Note: Descriptions are shown in the official language in which they were submitted.
1295558
-- 1 --
The present invention relates to a novel method of
synethsizing long chain DNA carrying information for synthesis of
specific proteins and, more particularly, it relates to a method of
synthesizing long chain DNA purely chemically, i.e. without the use
6 of enzymes, by a so-called solid phase method using blocks of 4 to 8
base sequences and using aminated CPG as a carrier.
It has been known that the synthesis of polypeptides using a
synthetic gene is possible by the steps of (1) synthesis of a
structural gene; (2) recombination of the gene into a suitable plasmid;
(3) transformation of a suitable host by the formed chimera plasmid;
and (4) obtaining the desired polypeptide by culturing the transformed
substance.
Recently, development of a DNA probe has attracted public at-
tention as a novel means for gene technology. This is a method of
identifying unknown DNA and RNA which is a transcribed product by a
hybridization of single stranded DNA and RNA which are known in the
art by utilizing the properties of DNA and RNA whereby they can form
duplexes, as in the relationship of a template and a casting. Since
very sensitive and prompt identification is possible by utilizing
the hybridization method, this method can be used for diagnosis of
diseases by finding the specific DNA and RNA in a gene level from the
blood and cells of patients and pathogenic bacteria. Accordingly,
~.
1~955~i8
-- 2
DNA has an important value as a diagnostic agent by virtue of its
utilization as a DNA probe.
When DNA is used as structural gene or a DNA probe, it has
known that the longer the base sequence of the DNA, the better it
functions as an information source or DNA probe. However, it
has also been know that the longer the base sequence, the more
difficult it is to synthesize DNA.
It is an object of the present invention to provide a
simplified process for synthesizing long chain DNA.
The conventional method for synthesizing DNA is as follows.
First, comparatively short DNA fragments with 10 to 20 basis
residues (i.e., base pairs) are chemically synthesized, and then
they are combined to prepare fragments having a total structure
of double stranded DNA having the desired information for peptide
synthesis, then the fragments are combined using an enzyme called
DNA ligase.
However, by such a method, only comparatively short
fragments with 1 (monomer), 2 (dimer) or 3 (trimer) bases are
manufactured prior to block condensation and it is not possible
to synthesize long chain DNA with 80 residues or the like.
In addition, in that method it is essential to use an enzyme
called DNA ligase. Therefore, in synthesizing double stranded
DNA as a gene, it is necessary that all base sequences
constituting double stranded DNA are synthesized at one time.
Accordingly, the above method is not so effective in a process of
synthesizing double stranded DNA.
The present inventors overcame the above technical
difficulty and succeeded in synthesizing DNA with approximately
46 bases by utili-
'.~
~ `` lZ~55~8
-- 3 --
zing a method called a triester method (among the so-called solid
methods) in which 1% polystyrene is used as a support and the compounds
of 4 (tetramer) or 5 (pentamer) bases are subjected to a repeated
condensation.
Even by such a method, however, the base numbers in the re-
sulting DNA are 50 at the largest and there is still a difficulty in
synthesizing DNA with chains of as long as 80 to 150 residues.
Following this success, the present inventors have further
carried out studies paying their attention to (1) the synthesis of
long chain DNA carrying as much gene information as possible and (2)
the synthesis under re advantageous conditions. The present invention
represents the culmination of those studies.
Characteristic features of the present invention are as
follows:
(1) ~nit numbers prior to bloc~ condensation are 4 to 8
(Octamer) bases;
(2) among the so-called solid phase methods,"triester
method" is used; and
(3) aminated CPG carrier is used as a support.
2~ The present invention will be further illustrated as here-
under:
i5~58
Each block prior to the condensation can be obtained in the
conventional way in which each base is subjected to a liquid
phase synthesis.
Animated CPG (controlled pore glass) (cf. Tetrahedron, 24,
747-750, 1983) used in the present invention is used as a carrier
in the solid phase method. To the amino group of this substance
is combined deoxythymidine which is changed to 3'-succinate by
usual method. This is used as a carrier for nucleoside. Each
desired block is extended, on this resin, to the direction of
5'-terminal successively. Mesitylene sulfonyl-3-nitrotriazolide
(MSNT) is used as a condensation agent. The resulting DNA is
single stranded and the complimentary strand DNA which is
necessary for preparation of duplet DNA can be easily obtained in
a similar way. Alternatively, each duplet DNA can be very easily
obtained by the use of DNA polymerase using short fragment (10
b.p. or so) which is complimentary with the 3'-terminal region of
the resulting single stranded DNA. The fact that DNA polymerase
can be used is one of the most advantageous aspects of the
present invention for the condensation reaction can be
accomplished without the aid of DNA ligase which has been widely
used in conventional methods.
The resulting duplet DNA is combined to give vector plasmid
by the known method, then transformed to bacteria such as
Escherichia coli, and the strain is cultured to afford the
desired polypeptide. In the above steps, various gene
technological means which have been already established can be
applied.
.,
129~5~8
It is possible in accordance with the present invention to
synthesize DNA with as long as 80 to 150 residues and,
therefore, polypeptides with 15 to 30 amino acids can be
synthesized by the known gene technological means. For
instance, the following polypeptides can be synthesized. They
are growth hormone-release inhibiting factor (Somatostatin,
containing 14 amino acids), stomach acid secreting stimulant
(Gastrin, containing 17 amino acids), duodenum ulcer remedy
(Secretin, containing 27 amino acids), stimulant for secretion of
growth hormone, insuline and blood sugar level increase
(Glucagon, containing 29 amino acids), morphine like agent (beta-
Endorphin, containing 31 amino acids), and hypercalcemia remedy
(Calcitonin, containing 32 amino acids), and the like.
In addition, the long chain DNA of the present invention is
applied not only for DNA base sequences of structural gene parts
but also for the manufacture of general DNA including regulatory
sites and specific sequences as well as for long chain DNA probe
recognizing their structures. Accordingly the present invention
can be positively applied for development of diagnostic agents.
According to the present invention, long chain DNA can be
synthesized simply and in large quantities. The long chain DNA of
the present invention can be effectively utilized as (1) a gene
information source concerning polypeptide synthesis and (2) a
source for application of the DNA probe in gene technology.
1295558
-- 6 --
Production of DNA has been 0.1 OD (1 OD is equivalent to
about 50 micrograms~ per one lot at best. However, in accordance
with the present invention, it is now possible to manufacture in
quantities as large as 30 to 50 OD per lot. Consequently, ex-
pansion of the utilizable field of long chain DNA as a gene and as
a DNA probe is now possible.
The following non-limitative exa~es describe the synthesis
of endorphin whose physiological activities such as central nervous
analgesic action and endocrine hormone action have been known.
(1) Synthesis of each block constituting base sequences
including endorphin gene.
The amino acid sequence of endorphins has been known and the
DNA base sequence corresponding thereto can be freely selected by ~;
referring t~ a table of coden usage. They are given as hereunder to-
gether with their relation between each block constituting DNA base
sequences used in the present invention. The upper, middle and lower
columns are each block (figures therein are block numbers), base
sequence~and corresponding amino acid sequence, respectively. Re-
stricted enzyme sites are given at both terminals of DNA base sequences.
Said sites are used in inserting plasmids.
129SS~8
-- 7 --
O a - Endolph ~
~13 12 11 ~ 10
5' ACCTGCAGCC CGT CGC TAC GGT GGT TTC ATG
I
Pst I 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 Gln Thr Pro Leu Val
5_-4 3
ACT TAA TAG GGCTGCAGGT
I
Thr STOP STOP Pst I
0a - [ Leu' ] - Endolphin
--13 ~ 16 15 10
5 ' ACCTGCAGCC ATG TAC GGT GGT TTC TTG
I
Pst I Met Tyr Gly Gly Phe Leu
~9 8 7 6
ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG
Thr Ser Glu Lys Ser Gln Thr Pro Leu Val
5_--4_~3
ACT TAA TAG GGCTGCAGGT
Thr STOP STOP Pst I
lZ955~8
-- 8 --
O- [Leu~ ] - Eodo~
-- 13 ~16 15 ~ 10
5 ' ACCTGCAGCC ATG TAC GGT GGT TTC TTG
I
Pst I Met Tyr Gly Gly Phe Leu
9 ~8 - 7 ~ 6 ~--
ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG
5_---17_-3 ~
ACT TTG TAG GGCTGCAGGT
I
Thr Leu STOP Pst I
O y - Eodo~oio
--13 ~ 12 ~ 11 ~ 10
5 ' ACCTGCAGCC CGT CGC TAC GGT GGT TTC ATG
I
Pst I 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 Gln Thr Pro Leu Val
5 ~17_-3
ACT TTG TAG GGCTGCAGGT
I
Thr Leu STOP Pst I
Among the blocks constituting the above endorphin genes,
the block 7 was synthesized by the steps as given below.
.,~.
129S558
bz bz ( I )
d (DMTr) Ae Ae CE
TEA bz bz ( II )
d (DMTr) Ae Ce CE
d (DMi r) Ae Ae o~ ¦ d (DMTr) Te Ce CE ( III )
bz bz TEA bz( bIzV )
d IAe Ce CE d (DMTr) Ce Ae CE
¦ MSNT bzBSA
d (DMTr) Te Ce o-
bz bz bz bz ( YII 1
d (DMTr) Ae Ae Ae ce CE ~ I ~Z bz
I TEA ( IX ) d lce Ae CE
1 MSNT
bz bz bz bz
d (DMTr) Ae Ae A~ Ce 0- . bz bz bz
( XI ) d (DMTr) Te Ce Ce Ae CE
¦ BSA ( X )
bz bz bz
d Te Ce Ce Ae CE
~ I ( XII )
¦ MSNT ( XIII )
bz bz bz bz bz bz bz
d (DMTr) Ae Ae Ae Ce Te ce Ce Ae CE
¦ TEA ( XIV )
bz bz bz bz bz bz bz
d (DMTr) Ae Ae Ae Ce Te Ce Ce Ae O~ ~ Block 7
DMTr: 4,4 - Dimethoxytolityl
bAZ N-Benzoyladenosyl
bcz N-Benzoylcytidylyl
T: Tymidylyl
e o-Chlorophenyl phosphate
BSA: Benzenesulfonic acid
TEA: Triethylamine
,~,.
lZ9~5X8
- 9a -
The accompanying drawings illustrate the present invention.
In particular:
Fig. 1 is X-ray autoradiogram showing the result of 20%
polyacryamide electrophoresis of deoxy 80 mer containing alpha-
endorphin gene synthesized after determination by Maxam-Gilbert
method.
Fig. 2 is X-ray autoradiogram showing the result of 8%
polyacrylamide electrophoresis of deoxy 80 mer containing alpha-
endorphin gene synthesized after determination by Maxam-Gilbert
method.
Fig. 3 shows the result of high performance liquid
chromatography (Nucleosil 300-7*C18) of deoxy 80 mer containing
alpha-endorphin gene synthesized after determination by Maxam-
Gilbert method. Ordinate and abscissa show absorbency and time,
respectively. Solvent system used was triethylamine acetate-
acetonitrile and the flowing speed was 1.0 ml/min. Nucleosil
300-7 is a registered trademark.
12955S8
-- 10 --
Other blocks (1-6, and 8-17) constituting endorphin type
genes can be synthesized by similar way. Each yield is given as
hereunder.
Blocks Base Sequences Yield (%)
. _
1 CAGG 77
2 GCTG 94
3 TAGG 70
4 TTAA 104
TGAC 93
6 TTGG 67
7 AAACTCCA 70
8 GAAGTCTC 62
9 ACTTCTGA 65
GTTTCATG 65
11 CTACGGTG 70
12 GCCCGTCA 65
13 ACCTGCA 65
14 GTTTCTTG 70
GTACGGTG 67
16 GCCAT 75
17 TTTC 95
25 (2) Endorphins genes synthesis:
alpha-Endorphin gene (deoxy 80 mer) containing restricted
enzyme sites was synthesized by a solid phase method as follows:
1. Deoxytymidine CPG resin is washed with CH2C12/MeOH.
2. Detritylation is conducted with 2% BSA/CH2C12 (this was
30 conducted repeatedly and promptly until colorization
disappears)
3. Subjected to azeotropic drying after substituted with
pyridine.
lZ955~8
-- 11 --
A solution of each block is added, subjected to
azeotropic drying, and MSNT and pyridine for
the reaction are added. Allowed to stand at
room temperature and washed with pyridine.
4. O.lM Dimethylaminopyridine/pyridine solution and acetic
anhydride are added, allowed to stand at room
temperature, and washed with pyridine.
The above procedure is conducted repeatedly, for 13 times
in total. Average yield of this reaction was 84~. Then the
resin is deprotected, at room temperature, with a solution of
O.lM tetramethylguanidine-pyridine aldoxime (cf. C. B. Reese, et
al: Tetrahedron Lett., 2727, 1978) in dioxane-water, then washed
with pyridine-water, the washing is concentrated in vacuo,
concentrated ammonia water is added thereto and the mixture is
warmed. Ammonia is evaporated therefrom and a part of the residue
is taken using dimethoxytrityl group as a target to calculate the
yield of the final stage.
The residual reaction solution is subjected to a reversed
phase (Clg silica gel for Prep 500*manufactured by Waters), ion
exchange (DEAE-20 toyopal)*, and reversed phase (Clg silica gel,
TSK-Gel*10-20 micrometers) open chromatographies to afford pure
alpha-endorphin gene (containing restricted enzyme sites) (-
deoxy 80 mer). Prep 500, DEAE-toyopal and TSK-Gel are
registered trademarks.
Purity was confirmed by HPLC (Nucleosil 300-7*Clg) and by
electrophoresis and its base sequences were confirmed by Maxam-
Gilbert method. The result is given in Fig. 1 to Fig. 3.
Nucleosil 300-7 is a registered trademark.
12~SS58
Similarly prepared were alpha-(Leu5)-endorphin gene (containing
restrictive enzyme site) (deoxy 77 mer), gamma-(Leu5)- endorphin gene
(containing restrictive enzyme site) (deoxy 77 mer) and gamma-endorphin gene
(containing restrictive enzyme site) (deoxy 80 mer).
(3) Svnthesis of duplex DNA and its combination with vector plasmid
Each one mole of deoxy 80 mer and synthetic nucleotide primer which is
complimentary with 3'-terminal of the former were mixed, heated at 65-C, and
cooled to room temperature to anneal the deoxy 80 mer and the primer. Then
E.coli polymerase I (Klenow fragment) was added by conventional means and
10 made to rect at 37~C for 30 minutes so that DNA was converted into double
stranded.
DNA was recovered as a precipitate in ethanol, made to react at 37~C for
30 minutes using T~ polynucleotidekinase, and both 5'-terminals of the double
stranded DNA were phosphorylated.
Then the vector plasmid pUC 8 DNA was scissored with a restrictive
enzyme Pst 1, added to the above double stranded DNA solution, made to react
at 16'C overnight with T~ DNA ligase, and the double stranded d 80 mer DN~ was
combined with the vector plasmid.
(4) Clonina of ~lasmids containinq endor~hin aenes.
The plasmid prepared as above described was transformed into E. coli JM
103 strain by conventional procedure, then selected using a deficiency
`~D
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1%95~8
- 13 -
of beta-galactosidase activity present in the pUC 8 as a target,
and plasmid molecules were collected by cloning from the strain.
It has been confirmed that plasmid in which endorphin gene
S was inserted into the correct orientation and position as desired
in accordance with Maxam-Gilbert method.
(5) Obtaining of endorphins.
Transformed E. coli JM 103 strain was precultured overnight
in an LB medium, planted in 2YT medium, and subjected to a shake
culture at 37C.
IPTG was added to the logarithmic productive phase stages
(initial, medium and final stages) to make it 0.5mM and synthesis
of endorphin was induced. After being induced by IPTG, fused
protein was extracted, and analyzed by HPLC whereupon it was
found that adequate quantity of protein production was observed
(1-5.0 x 105 molecules per cell) when induction was applied at
the initial stage of logarithmic productive phase.
The natural type alpha-endorphin and gamma-endorphin having
methionine residue in a molecule were treated with trypsin by
conventional procedures. Alpha-(Leu5)-endorphin and gamma-
(Leu5)-endorphin having leucine residue in place of methionine
were treated with BrCN. Each of the desired endorphin proteins
was subjected to a column chromatography according to the general
purification method of proteins whereupon each of them was
separated and purified.
The fact that each of the resulting endorphin molecules
exhibits desired amino acid sequence was confirmed by the fact
that they were identical with the samples already obtained by the
peptide synthesis by testing with HPLC using a reverse phase
carrier.
.~
