CN1024350C - Nucleic acid derivatives - Google Patents
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- CN1024350C CN1024350C CN88104038A CN88104038A CN1024350C CN 1024350 C CN1024350 C CN 1024350C CN 88104038 A CN88104038 A CN 88104038A CN 88104038 A CN88104038 A CN 88104038A CN 1024350 C CN1024350 C CN 1024350C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
Abstract
To execute a numerical input with good operability by executing an input by designating the scale of a measure which can be reduced and enlarged, displayed on a display screen. The device is constituted of a CRT display 10, a mouse 11, and a CPU 13. A measure is displayed on the display 10. The measure is constituted of a measure operating part 1, and numerical input parts 2-4. In the operating part 1, icons 5, 9 for the left shift and the right shift of the numerical input parts 2-4, icons 6, 8 for enlarging and reducing the numerical input parts 2-4, and the display 7 of a numerical value instructed with the mouse are displayed. As for the numerical data input parts 2-4, the value at every scale becomes smaller in order of the display parts 2, 3 and 4. At the time of an input operation, with respect to the measure displayed on the display 10, a numerical data is inputted by operating the icon and selecting it by the mouse 11. In such a way, a numerical data input device whose operability is improved is obtained.
Description
The present invention relates to have useful physiologically active and the low nucleic acid derivative of toxicity as medicine.
Nucleic acid is that the sugar of ribose etc. is combined on purine skeleton or the pyrimidine ring, is formed between linking to each other with chain between them by phosphoric acid.
RNA(ribonucleoside acid polymer in the nucleic acid) be chain-like macromolecule compound, it has ribose as sugar, and sugar moieties is connected with the diester linkage of phosphoric acid.Constitute nucleic acid alkali (for example inosine, adenosine, cytidine, uridine etc.) purine skeleton or two chains of pyrimidine ring part are linked to each other by hydrogen bond in so-called complementary mode, constitute spiral helicine three-dimensional arrangement.Can expect to have useful physiologically active owing to have the nucleic acid of two chains, so so far, existing a lot of research [Biochemieal and Biophysical Research Communications 58, (1974) etc.].
In this manual, wherein the Polyinosinic acid as synthetic double-stranded RNA: the polycytidylic acid derivative is referred to as " polyIpolyC " derivative, and it is constituted the unit is that Polyinosinic acid is referred to as " polyI ", and polycytidylic acid is referred to as " polyC ".
In recent years, people knew already that various skies or synthetic double-stranded RNA had Interferon, rabbit and produce energy people such as [Off ィ-Le De], Proc.Nat.Acad.Sci.U.S.581004(1967); [Off ィ-Le De]
Deng the people, wait the people, Proc.Nat.Acad.Sci.U.S.582102(1967); Off ィ-people such as Le De, Proc.Nat.Acad.Sci.U.S.61340(1968); People such as ィ テ Le, Proc.Nat.Acad.Sci.U.S.581719(1967); Deng the people, J.Gen.Physiol.56905(1970); People such as デ Network テ-Network, Methods in Enzymology78 291(1981)].
Above through arrangement, be summarized as follows table:
Table look-up as the nucleic acid derivative of interferon derivative
(I) homopolymer homopolymer complex body (making the double-strandednucleic acid polymkeric substance of matrix with polyIpolyC)
(1) modification alkali
Polyinosinic acid poly (5-bromo cytidylic acid), Polyinosinic acid poly (2-sulfo-cytidylic acid), poly (7-denitrogenation mix t-inosinic acid) polycytidylic acid, poly (7-denitrogenation mix t-inosinic acid) poly (5-bromo cytidylic acid).
(2) modified sugars
Poly (2 '-the nitrine t-inosinic acid) polycytidylic acid.
(3) modified phosphate
The Polyinosinic acid poly (Cytidine-5 '-thiophosphoric acid).
(II) checker multipolymer
Poly (adenylic acid (AMP)-uridylic acid).
(III) homopolymer multipolymer complex body
Polyinosinic acid poly (cytidylic acid, uridylic acid); Polyinosinic acid poly (cytidylic acid, 4-thiourdine acid).
The complex body of (IV) nucleic acid and polycation
Poly I-C poly-l-lysine (being referred to as " poly ICLC ").
(V) other
Polyinosinic acid poly (the rare basic cytidylic acid of 1-second).
As mentioned above, delivered many double-stranded RNAs in recent years, particularly done the report of the derivative of matrix with polyIpolyC.And, one piece of relevant people such as summary ェ デ Network ラ-Network of containing their a series of nucleic acid derivatives and structure-activity thereof is arranged, Texas Reports on Biology and Medicine 41 77(1982)].
In article, narrate nucleic acid polymers especially and will have Interferon, rabbit generation energy, then must be the above polymer of 4S surely.
Other physiological actions people of polyIpolyC are known that and have the cultivation effect that stops the zoografting cancer people such as [, Proc.Nat.Acad.Sci.U.S.62357(1969)] レ ウ ィ.
It is believed that, wherein do not include the organism immune activation effect that helps [カ--wait people, J.Immunol.104 1035(1970)].
As mentioned above, past attempts expectation, polyIpolyC is with its antivirus action and antitumous effect application clinically, but after recognizing that it has unforeseeable strong toxicity, is applied to the people and just has been a problem.Even like this, now, also do therapeutic test at derivative with polyIpolyC.
Levy has developed to have strong Interferon, rabbit and produces people such as active poly ICLC[Levy, Texas Reports on Biology and Medicine 41 653(1982)].
Above-mentioned polyIpolyC and derivative thereof can expect fully that really it has useful effect, but its toxicity also is irrefragable [people such as デ Network ラ-Network, Infect.Immuni., 6344(1972)] under many circumstances.In order to reach the purpose that is applied to organism, must think some ways.
The inventor, finds the length of the chain of double chain acid derivative is controlled in certain scope based on prior art in the years of researches process, and it just has necessary physiologically active, has reduced its toxicity significantly simultaneously, thereby has finished the present invention.
Therefore, the objective of the invention is to the length of the chain of double-strandednucleic acid polymkeric substance is limited in certain scope.
The inventor to commercially available or to have this thing of toxicity by common reaction synthetic polyIpolyC very interested, constantly study it and produces reason, thereby obtain certain achievement at first.
That is to say, the inventor studies as index simultaneously the cancer cells interception of defeated cancer mouse with to the mouse marrow stem cell obstacle, the result checks on the following fact, promptly the sedimentation of nucleic acid polymers be definite value and its molecular size distribution under the situation more than the 13S, then its existing activity is toxic again, in the time of in being in the 4S-13S scope, activity is arranged for it but toxicity significantly reduces, and then under the situation below the 4S, then it does not have toxicity, but activity has also almost disappeared.
PolyIpolyC is if below 4S, and then activity also disappears, and this view with people such as above-mentioned デ Network ラ-Network almost is consistent, but grinding still to have no talent so far and did about chain length and toxic relation.
Below this part thing to be that the present inventor is initial find, first factor that the toxicity of polyIpolyC and derivative thereof is reduced significantly is that the molecular size (chain length) nucleic acid polymers is controlled in certain scope.
Wherein, the distribution of the molecular size that should control, being preferably in 4S-13S(base number under the sedimentation definite value is about 50-10000) scope in.
The chain length of maximum distribution at this moment is usually near 100-600 base number.
As the unit of the molecular size of representing nucleic acid base pair (to call " bp " in the following text) commonly used, be to represent its bulk of molecule (10bp is expressed as the dichain polymer with 10 base pairs) by the base number of nucleic acid.In this manual, because also will handle dichain polymer nucleic acid polymers in addition, so use the term of " base number " for example to replace bp(, so-called " 10 base numbers then represent to have the nucleic acid polymers of 10 bases).
When the molecular size that will make nucleic acid is specific, then use common sedimentation definite value (S value) widely, the inventor organizes the double-stranded DNA (M13 phage segment) with known molecular size in contrast, adopt following high performance liquid chromatography (HPLC) or electrophoretic method with gel-filtration column, by comparing conversion, can try to achieve above-mentioned base number with its control group.
In the past, when the high molecular molecular weight of expression nucleic acid, once used sedimentation definite value (S value) widely.Now commercially available nucleic acid polymer is also with the S value representation.But, because gel electrophoresis, gel filtration chromatography, ion exchange chromatography etc. have been adopted in the progress of experimental technique in recent years, having established the means of determining molecular weight more accurately, can measure now its chain length.Therefore the problem that exists is the relation that S value representation and chain length are represented, and since during with the S value representation each nucleic acid molecule be to get the inherent value, therefore from the method as the molecular weight of representing nucleic acid, seeing on the meaning whether S value representation and chain length are represented can correctly adapt to, is not no problem.
In specification sheets of the present invention, when expression during molecular weight, according to the convention of so far nucleic acid chemistry, the inventor has also put down in writing the S value representation.But, because the S value representation is that the nucleic acid polymer is carried out method for measuring as group's (perhaps molecular conformation) of whole molecule, so be necessary more clearly to show the boundary of molecular weight distribution from now on, therefore the expression value of measuring based on chain length (being " base number " in this specification sheets) also record in the lump.
Can list the derivative of the derivative described in the table look-up of some above-mentioned nucleic acid derivatives through dimensioning (short chainization) as nucleic acid derivative of the present invention.At this moment, be derivative, and nucleic acid derivative of the present invention is characterised in that and has carried out specific to its molecular size distribution with basic structure identical with polyIpolyC.
Can list such as some following derivatives as nucleic acid derivative of the present invention.
(5-bromo cytidylic acid, S value are 4-13 to the Polyinosinic acid poly, and the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.Polyinosinic acid poly (2-sulfo-cytidylic acid), the S value is 4-13, the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.Poly (7-denitrogenation mix t-inosinic acid) polycytidylic acid, the S value is 4-13, the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.Poly (7-denitrogenation mix t-inosinic acid) poly (5-bromo cytidylic acid), the S value is 4-13, the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.
Poly (2 '-nitrine t-inosinic acid) polycytidylic acid, the S value is 4-13, the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.
Polyinosinic acid poly (Cytidine-5 '-thiophosphoric acid), the S value is 4-13, the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.
Poly (adenylic acid (AMP)-uridylic acid), S value are 4-13, and the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.
Polyinosinic acid poly (cytidylic acid, uridylic acid), the S value is 4-13, the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.
Polyinosinic acid poly (cytidylic acid, 4-thiourdine acid), the S value is 4-13, the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.
Poly I-C poly-l-lysine, S value are 4-13, and the molecule of maximum distribution shows with the base numerical table and is preferably 100-600.
Polyinosinic acid poly (1-vinyl cytidylic acid), the S value is 4-13, the molecule of maximum issue shows with the base numerical table and is preferably 100-600.
When preparing nucleic acid derivative of the present invention, the chain portion of phosphoric acid one side that can be by making polymer derivant listed in above-mentioned polymkeric substance and the above-mentioned table look-up disconnects, and makes it degraded.
Except that following thermal decomposition method, the alkali that can use RNA limit decomposition method and with the portion water solution of RNA lytic enzyme etc. as this method.
The feature of nucleic acid derivative according to the present invention, promptly control the distribution of the degraded molecular size that causes by the disconnection of above-mentioned phosphoric acid one pendant moiety, soon can make the people find effect of the present invention, promptly 1. can keep physiologically active together, 2. improve security.
The physiologically active of nucleic acid derivative of the present invention is exceedingly useful as medicine.As described below, nucleic acid derivative of the present invention has very strong antitumous effect.This effect only is in several physiological activities of having of nucleic acid derivative of the present invention.
In addition, as being the physiologically active of the nucleic acid derivative of the present invention of matrix with polyIpolyC, can listing TNF generation energy, Interferon, rabbit generation energy, ask that leukin 2 produces energy, giant bacteriophage activation energy, the activation to the NK cell, the effect of prevention tumor cell proliferation, the proliferation function of prevention human tumor cells in failing the cancer nude mice, the lung transferance of inhibition tumour cell.
Interferon derivative of nucleic acid derivative of the present invention and present polyIpolyC etc. etc. is compared has high security.Therefore, compound of the present invention is useful as antiviral agent, antineoplastic agent.
When being used as medicament administration nucleic acid derivative of the present invention, nucleic acid derivative of the present invention can be directly or as in pharmaceutically acceptable nontoxicity and inert carrier, contain for example 0.1%-99.5%, be preferably the medical component of 0.5%-90%, be applied to the animal that comprises the people.
As the thinner that aqueous, solid state or semi-solid are arranged, weighting agent and other prescriptions of carrier auxiliary agent with more than one.Medical component is preferably used with administration unit's form.Nucleic acid derivative of the present invention can be taken orally, organizes interior administration, topical or rectal administration.Yes uses with the formulation that is suitable for these medications, for example, and various oral preparation, injection, inhalation, eye drops, ointment, seat agent etc.Special hope is such as administration in the tissue, topical.
The consumption for the treatment of malignant tumor agent is preferably according to the adjustment such as nature and extent of situations such as patient's age, body weight, route of administration, disease, but usually, the adult uses the composition significant quantity of nucleic acid derivative of the present invention to be the 0.05-1000 milligram at every turn, preferably generally splash into the 5-100 milligram with intravenously.According to circumstances, also have at this enough below consumption, or on the contrary must be more than this.In addition, can with every day 1 time to the every day of administration for several times or with at interval 1 day to a few days administration.
The consumption of the therapeutical agent of innocent tumour or virus disease is preferably according to the nature and extent of disease, route of administration, patient's adjustment such as situation, but usually, and its consumption is 1/10th to 1/tens of an above-mentioned consumption as the treating malignant tumor agent.
In addition, nucleic acid derivative of the present invention can be with the following listed physiological response regulation substance B of various BRM(iologieal Response Modifiets) and following listed various carcinostatic agents use.At this moment, can expect to obtain the effect that multiplies each other mutually of these materials and nucleic acid derivative of the present invention and alleviate the side effect of following it fully.
[example of BRM]
Interferon, rabbit (α, β, γ),
Between leukin (IL1, IL2),
The CSF(colony stimulating factor),
The TNF(tumour necrosis factor),
LEVAMISOLE HCL, ベ ス チ Application, vitamin A acid (レ チ ノ ィ Star ァ シ De), LAK cell etc.
[examples of carcinostatic agent etc.]
The 5-FU(5-Fluracil), CDDP(Platinol vidarabine Ara-A Ara-C(cytosine arabinoside)))), endoxan, the pyridine of AZT(zidovudine).
The physiologically active of nucleic acid derivative of the present invention
The physiologically active of nucleic acid derivative of the present invention below will be described.The interception of the tumor proliferation in the defeated cancer mouse
Seek tumor proliferation interception in the defeated cancer mouse that has isotransplantation cancer Meth-A cell with following method.With Meth-A cell (3 * 10
5/ 0.2 milliliter) be dissolved in the physiological saline, give Balb/c mouse (5 age, male in week) subcutaneous injection.After 2 days,, give a corpse or other object for laboratory examination and chemical testing with two weeks of intravenous administration with the inferior on every Wendesdays course of treatment.After 2 days of final administration, take out the tumour cell part, measure its weight.It the results are shown in following table.Test corpse or other object for laboratory examination and chemical testing I poly IpolyC, its molecular size distribution is more than the 13S; Test corpse or other object for laboratory examination and chemical testing II polyIpolyC, its molecular size distribution are that 4S is above to 13S following (S value=8); Test corpse or other object for laboratory examination and chemical testing III polyIpoly(C
12, U) (that is,, replacing the polymkeric substance of cytidylic acid with the displacement of 1 uridylic acid) with respect to 12 of polyC cytidylic acids, its molecular size distribution is more than the 13S; Test corpse or other object for laboratory examination and chemical testing IV polyIpoly(C
12, U), its molecular size distribution is that 4S is above to 13S following (S value=9); Test corpse or other object for laboratory examination and chemical testing V polyIIpoly(C
20, S
4U) (that is, with respect to 20 of the cytidylic acids of polyC, replace the polymkeric substance of cytidylic acid with 1 4-thiourdine acid displacement), its molecular size distribution is more than the 13S; Test corpse or other object for laboratory examination and chemical testing VI polyIpoly(C
20, S
4U), its molecular size distribution is that 4S is above to 13S following (S value=9).
In addition, control group represents only to give the situation of physiological saline.
Corpse or other object for laboratory examination and chemical testing dosage number of elements hinders
(μ g/ mouse) %
Control group-10-
Test corpse or other object for laboratory examination and chemical testing I 100 10 83
*
Test corpse or other object for laboratory examination and chemical testing II 100 10 63
*
Test corpse or other object for laboratory examination and chemical testing III 100 10 73
*
Test corpse or other object for laboratory examination and chemical testing IV 100 10 34
*
Test corpse or other object for laboratory examination and chemical testing V 100 10 75
*
Test corpse or other object for laboratory examination and chemical testing VI 100 10 52
*
*: obvious errors P＞0.01.
Nucleic acid derivative of the present invention is tangible to the effect that stops the tumor proliferation in the defeated cancer mouse.The lung metastasis inhibition effect of tumour cell
By vein the portable melanomatous B16F10 cell 2 * 10 of homology
5Individual being transplanted in the C578L/6 mouse (male, 5 ages in week).The lung that calculating was transplanted after 2 weeks shifts joint number (colony number).A corpse or other object for laboratory examination and chemical testing before the B16F10 melanoma was transplanted 24 hours with intravenously administrable.Totally 9 examples.It the results are shown in following table.Test corpse or other object for laboratory examination and chemical testing I, test corpse or other object for laboratory examination and chemical testing II and control group are all with above-mentioned identical.
Corpse or other object for laboratory examination and chemical testing dosage colony number hinders %
（μg/kg）
Control group-85 ± 5
Test corpse or other object for laboratory examination and chemical testing I 5 25 ± 8* 71
50 11±3＊ 87
500 1±1＊ 99
Test corpse or other object for laboratory examination and chemical testing II 5 58 ± 8* 33
50 24±7＊ 72
500 13±2＊ 84
*: obvious errors P＜0.01.
The tumour cell lung metastasis inhibition effect of nucleic acid derivative of the present invention is clearly.
The security of nucleic acid derivative of the present invention
The security of nucleic acid derivative of the present invention below will be described.Cytotoxic effect to mouse marrow stem cell.
Seek nucleic acid derivative of the present invention to the mouse marrow stem cell cytotoxic effect.Test corpse or other object for laboratory examination and chemical testing I, test corpse or other object for laboratory examination and chemical testing II, test corpse or other object for laboratory examination and chemical testing III, test corpse or other object for laboratory examination and chemical testing IV, test corpse or other object for laboratory examination and chemical testing V, test corpse or other object for laboratory examination and chemical testing VI are all same as described above, and control group gives physiological saline.
Annotate in the Balb/c mouse as a corpse or other object for laboratory examination and chemical testing (8 ages in week, male, 5 every group) 0.5 milligram/kilogram dosage is quiet, after 24 hours, take the medullary cell of this mouse.After cell fixation, do Jim Sa (Giemsa) dyeing.Examine under a microscope the cell of this smear preparation,, calculate velocity of variation by following formula based on the netted erythrocytic number that occurs separately.It the results are shown in following table.
Velocity of variation=((control group)-(respectively testing a corpse or other object for laboratory examination and chemical testing))/((control group)) * 100
Velocity of variation
Test corpse or other object for laboratory examination and chemical testing I 61
Test corpse or other object for laboratory examination and chemical testing II 0
Test corpse or other object for laboratory examination and chemical testing III 59
Test corpse or other object for laboratory examination and chemical testing IV 0
Test corpse or other object for laboratory examination and chemical testing V 30
Test corpse or other object for laboratory examination and chemical testing VI 0
The security of nucleic acid derivative of the present invention is high to be clearly.
Acute toxicity test
Acute toxicity test is made of the Balb/c mouse.Once give a corpse or other object for laboratory examination and chemical testing medicine with vein,, calculate LD by the life or death of mouse after the week
50It the results are shown in following table
L D
50
Method is administered systemically
Test corpse or other object for laboratory examination and chemical testing I test corpse or other object for laboratory examination and chemical testing II
The balb/c intravenous injection
35mg/ky ＞150mg/kg
Test corpse or other object for laboratory examination and chemical testing II compares with test corpse or other object for laboratory examination and chemical testing I, and toxicity has reduced, and clearly illustrates that, chain length and toxicity have extremely confidential relation.
Embodiment below with reference to the preparation method of relevant nucleic acid derivative of the present invention illustrates in greater detail the present invention.
Figure 1 shows that the gel-filtration elution profile of the HPLC of nucleic acid derivative of the present invention.
Ordinate is represented the absorbancy in 260 millimicrons; Abscissa is represented tension force time and the bp corresponding with it.
Among Fig. 1, (1) representative test corpse or other object for laboratory examination and chemical testing I, (2) representative test corpse or other object for laboratory examination and chemical testing III, (3) representative test corpse or other object for laboratory examination and chemical testing IV, (4) representative test corpse or other object for laboratory examination and chemical testing II, (5) representative test corpse or other object for laboratory examination and chemical testing V, (6) representative test corpse or other object for laboratory examination and chemical testing VI.
Embodiment
(1) preparation of double chain acid derivative
Make commercially available polyC(S value for 6-12) and the polyI(S value be 6-12) be dissolved in the tris buffer that contains 50 mmoles, make it to be the concentration of 10-12 mg/ml.In water-bath, temperature is gently brought up to 68 ℃ from room temperature,, be placed into temperature and fall only room temperature after about 10 minutes in insulation under 68 ℃, preserve down at 4 ℃ again.
Frost drying obtains 1.2 gram white solids by the solution of last gained.
This material is above-mentioned test corpse or other object for laboratory examination and chemical testing I (the S value is 13-24).
In the preparation of double chain acid derivative, by using poly(C
12U) multipolymer substitutes polyC, according to above-mentioned identical method, can be prepared into polyIpoly(C
12, U) [test corpse or other object for laboratory examination and chemical testing III (the S value is 13-24)].In addition, by using poly(C
20S
4U) multipolymer substitutes polyC, according to above-mentioned identical method, can be prepared into polyIpoly(C
20, S
4U) [test corpse or other object for laboratory examination and chemical testing V(S value is 13-24)].
Above-mentioned method had become general method already, all can similarly modulate with polyIpolyC all nucleic acid derivatives listed in the above-mentioned table look-up.
(2) degraded
The examination corpse or other object for laboratory examination and chemical testing I of 1 gram by above-mentioned (1) gained is dissolved in 120 ml waters, wherein adds 5M30 mL of saline and 150 milliliters of methane amides again.Become uniform solution through vigorous stirring.After 80 ℃ make this reaction soln heating 8 hours,,, obtain 0.95 gram white solid through lyophilize to the abundant dialysis of water.
This solid is test corpse or other object for laboratory examination and chemical testing II (the S value is 8).
The test corpse or other object for laboratory examination and chemical testing III of 1 gram by above-mentioned (1) gained is dissolved in 120 ml waters, wherein adds 5M30 mL of saline and 150 milliliters of methane amides again.Become uniform solution through vigorous stirring.Make this reaction soln heating after 12 hours at 60 ℃,,, obtain 0.95 gram white solid through lyophilize to the abundant dialysis of water.
This solid is test corpse or other object for laboratory examination and chemical testing IV (the S value is 9).
The test corpse or other object for laboratory examination and chemical testing V of 1 gram by above-mentioned (1) gained is dissolved in 120 ml waters, wherein adds 5M30 mL of saline and 150 milliliters of methane amides again.Become uniform solution through vigorous stirring.Make this reaction soln heating after 12 hours at 60 ℃,,, obtain 0.95 gram white solid through lyophilize to the abundant dialysis of water.
This solid is test body VI (the S value is 9).
These test corpse or other object for laboratory examination and chemical testing are shown in Fig. 1 through the figure of the high performance liquid chromatography wash-out of gel filtration method.Adopt tsk gel G-DNA-PW gel-filtration carrier post resin, with containing 1 mmole EDTA and 0.3M brinish 0.05M hydrochloric acid-tris buffer (pH7.5) carries out wash-out.Elution speed is 0.2 ml/min, and each elution time writes on elution profile top (abscissa) respectively.With 260 millimicrons of detections of uv-absorbing ripple, ordinate zou is depicted as full scale 0.04 optical density(OD) (OD).Optical density in 1 milliliter.
Tag value shown in the abscissa bottom (bp) expression DNA base number.
The hydrolysis segment of this mark by the control enzyme of M13 phage DNA.
Test corpse or other object for laboratory examination and chemical testing I [among the figure (1)] is very similar with the elution profile of test corpse or other object for laboratory examination and chemical testing III [among the figure (2)] and test corpse or other object for laboratory examination and chemical testing V [among the figure (5)], all appears at and uses sample after 34 minutes.This is the clear area of post, and learns, the most base number of tool is more than 6000.
In addition, also learn through PD test corpse or other object for laboratory examination and chemical testing II [among the figure (4)], and the test corpse or other object for laboratory examination and chemical testing VI [among the figure (6)] molecular size distribution be peak value with maximum molecule number about 150 and about 600 respectively, be distributed in certain scope.
If, be consistent as unit by " base number " and " bp " in Fig. 1 with " base number " expression molecular size.
With polyIpolyC is example, and the example that is had the material of various molecular dimensions by reaction times that changes said degraded reaction and temperature of reaction acquisition is shown in following table.
Maximum distribution distributes
The temperature of reaction reaction times
(base number) (base number)
-0 hour-＞10000
80 ℃ of 24 hours 30 10-55
80 ℃ of 16 hours 100 50-800
60 ℃ of 8 hours 1000 200-6000
60 ℃ of 12 hours 600 50-5000
Above-mentioned degraded be to narrate with regard to double-stranded RNA, but according to identical operational condition, also can carry out degraded to single stranded RNA.
Claims (1)
1, the preparation method of nucleic acid derivative, it is characterized in that after annealing, carrying out degraded with the alkali qualification decomposition method of thermal decomposition method, RNA and the portion water solution of RNA lytic enzyme, from being to make double-stranded RNA the nucleic acid derivative of matrix with RNA, the molecule of maximum distribution in all molecular size distribution of base, show with the base numerical table, concentrate on 50-10, in 000 the scope.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16743/87 | 1987-07-03 | ||
| JP16743387 | 1987-07-03 |
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| Publication Number | Publication Date |
|---|---|
| CN1030423A CN1030423A (en) | 1989-01-18 |
| CN1024350C true CN1024350C (en) | 1994-04-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN88104038A Expired - Fee Related CN1024350C (en) | 1987-07-03 | 1988-06-29 | Nucleic acid derivatives |
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| KR (1) | KR890002225A (en) |
| CN (1) | CN1024350C (en) |
| AT (1) | AT397657B (en) |
| AU (1) | AU1864388A (en) |
| CH (1) | CH676122A5 (en) |
| DE (1) | DE3822405A1 (en) |
| DK (1) | DK368988A (en) |
| ES (1) | ES2010286A6 (en) |
| FI (1) | FI883162A (en) |
| FR (1) | FR2617486A1 (en) |
| GB (1) | GB2207432A (en) |
| HU (1) | HU199504B (en) |
| IL (1) | IL86863A0 (en) |
| IT (1) | IT1224503B (en) |
| NL (1) | NL8801663A (en) |
| NO (1) | NO882949L (en) |
| PT (1) | PT87902A (en) |
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| DE69029738T2 (en) * | 1989-10-11 | 1997-08-14 | Hem Pharma Corp | PROTECTION AGAINST SHOCK DUE TO DAMAGE FROM DOUBLE-STRANDED RNA'S |
| JPH05163160A (en) * | 1991-12-13 | 1993-06-29 | Snow Brand Milk Prod Co Ltd | Nutrient preparation for prevention and treatment of infectious disease caused by immune depression |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ194880A (en) * | 1979-09-17 | 1983-07-15 | Merck & Co Inc | Interferon-inducing composition containing modified polyriboinosinic-polyribocytidylic acid |
-
1988
- 1988-06-26 IL IL86863A patent/IL86863A0/en unknown
- 1988-06-27 GB GB08815263A patent/GB2207432A/en not_active Withdrawn
- 1988-06-29 CN CN88104038A patent/CN1024350C/en not_active Expired - Fee Related
- 1988-06-29 CH CH2465/88A patent/CH676122A5/fr not_active IP Right Cessation
- 1988-06-30 NL NL8801663A patent/NL8801663A/en not_active Application Discontinuation
- 1988-06-30 ZA ZA884700A patent/ZA884700B/en unknown
- 1988-07-01 DK DK368988A patent/DK368988A/en not_active Application Discontinuation
- 1988-07-01 HU HU883460A patent/HU199504B/en not_active IP Right Cessation
- 1988-07-01 SE SE8802479A patent/SE8802479L/en not_active Application Discontinuation
- 1988-07-01 ES ES8802077A patent/ES2010286A6/en not_active Expired
- 1988-07-01 FI FI883162A patent/FI883162A/en not_active IP Right Cessation
- 1988-07-01 AU AU18643/88A patent/AU1864388A/en not_active Abandoned
- 1988-07-01 IT IT8848146A patent/IT1224503B/en active
- 1988-07-01 PT PT87902A patent/PT87902A/en unknown
- 1988-07-01 NO NO88882949A patent/NO882949L/en unknown
- 1988-07-01 FR FR8808937A patent/FR2617486A1/en active Pending
- 1988-07-01 DE DE3822405A patent/DE3822405A1/en not_active Withdrawn
- 1988-07-02 KR KR1019880008257A patent/KR890002225A/en not_active Application Discontinuation
- 1988-07-04 AT AT0172788A patent/AT397657B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| GB8815263D0 (en) | 1988-08-03 |
| SE8802479L (en) | 1989-01-04 |
| NO882949D0 (en) | 1988-07-01 |
| ES2010286A6 (en) | 1989-11-01 |
| DK368988A (en) | 1989-01-04 |
| ZA884700B (en) | 1989-03-29 |
| ATA172788A (en) | 1993-10-15 |
| IT8848146A0 (en) | 1988-07-01 |
| DE3822405A1 (en) | 1989-01-12 |
| KR890002225A (en) | 1989-04-10 |
| DK368988D0 (en) | 1988-07-01 |
| PT87902A (en) | 1989-06-30 |
| AU1864388A (en) | 1989-04-27 |
| IL86863A0 (en) | 1988-11-30 |
| FI883162A (en) | 1989-01-04 |
| HUT48271A (en) | 1989-05-29 |
| IT1224503B (en) | 1990-10-04 |
| NO882949L (en) | 1989-01-04 |
| HU199504B (en) | 1990-02-28 |
| CN1030423A (en) | 1989-01-18 |
| GB2207432A (en) | 1989-02-01 |
| AT397657B (en) | 1994-06-27 |
| SE8802479D0 (en) | 1988-07-01 |
| FI883162A0 (en) | 1988-07-01 |
| CH676122A5 (en) | 1990-12-14 |
| FR2617486A1 (en) | 1989-01-06 |
| NL8801663A (en) | 1989-02-01 |
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