JPH03500169A - Chemical production method and biological use of labeled oligonucleotides - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Chemical production method and biological use of labeled oligonucleotides The present invention provides a method for chemically synthesizing labeled oligonucleotides and a method for chemically synthesizing labeled oligonucleotides. related to the biological use of froid as a cryoprobe (sonde froid). Ru.

The invention more particularly relates to known DNA fragments or RN^ fragments. DNA fragments that are complementary to Involved in the synthesis of fragments.

Detection of gene sequences uses high-temperature probes labeled with radioactive isotopes. A probe called ``chaude'' has been used for a long time. However, Autora Although the method of geography is quite sensitive, it is time-consuming, expensive, and dangerous. The disadvantage is that it deals with radioactive materials.

Therefore, over the past five years, a new type of probe called a cryogenic probe that does not use radioactive elements has been developed. Research on methods for detecting nucleic acid sequences using microtubes has gradually become of great interest.

For example, oligonucleotide probes can be synthesized with biotinylated nucleotide triphosphates (n ucl! Enzyme labeling with otide triphosphate biotiny16) There are already a number of kits available on the market for this purpose. These biotinylated proteins detection of peroxidase, alkaline phosphatase or galactose Enzyme systems such as sidases, or colorimetry by fluorescence or chemiluminescence The system is activated by biotin, a protein that has an extremely high affinity for biotin. It is carried out by binding to trebutavidin.

For extremely precise detection, probes of defined composition are regulatively labeled at defined locations. It is believed that the use of

The inventors have continued to study this and have developed a method to improve the synthesis of cryogenic probes. uttered. The gist of this method is that when constructing an oligonucleotide sequence, It consists in incorporating modified nucleotides in place of the bases normally present in it.

Through these modified bases, while retaining the same hydrogen bonding potential as regular bases, Then, a substituted strand is introduced and a label is immobilized on this strand.

Therefore, the present invention provides a method for producing oligonucleotides by chemical synthesis. , provides a method by which a labeling unit can be incorporated in one or more sites in a defined manner. .

In the main text and claims of this specification, the units of the sequence that are the subject of the synthetic method of the present invention (motif) are identified by the names of the bases of these units.

These units may be nucleosides and nucleotides, including ribose and/or 2 - May contain deoxyribose as sugar.

Although the method of the present invention is generally used for the synthesis of oligodeoxynucleotides, Of course, it can also be used for the synthesis of corresponding oligoribonucleotides.

The chemical synthesis method of the present invention is characterized by comprising the following steps. - When chemically synthesizing the oligonucleotide sequence to be constructed using the solid phase method, Instead of one or more cytosines that should be present in the sequence, position 4 is an activating group. incorporating one or more substituted thymines, respectively; - The constructed oligonucleotide sequence has the formula R-^-R.

[In the formula, R and Ro are the same and represent -NH2 group, -SH group or 10FI group; or R and Ro are different from each other and one is -NF1. group, SO group or 10 H group, and the other represents -Nu-X group, S-X group or 10-X group, provided that X is a group that can function as a label for the molecule, A is an alkyl chain having 4 to 20 carbon atoms, if necessary O, N or S. containing one or more active 5-methyl-cytosine with a substituted chain at the 4-position is formed in place of thymine, and the compound When R-^-R゛ does not contain a labeling group, the -methyl-cytosine substituted chain is Introducing a labeling group.

Replacement of cytidine with activated thymidine allows for localization of the sequences constituting the unit to be labeled. You will be able to place it anywhere. Therefore, fully determined structures and substitutions A house acid having the following properties is formed. In contrast, labeled oligos produced by conventional enzymatic synthesis In the production of nucleotides, nucleotides are incorporated unregulated from the primer.

The method of the present invention also provides great flexibility in replacing the incorporated units with chains of various structures. immobilization of various labels is possible and therefore a single activation sequence. Multiple probes can be obtained, each with a different strand or a different label.

The synthesis method of the present invention further provides an origin in which the 5° terminal OH and the 3° terminal OB remain free. It also has the advantage that oligonucleotides can be obtained. Such oligonucleotides can be inserted into a biological vector, for example.

Another advantage of the method of the invention is that a large number of DN^ fragments can be prepared quickly and at low cost. The point is that automation of manufacturing is possible.

The step of incorporating an activated thymine unit at position 4 involves converting the oligonucleotide sequence into During construction, this arrangement is carried out by placing the units of the desired arrangement on a final substrate fixed to a solid support. The first nucleotide or nucleoside is linked sequentially, with a cytosine unit at the 4th position. by the desired substitution with thymine units activated with .

The term "solid support" refers to at least one specific support capable of containing nucleic acids in solution. There are many supports that can be used, referring to any support that can remain insoluble in a constant medium. Ru. Specific examples include organic polymers of silica, glass beads, and the like. this These supports are used in reactions where separate molecules are immobilized directly or through links. It is advantageous to carry out a prior (functionalization) treatment in order to impart functional groups that can be used. this kind of Some supports are commercially available. A specific example is polyacrylic morpholide. For example, it is commercially available under the trade name ENZACRYL K-2. Things can be mentioned. Polyacrylamide, polystyrene or cellulose are also of this type. 6. Can be used as a support for Linking of nucleosides or nucleotides is done unit by unit or by nucleotide group. , the construction of the sequence is carried out using known solid-phase methods that elongate the nucleotide values, especially phosphor Reester method or phosphoramidites method and It is advantageous to use the method called Methods of this kind are e.g. It is described in documents (1) to (6) in the reference list.

The condensation is carried out in the presence of a binder, the binder is preferably used in excess, the binding rate , it appears that condensation of activated thymidine units does not significantly reduce these units. I understand.

A method of activating the 4th position of thymine is to add triazole or nitrotin to this apex. Immobilizing heterocyclic derivatives of the type lyazole or tetrazole, imidazole There is a way.

One of the preferred activating functional groups that facilitates the formation of desired activated thymine consists of a 1,2,4-)riadyl group.

The procedure for fixing this group to thymine is described, for example, in References (7), (8), or (9). Perform as described.

Other methods for activation of thymine apex 4 may also be used, for example as described in Refs. (10) and (1). It can also be carried out using an acid chloride according to the method described in 1).

In the second step of the method of the invention, one synthesized oligonucleotide sequence, i.e. The above sequence containing an activated thymine unit at the desired site is combined with a compound of formula R-^-R゛. Make it react.

Advantageously, diaminoalkanes, glycols, such as α,ω-amino alcohols using difunctional compounds.

Suitable diaminoalkanes include ethylene-diamine, 1-6-diamithexane , 1-10-diaminodecane or spermine.

Replacement of activated thymine with diaminoalkanes in organic solvents such as pyridine , preferably with an excess of amine.

The oligonucleotide sequence formed, i.e. the sequence with methyl-cytosine substitution units. The columns are separated from the supports that fixedly supported them during construction and removed from the brackets. Unlock. Advantageously, the base and phosphate used in the solid phase synthesis Using common methods for removing protecting groups from.

More specifically, the phosphate group can be used, for example, with THCP^0 and concentrated aqueous ammonia. Unblock using in sequence.

Operating at room temperature, TM is allowed to react with P^0 for approximately 14 hours. Concentrated ammonia water When left to act for about 2 to 7 hours, especially 5 hours at a temperature above 50°C, preferably about 50°C. It's advantageous.

The DNA fragments are then purified by passing them through one or more ion exchange columns and their Afterwards, it is deprotected using, for example, acetic acid, and preferably purified again.

If the substituted steel does not contain a labeling group, one or more chains substituted in the 4-position oligonucleotide sequences containing several methyl-cytosine units as the label for the molecule. reaction with a group that can function as

Commonly used groups include peptides, habuten, colored or fluorescent groups, For example rhodamine, fluorescein or dansyl, or phosphatase Examples include enzymes such as peroxidase.

Preferred labels consist of peptides and oligonucleotides containing substituted steel at the amino end group. Combines with chido.

More specifically, activated forms of biotin, such as biotinyl-N-hydroxy Used in the form of succinimide ester.

Biotinylation of aminated oligonucleotides is carried out in an organic solvent of the DMF type. The activated form of biotin acts on these oligonucleotides in basic medium. Preferably, this is carried out by

The oligonucleotide is therefore more particularly at a pH of about 8-10, preferably 9. Dissolve in buffer.

This reaction is advantageously carried out using an excess of activated biotin.

The ratio of oligonucleotide concentration to activated biotin concentration should be approximately 10-'/1. The reaction temperature is below room temperature and can vary from about 0°C to 25°C. reaction mixture Maintain at about room temperature for 3-8 hours, preferably about 6 hours, then 8-20 hours. Good results when maintained at about 0°C for an hour, preferably about 12-14 hours. is obtained.

Labeled DNA fragments produced by the method of the present invention can be used for biological purposes. Refine it as follows.

Removal of the salts formed during the synthesis is performed using one or more ion exchange resin columns, SI! Advantageously, it is carried out using a phadex II column or a Biogel column.

Other features and advantages of the invention include the detection of cloned genes for antithrombin III; The following non-limiting practices related to the production of biotinylated DNA probes used in Let's make it clear with an example.

Abbreviations used in these examples have the following meanings: tT: 2°-deoxythymidine substituted at the 4-position with 1.2.4-) lyazole, DMTr Nidimethoxytrityl, is the following formula Thymidine substituted with the group p shown by, TPSNT: 2,4.6-) sopropyrubenzenesulfonyl-3-nitro 1.2.4-) riazole, TMCP^0: pyridine-aldoximate-tetramethylguanidine, DCCI rainbow cyclohexyl carbodiimide, DCll rainbow cyclohexyl urea, BNHS: biotinylated hydroxysuccinimide, TEAB: bicarbonate ethyl ammonium.

Example 1: cDNA DNA fragment of human antithrombin III (or AT II) Synthesis of complementary biotinylated probes.

In an experiment previously conducted by the present inventors, a probe labeled with an enzyme at the 5° position with 32p was used. cDN^ can be isolated from ATIII inserted into the plasmid. Ta.

These cDN^ were sequenced and the corresponding mRN^ sequences were described.

Based on this known sequence, the following three biotinylated probes were phosphotriated. Synthesized manually by Stell solid phase method: C★=5-methyl, 2°-deoxycytidine; Biot=biotinyl; B=4th place (Nt(CH2)+.-NR-Biot) of C★.

This synthesis involves three steps: 1) Chemistry of the triazolized DNA sequence. synthesis, 2) substitution of the 1.2.4-) riazolyl group of tT with decanediamine, 3) Biotinylation of aminated DN^ sequences.

In the synthesis of probe 1, these steps are carried out as follows, and the support is e.g. Resin-like polyacrylamide sold under the trademark Enzacryl Km type of resin used. The thymidine substitution rate was 1301 mol per 1 g of support. be.

The substitution rate of this support, that is, the amount of initial nucleoside immobilized on the support, is determined by acid treatment. C)12c12/CH,011 liberated by chemical reaction (2% benzenesulfonic acid) : Determination of DMT” cation in 70-30 mixture by 507 nm UV I evaluate it.

- Formula I below Composite the units shown by: Completely protected thymidine D M T r T [F] 4g (5.07 mmol) Evaporate with 1.4 g (20.3 mmol of 1.2.4-) lyazole in pyridine. Process.

Dissolve the residue in distilled pyridine 25-1 and place the flask in a water bath.

2.5 g (10.14 nmol) of 0-chlorophenylphosphodichlororesort The reaction medium is stirred for 10 minutes at 0° C. and then kept at room temperature for 48 hours.

After evaporation to dryness, the residue was taken up in CH2Cl, washed with concentrated NaFICO2, and Rinse with H,0 until pH p)I, dry with NaSO, filter, then evaporate. Process.

The product obtained was dissolved in a minimum amount of silver in CH, CL and repeatedly dissolved in petroleum ether for 3 Precipitate again.

Silica column and S! Purify DMTr several times with a phadex LEI20e column. (t) Then, 1.7 g of ℃ is obtained (yield 40%).

Thin layer chromatograph 4 (CHzClz, Neon:95-5) Rf=0.4 Synthesis of 8°-triazolized DNA sequences riMTr 120 mg, i.e. 15.5 pmol of support T was mixed with 2% benzenesulfonic acid ( Detritylation with ^BS).

MTr 〇 (6eq,) to nucleoside C with triethylamine-pyridine-〇, 0:1 -3-1 mixture (TPE) for 20 minutes, then 1 The functional group 5′ of thymidine was activated with 8 eq of TPSNT and fixed on the support. Connect to OB.

The binding rate of the cationic DMT” released by treatment with benzenesulfonic acid was determined by Evaluation was made by quantification using 507 nm UV.

The binding rate for this first binding is 100%.

The binding rate of nucleosides is 59% for DMTrCAT, 65% for DHTrCT^, DMTrCTC is 86% and DHTrTTC is 93%.

2) tT's 124-)1 sol 1 ru! The second step is diamine. 61 g (7,911 + 1 ol) of resin was mixed with 120 ffiM decanediamine pyridine. Suspend in 610 ul (10 eq) of solution.

The reaction medium is maintained at room temperature for more than two days. After dehydrating the resin, rinse with pyridine. Completely remove residual diamine.

This aminated sequence is excised from the support using methods commonly used in solid-phase synthesis. Unblock processing is performed by

Water-dioxane: 1.1 ml of a solution of P^0 dissolved in 187H in a 171 mixture (1.1 mmol) In addition, unblock the oligonucleotide. Use 10 eq of TMCP^0 for each phosphate to be extracted. That is, the above D For NA sequences, 150 eq of IMTNII; Ph3 is used.

After standing overnight at room temperature, the reaction medium is evaporated to dryness.

Add 31 ml of concentrated aqueous ammonia (20%) and heat to 50° C. for 5 hours.

The resin is separated by filtration, rinsed, and the V solution is concentrated, then eluted with H2O. The resulting product was lyophilized and separated through a Dowex NFI4” resin column. triethylammonium acetate 10-2 using a Nucleosile column for Change the acetonitrile in M buffer from 5 to 25% or from 5 to 50% in 20 minutes. Purify in a DMTr step by HPLC with gradient elution.

Perform detritylation of the sequence with 80% acetic acid in H20 for 20 min at room temperature, evaporate the acid. and then removed by two or three coevaporations with toluene.

The residue is dissolved in l'+20, washed with ethyl ether and concentrated.

The fully deprotected product was purified by HPLC on a preparative Nuc1Mosil’ column. and then tested on an analytical Nue I6os iI' column.

3) Amino DNA vinyl -Synthesis of biotinyl, tohydroxysuccinimide (B, N, H, S) esters Growth The protocol used was published by BAYER and WILC) IEK. Also described in Enzymol, (1974), 34, 265-7 It is. Biotin 250+eg (1ms+) in 3ml DMF passed through alumina ol) and 150+eg (1,3 eq,) of N-hydroxyl was added to this solution. Add succinimide and 412w1g (2eQ,) of DCCI.

The reaction medium has the form of a white suspension. Heat this to 50℃ for 30 minutes, then Stir for 20 h at room temperature. Filter the dicyclohexylurea precipitate formed. , store the furnace solution at 0° C. for about 14 hours. The insoluble part of the DCU formed is removed by filtration. and concentrate the P solution. BN) Is is recrystallized with a large amount of isopropanol become

The product is 2f1w11! can get. This is equivalent to an esterification rate of 77.6% .

- Amination probe of biotinylated IDO according to formula 3 below , mixed with 20 hl of n2o and 10 hl of NaHCOs buffer y (pH 9) dissolve in substances. 1 mg (2,93 umol) of biotinyl, N-hydroxy Add succinimide ester to 40 μm DMF. 6 hours at room temperature and 0 After standing overnight at °C, DNF was evaporated and the resulting product was dissolved in 0.05M TEAB. Pass through SL'phadex G106 column eluted with buffer.

The probe is then used for analysis or separation with NuclI! oisl’! 'it in column, Purify by C.

After purification, transfer the oligonucleotide to a Dowex 50 wN) 14” e cartridge. ammonium in the ammonium form and then for analysis with Nucll! with oil column Test by FIPLC.

The biotinylation rate is 21%-23%. 5’Biot=3.18DO0 Example 2 Probes 2 and 3 are manufactured by performing the same operations as above.

Probe 2: 3' NFIz=5.23DO3' Biot; 1.1240 0 Efficiency = 21.5% Probe 3: 5° 3’ NHZ: IDOS’ 3’ Bi ot=0.228 DO efficiency=22.8% Example 3: Biotinylated probe dots Using the above-mentioned probe, two types commercially available from BRL Co., Ltd. The performance of the biotinylated DNA detection kit was investigated. These kits are as follows It is.

1. Nucleic acid detection product, ver. 8239S^.

2, “ENE on Blu”, ref, 8279 S^.

In these two enzyme detection methods, biotin is known as avidin or streptavidin. It was developed based on the fact that it has an extremely high affinity for proteins. It is something that

Initial color development was performed on dot plots of biotinylated probes.

Probes were added to nitrocellulose at various concentrations (Loop import. 1-0.5 °C mol). and then fixed by passing in an oven at 80° C. for 1 hour.

0, IM) Lis-HCI (pH 7,5); 3% in 0.158 NaCl buffer Filters are blocked with bovine serum albumin for 2 hours at 55°C.

In the detection kit used, color development occurs in two or three stages.

The first commercially available detection kit 1 follows a three-step protocol.

1, combination with streptavidin, 2. Binding with biotinylated alkaline phosphate; 3. Nitro blue tetrazo (NST) and 5-bromo-4-chloro-3-indonylphosphate ( Colored enzyme reaction with BCIP).

Because streptavidin has the property of binding four biotin molecules, the amplification system You can get Gunar.

In the second kit 2, color development occurs in two steps.

1. Streptavidin-alkaline phosphatase binding; 2. Colored enzyme reaction with NBT and BCIP.

Visually evaluate the intensity of color development.

Each color of synthetic biotinylated 70-b was biotinylated with an enzyme and various The color development of IIIRL DNA placed on nitrocellulose was compared with that of The sensitivity of each DNA detection is determined by this.

For biotinylated probes placed on nitrocellulose, 50f-01 DNA was colored.

-Manufacture of nitrocellulose filters ^Target plasmid DN^ with 1.6kb inserted corresponding to cDN^ of III and pBR322 control DN^ were placed on nitrocellulose.

These DNAs, which had been linearized in advance with the restriction enzyme old ndlll, were diluted 2-fold to 1 By heating to 100°C for 10 minutes to obtain a concentration range of 0 to 0.01,11. denatured and added 0.58 Na0ff over 20 minutes at laboratory temperature, then 0.38) IcI in water, 0.3N NaCl, 0, 15M Tris FI CI (pH 8), neutralized with 0.1H sodium citrate and each dilution 7.5.1 were placed on the cellulose. Air dry the filter for 1 hour, then It was dried for 2 hours in an oven at C.

- bibrid formation The nitrocellulose filter was filtered with 6xNET (1xNET=0.15M NaCl, 0.05M Tris tlcI (pH 7,5), O, OOIM EDT^) , 5x Denhardt's (100 Denhardt's: 0.02% bovine blood Clear albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 5 % dextran sulfate, 0.1% SDS and 1100p/+sl sonicated solution. Semen DNA was used for pre-bibrid formation at 37°C for 2 hours. bibri Code formation is performed by polynucleotide kinase at 5° [32pl-labeled probe]. or biotinylated probe at 100 np-1 per molecule. /ml in the same solution at 37°C for 20 hours. Store these filters at 4°C. Washed 3 times for 15 minutes in 6SSC, once for 5 minutes at 40°C, then washed in the oven. It was subjected to autoradiography or color reaction.

In the method of the present invention, an oligonucleotide that can be used as a probe to detect a complementary sequence of a nucleic acid is used. Gonucleotides can be produced chemically. By the way, streptavidin -The detection limit of the alkaline phosphatase kit is approximately 15 fentomoles. The probes of the present invention may also contain complementary molecules contained in a solution that contacts molecules on a support. It is also extremely useful when separating target polynucleotides. Such detection or The separation is carried out under conditions such that reciprocal hybridization occurs. Proteins that interact with DNA fragments to form heavy chains You can also refine the quality.

The inventors found that when a labeling group was introduced on the 5° side, this group was placed on the 3° side. We discovered that a more stable duplex can be obtained during hybridization.

11e1 <1>) R. Koster et al., Tetrahedron, 40, 103. t9 84゜(2) H, Caruthers et al., Tetrahedron Lett ers, 21,719.1980゜(3) M, J, Galt et al., Nuclei c Ac1ds Re5search, 10.6243.1982゜(4) K, , Itakura et al., Hue! eie Ac1ds Re5arch, 8,5 507, 1980゜(5)^, Rosenthal et al., Tetrahedron Letters, 24,1691,1983゜<6)　C0, Brush et al., Nucleic Ac1ds Re5earch, 10,6295,198 2゜<7　)　C, B, Reese et al., Tetrahedron Letter s, 21,2265,1980゜<8) I'1. L, Sung, J, Org. Chem, 47, 3623, 1982゜(9) C, B, Reese et al., Nu cleic Ac1cls Re5earch, SymposiumSerie s7. Kosterli, p, 5゜(10) C, B, Reesefl! t , Tetrahedron Letters, 22,4755, 1981゜(1 1) R, ^, Jones et al., Tetrahedron Letters, 40 ,3.1984゜International Investigation Report S^　24077

Claims (10)

[Claims] 1. A method for chemically producing a labeled oligonucleotide, the method comprising: - When chemically synthesizing the oligonucleotide sequence to be constructed using the solid phase method, Instead of the cytosine that would be present in the column, one or more activating groups are substituted at position 4. incorporates multiple thymines, - The constructed oligonucleotide sequence has the formula R-A-R' [wherein, R and R' are the same and represent -HH2 group, -SH group or -OH group; , or R and R' are different from each other and one is -NH2 group, -SH group or -OH group, and the other represents a -NH-X group, -S-X group, or -O-X group, provided that is a group that can function as a label for the molecule, A is an alkyl chain having 4 to 20 carbon atoms, optionally containing O, N or S. containing one or more active 5-methyl-cytosine with a substituted chain at the 4-position is formed in place of thymine, and the compound When R-A-R' does not contain a labeling group, -methyl-cytosine is substituted with A method comprising the step of introducing a labeling group. 2. The operation of activating the 4-position of thymine is useful for triazoles, especially 1,2,4-triazoles. zole, nitro-triazole or tetrazole, imidazole type A claim characterized in that it includes an operation of fixing a heterocyclic derivative or an acid chloride at the apex. The method described in claim 1. 3. Attachment of nucleosides or nucleotides to form a chain to a solid support After fixation of one unit of The method according to claim 1 or 2, wherein: 4. The compound of formula R-A-R' is a diaminoalkane, especially 1-10-diaminodecane. , glycol or α,ω-amino alcohol. 3. The method according to any one of Item 3. 5. Replacement of activated thymine with diaminoalkanes in organic solvents such as pyridine According to claim 4, preferably carried out using an excess amount of amine. the method of. 6. Supporting oligonucleotides containing a methyl-cytosine unit with a substituted strand at position 4 Separate from the body, then unblock, and subject to one or more types of refinement 6. A method according to any one of claims 1 to 5, characterized in that: 7. oligonucleotide sequences containing one or more substituted methyl-cytosine units. , characterized in that it is reacted with one or more groups capable of functioning as a label for the molecule. 7. A method according to any one of claims 1 to 6. 8. The labeling group can be a peptide, a hapten, a colored group or a fluorescent group, e.g. damin, fluorescein or dansyl, or alkaline phosphatase or peroxidase. How to put it on. 9. 8. The method according to claim 7, wherein the labeling group is biotin. 10. An oligonucleotide produced by the method according to any one of claims 1 to 9. Use of DNA as a low-temperature probe for detecting complementary sequences of nucleic acids.