JPS6157596A - Method of labeling polydeoxyribonucleotide or polyribonucleotide with an enzyme - Google Patents

Abstract

PURPOSE:DNA or RNA which has phosphonic acid bondage bearing a functional group as a part of phosphate bonds is allowed to react with a covalently bondable enzyme for labelling at the functional group whereby the titled compound labelled with an enzyme which is used for clinically diagnostic purposes with high detection sensitivity is obtained. CONSTITUTION:The objective compound labelled with an enzyme is obtained by reaction of a polydeoxyribonucleotide or polyribonucleotide which has at least one of phosphoric bond bearing a functional group of the formula (R is 1-20C aliphatic hydrocarbon, aromatic hydrocarbon; X is a functional group which may be protected) among its phosphate bonds with a covalently bondable enzyme for labelling. The functional group linking to the phosphonic bond is, e.g., thiol or amino.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides that at least one phosphoric acid bond has the formula CI) op-0-CI) -X (wherein B has 1 to 200 carbon atoms) aliphatic hydrocarbons or aromatic hydrocarbons, which may have branched chains; The present invention relates to a method for enzymatically labeling a polynucleotide (hereinafter collectively referred to as a polynucleotide), which comprises reacting a covalent enzyme labeling compound with a phosphonic acid-bonded functional group of a polylysed nucleotide (hereinafter collectively referred to as a polynucleotide), and which uses the enzyme obtained by the present invention. A labeled polynucleotide having a specific base sequence is used to identify and extract a target gene, and can be widely used in fields such as genetic engineering, clinical diagnosis, and food.

(Background of the Invention) As genetic engineering research has become more active in recent years, polynucleotides having specific base sequences have come to be used to detect genes necessary for producing useful substances.

For example, in the field of clinical diagnosis, immunological and biological methods are currently used; however, although the test times for the former are generally short (a few minutes), they often give ambiguous results due to cross-reactivity and interference effects. , and the latter requires a long time for culture.
- It has disadvantages such as:

On the other hand, the polynucleotide method, which diagnoses diseases by detecting genes, generally takes several hours, but it has very high detection sensitivity and specificity.
It also has the advantage of having extremely small errors and can detect not only infectious diseases but also latent viruses that cannot be detected by immunological methods, so there is a demand for the development of polynucleotides suitable for this purpose.

(Prior Art) When detecting a target gene by a hybridization method using a polynucleotide, it is necessary to label the polynucleotide. Drifting can be done using methods using radioactive isotopes and optical labeling methods ζ
This is broadly classified. Currently, the method using the radioactive isotope sip is the most used, and Japanese Patent Application Laid-Open No. 170496/1983 describes the use of ATP (γ-5tp) and a kinase to attach a phosphate labeled with sxp to the 5'-terminal group of a polynucleotide. A method is disclosed in which a group is introduced, the film is exposed to β-rays emitted from the lap, and the presence of the target gene is detected by the black spots. Although labeling with radioisotopes is superior in terms of sensitivity, its use requires (1) skill in handling; 01) strict legal regulations and requires special facilities and measuring equipment; (fil) There is a health problem; (iv) There is a problem with disposal after use; (V) Pre-purchase is made according to the half-life, so the experiment date is around that time. There are some drawbacks, such as being restricted by In response to this, an optical labeling method was proposed as a g recognition method that does not use radioactive isotopes, and was published in JP-A-58-23795 and JP-A-58-4.
Publication No. 0099 discloses a method using chemiluminescence, bioluminescence, and fluorescence. However, as a photolabeling method, d-O-triphosphate, which has biotinylated at the C-5 position as a thymidine or uridine analogue, is incorporated into polynucleotides by enzymatic nick translation. Method for labeling and detecting target genes [Proceedings o (Nati
onal Academy of8clenceof
the Unlted 8tate of Amerl
ca, Volume 80.

No. 4045 (1983)] has been put into practical use.

(Problems to be Solved by the Invention) Nucleic acids are composed of three components: nucleobases, sugars, and phosphoric acids, and each component can be photolabeled, but in this case, it is possible to label the polynucleotide as a target. It is necessary to introduce a labeling compound so as not to impair the ability to hybridize with the gene. However, when photolabeling a nucleobase moiety, it is possible to introduce a labeling compound into the C-5 position for zerimidine bases, but for purine bases, it is stereochemically possible to introduce a labeling compound at the C-5 position. -a position and N-7
position only, and when introduced at the C-8 position, the base and sugar are 8yn
In general, if the double parallel is right-handed to adopt a right-handed doublet conformation, the height-lifting ability is lost, and when introduced at the N-7 position, the nitrogen becomes quaternary and forms a glycosidic bond between the sugar and the base. It is extremely difficult to label all purine bases because the so-called depurination reaction, in which the purine bases are cut off, is likely to occur. Therefore, photolabeling of the nucleic acid base portion is effective only in the case of rimidine bases, and when the base sequence of the -rinucleotide consists of only purine bases, it is difficult to photolabel. Next, when photolabeling sugar moieties, only the hydroxyl groups at both ends can be used with polydeoxynucleotides, and although theoretically the 2'-position hydroxyl group can also be used with polylysate nucleotides, it is necessary to photolabel them at this position. When a molecule as large as a compound is introduced, it does not affect hybrid formation, and as with 4-lideoxylyse nucleotides, only the hydroxyl groups at both ends can be used, resulting in a decrease in detection sensitivity.

(Means for Solving the Problems) The present inventors conducted various studies to improve the detection sensitivity of polynucleotides labeled by optical labeling, and as a result,
We have discovered that by introducing a photolabel compound into the phosphate moiety of a nucleic acid component, it is possible to introduce any number of photolabel compounds into any position of a polynucleotide;
By selecting a phosphonic acid bond having a functional group instead of a phosphoric acid bond and reacting the functional group of this phosphonic acid bond with a photolabeling compound to photolabel the phosphonic acid bond,
(a) The removal of the labeled compound or the cleavage of the internucleotide bond in the deprotection step after polynucleotide synthesis by the synthetic method, which occurs when photolabeling the phosphate bond, (
b) It also solves the problem of stereochemical inconsistency of the phosphorus atoms in the obtained photolabeled polynucleotide due to the chirality of the phosphorus atoms, and also has a covalent bond that specifically reacts with functional groups as a photolabeled compound. The present invention was completed by discovering that the intended purpose could be achieved by using an enzyme-labeled compound.

(Enzyme labeling of polynucleotides) Enzyme labeling of polynucleotides of the present invention involves replacing the phosphate bonds of polynucleotides with phospho/acid bonds having a functional group, and reacting this functional group with a covalent enzyme labeling compound. Enzyme labeling is achieved by vomiting.

In the reaction between phosphonic acid and an enzyme-labeled compound, a functional group that specifically reacts with the enzyme-labeled compound is bonded to a phosphorus atom by sandwiching an aliphatic hydrocarbon or an aromatic hydrocarbon, which may have a branched chain. After that, this functional group is reacted with an enzyme labeling compound. The functional group to be removed is usually a thiol group or an amino group, but is not limited to these as long as it is a functional group that specifically reacts with the enzyme labeling compound. Since these functional groups may cause side reactions during the synthesis of nucleotides, it is desirable to protect them with a protecting group, and the protecting groups are removed at the same time as the protecting groups for the base and hydroxyl groups of the nucleotide in the final deprotection step. It is fine as long as it can be done.

The enzyme labeling compound used in the present invention is a functional group having a specific covalent bond with a functional group having a phosphonic acid bond at one end;
That is, a crosslinking agent that has a maleimide group or a dithio group and a functional group that reacts with the amino group or mercapto group in the enzyme at the other end, and an enzyme that has an amine group or mercapto group, such as galactosidase, alkaline phosphatase, glucose oxidase, and peroxidase. , luciferase, catalase, etc., but the combination of enzyme and crosslinking agent is not limited to these.

Enzymatic labeling of polynucleotides can further improve detection sensitivity by not only enzymatically labeling both ends of the polynucleotide but also enzymatically labeling the internucleotide bonding region.

Enzyme labeling methods include synthesizing a polynucleotide having a phosphonic acid bond at any position and reacting it with an enzyme labeling compound to label it with an enzyme, and reacting a phosphonic acid with an enzyme labeling compound. The resulting enzyme-labeled phosphonic acid group may be introduced and enzyme-labeled using a method similar to that used for synthesizing polynucleotides using ordinary phosphate groups. It is necessary that the transformed polynucleotide is stereochemically hybridizable with the target gene.

The synthesis of the phosphonic acid used in the present invention was carried out by Houb
en Wegls Methoden der Org
anischenChemie, E, Mulle
r, Editor, Mol, XL/1.

2, Georg Thieme Verlog,
8tuttgart.

(1963-64), Arbuz
ov) Synthetic methods using reactions [Journal of the Society of Synthetic Organic Chemistry,
Vol. 39, p. 918 (1981)] has few side reactions and a good yield.

The polynucleotide in the present invention consists of a nucleoside (compound ■ of reaction formula [l]) and a phosphonic acid having a functional group (
A phosphonated monomer ( Compound (1) of reaction formula [I] is obtained. The monomer represented by compound ■ is a nucleoside with an unprotected 5'-hydroxyl group (
Compound (2) of reaction formula CI) is condensed in the presence of a condensing agent to form a dimer (compound (2) of reaction formula [I]). In compound ①, 几3 is different from the protecting groups of X, R1, R'' and is preferably a protecting group that can be selectively removed.
It may be a protecting group that can be removed under the same conditions as the protecting group of
Further, depending on the case, a phosphoric acid group having a protective group may be used.

The dimeric phosphonic acid phosphorus atom shown in compound (■) has chirality because all the groups bonded to it are different, and it can be expressed as a mixture of two diastereomers, the R-form and the 8-form, using the absolute representation method. However, these two diastereomers can be separated by silica gel chromatography to form a stereochemically single dimer. When R1 of the dimer represented by compound ■ is a phosphoric acid group having a protecting group,
Since there are two phosphorus atoms with chirality and four diastereomers are generated, making it difficult to separate stereoisomers, 几3 is a dimer with a free 3'-terminal hydroxyl group (reaction formula [ A protecting group that can be induced in compound (I)), i.e., X
, R1, R+'' and can be selectively removed, or a protecting group that can be removed under the same conditions as the protecting group for R1. In the latter case, 3'
After making the hydroxyl groups at both terminals of dar into a free form, only the terminal hydroxyl groups of dar are reprotected to form a dimer represented by compound (2). Compound (1) is converted into a dimer (compound (2) of reaction formula (I)) by phosphorylating the 3'-terminal hydroxyl group. Compound (2) can also be derived from compound (2) in which R8 is a phosphoric acid group with a protecting group, and in this case, the chirality of the 3'-terminal phosphoric acid group of compound (2) disappears, resulting in two diastereomers. It can be separated into 8-form and 8-form by silica gel or reverse phase silica gel chromatography.

Reaction formula CI) ■ ■ ■ ■ ■ (In reaction formula CII, B is a base residue that may have a protecting group, and moiety is a hydrogen atom or a protecting group such as a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, 2 is a hydrogen atom or a hydroxyl group that may have a protecting group, Rj is a protecting group that is different from the protecting groups of X%R1 and R2 and can be selectively removed, or is removed under the same conditions as the protecting group of phosphorus with a protecting group or a protecting group, 几4 indicates a phosphoric acid protecting group,
is the same as above. ) Next, a monomer in which the phosphonic acid of the monomer represented by compound ■ is protected with a phosphoric acid protecting group Bs (compound ■ of the reaction formula [■]
), then remove the protective group in compound (■) under acidic conditions to obtain a monomer with a free 5'-hydroxyl group (reaction formula [■]
Then, the monomer represented by the compound ■ and the monomer represented by the compound ■ are reacted in the presence of a condensing agent to form a dimer (the compound [phase ]).

Note that this compound [phase] can also be obtained by phosphonication of a dimer scaffolded by compound (1).

Reaction formula CI [) ■ ■ O■ (In reaction formula [■], 几S represents R4,!: M - or a different phosphoric acid protecting group, B, X.

RlW and R2 are the same as above. ) DNA and RNA each consist of four types of nucleosides, and by preparing in advance 16 types of dimers represented by Compound ■, Compound ■, or Compound O, it is possible to synthesize 4-nucleotides of any base sequence. It is. These dimers can be produced using solid phase or liquid phase methods (Nucleic AcIDs).
Research, Vol. 11, pp. 5189 and 6225 (1983), Biochemistry
, Vol. 20, p. 1874 (1981)] for the synthesis of poly-4 nucleotides with desired base sequences, and compound
n Letters, Volume 24, Page 1019 (19
[1983]], the 3'-terminal hydroxyl group can be used in the synthesis of 4-nucleotides while remaining free. The number of phosphonic acid bonds contained in a polynucleotide can be freely selected depending on the polynucleotide chain length, detection sensitivity, hybrid performance, etc. When the monomer represented by compound ■ or the dimer represented by compound [phase] is used as a raw material, it is difficult to unify the stereochemistry, but it is difficult to unify the stereochemistry, but it is possible to convert all phosphate bonds into phosphonate bonds. Alternative polynucleotides can be obtained.

However, considering the ease of purification, it is preferable to use a compound [phase]. The chirality of the 91-horn atoms does not have to be unified as long as it does not affect hybridization with the target gene, but it is stereochemically consistent.

Synthesis of a polynucleotide can be achieved by using as a raw material either the diastereomer of the 8-unit compound (1) or the dimer shown by the compound (2) separated into 8-units. This problem is solved because the phosphoric acid bond between dimers becomes a diester after complete deprotection and its chirality disappears. In this case, the phosphonic acid bond is at most 1
It will be introduced separately (Journalof
Blogical Chemistry,!
Volume 255, page 9659 (1980), Bioche
mistry, vol. 21, p. 2507 (1982
Year)〕.

In addition, the binary entities or polynucleotides represented by the reaction formula CI) and the reaction formula CI[] are represented by the reaction formula Cl) -
It can also be synthesized by the Arpschiff reaction from a phosphite intermediate as shown in (1), or by using the phosphite method using an alkyl dichlorophosphone as a phosphonic oxidizing agent as shown in reaction formula CI)-(2). However, since the former may decompose during the Arpschiff reaction, the latter is preferable.

Reaction formula (ff[) Roux几2 is the same as above. ) The polynucleotide containing a phosphonic acid bond obtained by the above method and having a specific base sequence is purified by removing all protecting groups by a normal deprotection operation, and then the polynucleotide is purified before or after hybridization with the target gene. The polynucleotide is labeled with an enzyme by a reaction with a covalent enzyme labeling compound that specifically reacts with the phosphonic acid bonding functional group in the polynucleotide. This reaction progresses extremely easily, by heating the polynucleotide and enzyme-labeled compound at or below room temperature.
Enzyme labeling can be easily completed by stirring.

In addition, the polynucleotide in the present invention is composed of two or more monomers having at least one phosphonic acid bond, and the chain length and base sequence of the nucleotides are not limited and can be freely changed according to the purpose. can be selected.

Example 1 [Preparation of alkaline phosphatase labeled compound] Alkaline phosphatase 10 mgt-0.1 mol sodium disodium phosphate buffer 7. o) Add 1.5 mg of N-hydroxysuccinimide ester of N-(4-calzoxycyclohexylmethyl)maleimide dissolved in 0.1 m of dioxane in 5 portions at 5 minute intervals. , react at 30°C. Sephadex G
-25 column with Q, l mol sodium phosphate buffer p containing Immol ethylenediaminetetraacetic acid.
The alkaline phosphatase-active portion eluted with H7,0) is concentrated in cold to obtain an alkaline phosphatase-labeled compound.

[Synthesis of decamer] 2 obtained by Arpschiff reaction of 2-N-benzoylethyl bromide and tris(trimethylsilyl)phosphite
-N-benzoylethylphosphonic acid dilysine salt 40 m
mof and dar〇-dimethoxytritylthymidine 2
0 mmol, DOWEX pyridinium resin 70.5
g and dicyclohexylcalzediimide 200 mmol
After reacting in 100 d of pyridine at 37°C for 3 days, 100 ml of cyanethanol was added and the reaction was continued for another 2 days. After the reaction is complete, add 200 ml of hydrated Zerysine.
M is added, dicyclohexylurea is filtered off, and the filtrate is concentrated. The residue t-2504 was dissolved in chloroform, purified by silica gel chromatography (eluent: methanol-chloroform), and then dropped into Beyasan to powder.

The cyanoethyl group was then removed by treatment with triethylamine in acetonitrile, yielding 0-dimethoxytritylthymidyl-3'-0-(2-N-benzoylethyl).
The phosphonate triethylammonium salt is obtained.

Next, timidium was supported via succinic acid (100 Amo
5'-0-dimethoxytritylthymidyl-3'-0-(2-N-benzoylethyl)phosphonate triethylammonium salt was added to Nu
According to the method described in clelcAcids Re5aarch, Vol. 11, p. 6225 (1983), 101L was synthesized by sequential condensation nine times. Polystyrene m fat t1orttt was shaken with ethylenediamine-ethanol (1:1) for 10 hours, filtered, and the filtrate t-
After condensing and purifying with reverse phase (Cta) silica gel chromatography (water-acetonitrile linear concentration gradient), treating with 80% acetic acid to remove the 5'-terminal protecting group, a portion was purified using reverse phase (Cta) silica gel chromatography (water-acetonitrile linear gradient). ) Purified with silica gel chromatography to obtain a decamer 100D (260
nm) was obtained.

[Labeling 10 with alkaline phosphatase in the body] This 10
50D t-0.5 mol sodium borate buffer pHs, s) 3. e- and acetonitrile/l/1. It is dissolved in OWLt, mixed with the alkaline phosphatase labeled compound, and reacted at 4° C. for 5 hours. The reaction solution was purified using a Sepharose 6B column.
Q, 1 m mo + salt o≠Q, l mo! Elution was performed with 1 mol sodium chloride phosphate buffer (pH 7, O) to obtain a fraction having ultraviolet absorption of the nucleic acid portion and alkaline phosphatase activity.

Patent Applicant Organic Synthetic Pharmaceutical Industry Co., Ltd. Procedural Amendment October 31, 1980 Patent Application No. 178917 of 1982 20 Title of Invention Method for Enzyme Labeling of Polydeoxyribonucleotides or Polyribonucleotides 3 Person Making Amendment Relationship to the case Patent applicant 2-17-4 Kyobashi, Chuo-ku, Tokyo Representative: Masao Tamashige 4, agent 5, subject of amendment 6, contents of amendment (1) Specification No. 1 page 11 line 8 r Vegls J
Corrected to i'weylsJl.

(2) rVerlogJ on the 10th line of the same page in the same book as WVer
Correct to lagJl.

(3) Set rlo ODJ on page 23, line 13 of the same book to “1
Corrected to 0.

(4) "5 oD" in line 16 of the same page of the same book is corrected to "r5 tatedan."

that's all

Claims (1)

[Claims] 1. At least one phosphate bond has the formula [I]▲A mathematical formula, a chemical formula, a table, etc.▼[I] (In the formula, R represents a branched chain having 1 to 20 carbon atoms. of polydeoxyribonucleotides or polyribonucleotides which are phosphonic acid bonds having a functional group represented by 1. A method for enzymatically labeling polydeoxyribonucleotides or polyribonucleotides, which comprises reacting a covalent enzyme labeling compound with a phosphonic acid bonding functional group. 2. The enzyme labeling method according to claim 1, wherein the functional group X, which may have a protecting group, is a thiol group or an amino group.