag -

• Block 7

DMIr:

bz.

A * bz. C * T:

2 =

BSA: TEA:

4,4 '-dimethoxytrityl N-benzoyladenosyl

N-benzoylcytidylyle Tymidylyle

O-chlorophenyl phosphate benzenesulfonic acid triethylamine y

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Other blocks (1-6, and 8-17) constituting endorphin-like genes can be synthesized in a similar manner. The yields are given below.

Blocks

Basic sequences

Yield

1

CAGG

77

2

GCTG

94

3

TAGG

70

4

TTAA

104

5

TGAC

93

6

TTGG

67

7

AAACTCCA

70

8

GAAGTCTC

62

9

ACTTCTGA

65

10

GTTTCATG

65

11

CTACGGTG

70

12

GCCCGTCA

65

13

ACCTGCA

65

14

GTTTCTTG

70

15

GTACGGTG

67

16

GCCAT

75

17

TTTG

95

(2) Synthesis of endorphin genes:

The alpha-endorphin gene (mother 80 deoxy) containing restriction enzyme sites was prepared by synthesis by a solid phase method as follows:

1. A CPG deoxythymidine resin is washed with CH2Cl2 / MeOH.

2. Deteritylation is carried out with 2% BSA / CH2Cl2 / MeOH (this is carried out repeatedly and rapidly until the coloration disappears).

3. Azeotropic drying is carried out after substitution with pyridine.

A solution of each of the blocks is added in pyridine, subjected to azeotropic drying and MSNT and pyridine are added for the reaction. It is left to stand at room temperature and washed with pyridine.

4. Add a 0.1M dimethylaminopyridine solution / pyridine and acetic anhydride, allow to stand at room temperature and wash with pyridine.

The above operation is carried out several times, 13 times in total. The average reaction yield is 84%. Then, the resin is deprotected, at room temperature, with a solution of tetramethylguanidine 0, 1M-pyridine aldoxime (see CB Reese, et al .: Tetrahedron Lett., 2727.1978) in dioxane-water, then washed with pyridine-water, the washing liquid is concentrated in vacuo, concentrated ammonia is added thereto and the mixture is heated. The ammonia is evaporated and part of the residue is taken using the dimethoxytrityl group as a target to calculate the yield of the final stage.

The residual reaction solution is subjected to open chromatography in reverse phase (Ci8 silica gel for Prep 500 manufactured by Waters), by ion exchange (DEAE-toyopal) and in reverse phase (Qs silica gel, TSK 10 gel— 20 microns), which gives the pure alpha-endorphin gene (containing restriction enzyme sites) (= 80 mother deoxy).

Purity is confirmed by HPLC (Nucleosil 300-7 Ci s) and by electrophoresis, and its basic sequences are confirmed by the Maxam-Gilbert method. The result is given in Figures 1 to 3.

The alpha- (Leu5) -endorphin gene containing a restriction enzyme site) (mother deoxy 77), the gamma- (Leu5) -endorphin gene (containing a restriction enzyme site) are prepared in the same way. mother deoxy 77) and the gamma-endorphin gene (containing a restriction enzyme site) (mother deoxy 80).

(3) Synthesis of duplicate DNA and its combination with the vector plasmid.

Each molecule of 80 mother deoxy and io of synthetic nucleotide primer which is complementary to the 3'-terminal element of the first are mixed, they are heated to 65 ° C. and they are cooled to room temperature to cook the mother 80 and the primer. Then E. coli polymerase I (Klenow fragment) was added by conventional means and reacted at 37 ° C for 30 minutes, so as to transform the DNA into its double strand form.

The DNA is recovered as a precipitate in ethanol, reacted at 37 ° C for 30 minutes using polynucleotidekinase T4 and the two 5'-terminal elements of double stranded DNA are phosphorylated.

The DNA of the vector plasmid pUC 8 is then cut with a restriction enzyme Pst 1, it is added to the above double-stranded DNA solution, it is reacted at 16 ° C. overnight with T4 DNA ligase, and double stranded mother deoxy-80 DNA is combined with the vector plasmid.

(4) Cloning of the endorphin gene containing the plasmid.

The plasmid prepared as above is transformed into an E. coli JM 103 strain by conventional means, then it is chosen using a deficiency of beta-galactosidase activity present in pUC 8 as target, and it is collected. plasmid molecules by cloning from the strain.

It is confirmed that the plasmid into which the endorphin gene has been inserted in the correct orientation and position is as desired according to the Maxam-Gilbert method.

(5) Preparation of endorphins

A strain of E. coli JM 103 transformed is precultured 40 overnight in LB medium, it is transplanted into 2YT medium and subjected to a culture shaken at 37 ° C.

IPTG is added to the stages of the logarithmic productive phase (initial, middle and final stages) to make it 0.5 mM, and the synthesis of endorphin is initiated. After being induced by IPTG, the condensed protein is extracted and analyzed by HPLC, and it is found that an adequate amount of protein has been formed (1-5.0 x 105 molecules per cell) when induction is applied at the initial stage of the logarithmic productive phase. As regards natural endorphin alpha and gamma endorphin having a methionine radical in a molecule, they are treated with trypsin in the conventional manner. As regards alpha- (Leu5) -endorphine and gamma- (Leu5) -endorphine having a leucine radical in place of methionine, it is treated with BrCN. In any case, each of the desired endorphin proteins is subjected to column chromatography in accordance with the general protein purification process, after which each of them is separated and purified.

The fact that each of the endorphin molecules obtained has the desired amino acid sequence is confirmed by the fact that they are identical to samples already obtained by peptide synthesis in the test by HPLC using a reverse phase support.

65 FIG. 1 is an autoradiogram showing the result of an electrophoresis on polyacrylamide containing 20% deoxy 80 mer containing the alpha-endorphin gene prepared by synthesis after determination by the Maxam-Gilbert method.

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FIG. 2 is an autoradiogram showing the result of an electrophoresis on polyacrylamide at 8% of the mother 80 deoxy containing the alpha-endorphin gene prepared by synthesis after determination by the Maxam-Gilbert method.

FIG. 3 shows the result of a high performance liquid chromatography (Nucleosil 300-7 Ci s) of the mother deoxy 80 containing the alpha-endorphin gene prepared by synthesis after determination by the Maxam- method

Gilbert. On the ordinate and on the abscissa, we have respectively the absorbance and the time. The solvent system used is triethylamine-acetonitrile acetate and the flow rate is 1.0 ml / minute.

The present invention is not limited to the exemplary embodiments which have just been described, it is on the contrary liable to modifications and variants which will appear to those skilled in the art.

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1 sheet of drawings

Claims (2)

672 791 2 CLAIMS 1. A method of synthesizing a long chain DNA, according to which a plurality of oligonucleotide blocks are linked together, in a predetermined order, by purely chemical means, by a phosphotriester condensation method, in solid phase, using as a support a glass with amine controlled porosity, characterized in that the starting oligonucleotide blocks comprise 4 to 8 bases and in that the condensation is carried out using mesitylene sulfonyl-3-nitrotriazolide as a condensing agent. 2. Method according to claim 1, according to which the 5 ′ terminal regions of the oligonucleotide blocks are protected by means of dimethoxytrityl protecting groups, characterized in that benzenesulfonic acid is used as detityity agent.