JPH0460600B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a composition containing an oligonucleotide having an aliphatic amino group and a method for producing the same.

BACKGROUND OF THE INVENTION Oligonucleotides are short polymers consisting of linear sequences of four nucleotides in a predetermined order. Nucleotide subunits are linked by phosphodiester bonds, linking the 3' hydroxyl portion of one nucleotide to the next.
Attach to the 5' hydroxy moiety. An example of an oligonucleotide is 5'ApCpGpTpApTpGpGpCp3'. The symbols A, C, G and T refer to the character of the purine or pyrimidine base attached to the 1-position of the deoxyribose. That is, A is adenine,
C is cytosine, G is guanine, and T is thymidine. The symbol p indicates a phosphodiester bond. The partial structure of the oligonucleotide is shown in FIG.

The single-stranded oligonucleotide of the present invention is further characterized by being homogeneous with respect to the sequence of nucleoside subunits, and furthermore having a uniform molecular weight.

Synthetic oligonucleotides are powerful weapons in modern molecular biology and recombinant DNA technology.
The uses for these molecules are many, including (a) as probes for the isolation of specific genes based on the protein sequence of the gene product, (b) for directing in vitro mutations of desired genes, and (c) (d) As a primer for DNA synthesis on a single-stranded template, (d) as a step in total gene synthesis, and many other uses;
Wm.R.Bahl et al.
Biology, Vol. 21, p. 101 (1978).

Therefore, considerable efforts are being made to develop efficient chemical synthesis methods for this type of oligonucleotide. A brief description of these methods developed to date is provided by Crockett, GC, Aldrihimica Acta, Vol.
16(3), pp. 47-55 (1983). The best method currently available utilizes phosphoramidite derivatives of nucleosides in combination with solid phase synthesis [Mateuchi et al., Journal American.
Chemical Society, Vol. 103, p. 3185 (1981) and Porkage et al., MH Tetrahedron Letters, Vol. 22(20), pp. 1858-1862 (1981)]. Oligonucleotides having a length of up to 30 bases can be produced according to such conventional methods, and molecules as long as 50 bases have also been produced. Devices using this technique are also currently commercially available.

Other reports on DNA derivatization can also be found in the literature. Modified nucleoside triphosphates have been developed in which a piotin group is attached to an aliphatic amino group at the 5-position of uracil [Langer et al., Bros. National Academy of Sciences, USA Vol. 78, No. 6633]. −6637
Page (1981)]. This nucleotide derivative is double-stranded
Effectively integrated into DNA. Once incorporated into the DNA, it is bound by an anti-biotin antibody, which can then be used to detect it by fluorescent or enzymatic methods. DNA with biotin-linked nucleosides incorporated by the method of Langer et al. is fragmented into smaller single- and double-stranded versions, which are heterogeneous in sequence of the nucleoside subunits and
They have different molecular weights. Draper and Gold [Biochemistry, Vol. 19, pp. 1774-1781 (1980] reported the introduction of aliphatic amino groups by a bisulfite-catalyzed transamination reaction and subsequent reaction with a fluorescent label. In the report of Draper and Gold, the amino group is directly attached to the pyrimidine base. The amino group attached in this way suppresses hydrogen bonding and for this reason these materials are not useful for hybridization etc. Chu et al. [Nukreitsk Assisted Research, No. 11(1)
8), pp. 6513-6529 (1983)] report a method for attaching amines to the terminal 5' phosphates of oligonucleotides or nucleic acids.

Methods for covalently attaching other chemicals to synthetic oligonucleotides are desirable for a number of reasons. Fluorescent dyes attached to oligonucleotides allow radioisotopes to be excluded from the research, diagnostic and clinical methods in which they are used, and improve longevity and availability. Synthesis of fluorescently labeled oligonucleotides, as described in the applicant's patent application for a DNA sequencing device,
Enables automation of DNA sequencing methods. The development of appropriate techniques and instrumentation for the detection and use of fluorescently labeled oligonucleotides will enable automation of other experimental and clinical techniques that are currently cumbersome.
For example, Schultz et al.
104, p. 6861 (1982) and the journal by Hertzberg et al.
American Chemical Society, Volume 104,
Coupling of DNA cleaving agents, such as those disclosed on page 313 (1982), allows for the creation of synthetic restriction enzymes, the specificity of which is dictated by the oligonucleotide sequence.

Problems to be Solved by the Invention The present invention provides a general method for introducing free aliphatic amino groups into synthetic oligonucleotides. This amino group reacts easily and specifically with various amino-reactive functional groups, thereby allowing covalent attachment of a wide variety of chemicals.

SUMMARY OF THE INVENTION In summary, the present invention consists of a novel aliphatic amino-derivatized single-stranded oligonucleotide conjugated to a detectable moiety, the detectable moiety being a chromophore, a fluorescent agent, a protein, It is a "label" for enzymes, etc.

Additionally, the present invention encompasses novel oligonucleotides into which at least one amino-derivatized nucleoside has been inserted via a phosphoramidite precursor.

In another aspect, the invention includes a method of synthesizing an oligonucleotide on a solid support, wherein the oligonucleotide is reacted with a protected amino-derivatized nucleoside phosphoramidite.

In one aspect, the present invention provides that the synthesized molecule 5′-
Trifluoroacetamide-5'-deoxy-3'-
N,N-diisopropylphosphoramidothymidine (Figure 2) and its addition to the 5' end as the last addition in solid phase oligonucleotide synthesis. Upon cleavage and deprotection of the oligonucleotide, the trifluoroacetyl group is hydrolyzed to free the aliphatic amino group of the oligonucleotide.
Leave at the 5′ end. This amino-derivatized oligonucleotide can then be reacted with any of a wide variety of amino-reactive molecules to generate the corresponding oligonucleotide derivative. this is,
This is a special example of the general method of using modified nucleosides that have a protected aliphatic amino group on the base moiety rather than the 5' carbon. Molecules of this type make it possible to incorporate several free amino groups into the interior of the oligonucleotide and at desired positions in the oligonucleotide sequence.

It is an object of the present invention to provide new reagents and techniques that can be used for DNA sequencing.

A further object of the invention is to provide improvements in DNA hybridization for the detection of genetic diseases and other purposes.

These and other objects and advantages of the present invention include:
It will be clear to those skilled in the art from the following description.

The method used to introduce aliphatic amino groups into oligonucleotides is to synthesize 3' phosphoramidite derivatives of nucleoside analogs containing protected aliphatic amino groups. This phosphoramidite can then be reacted with oligonucleotides synthesized on a solid support in a manner similar to the reaction of underivatized nucleoside phosphoramidites. Cleavage from the solid phase and deprotection of the base moiety and aliphatic amino group provides an amino-derivatized oligonucleotide.

Synthesis of Example 1: Figure 3 shows the synthesis of the compound from a commercially available compound (thymidine). Compound~
The synthesis of is described by Horowit et al., Journal of Organic Chemistry, Vol. 27, No.
3045-3048 (1962), and also Gibbs and Orgel, Journal.
Carbohydrates - Nucleotides and Nucleotides, Vol. 3 (5 and 6), pp. 315-334 (1976). The compound and its synthesis are as follows.

Example 5'-Trifluoroacetamide-5'-deoxythymidine (): 1.25 g (5 mmol) of 5'-amino-5'-deoxythymidine was dissolved in 25 ml of dry dimethylformamide. To this was added 1.3 ml (10 mmol) of S-ethyltrifluorothioacetate (Aldrich). The reaction was stirred gently at room temperature. TLC of the reaction mixture on silica gel F-254 plates performed with MeOH:acetone 1:1 shows a single spot of product detected by short wavelength UV. This product is
It has high mobility in this solvent system compared to the poorly mobile starting compound.

The reaction mixture was rotary evaporated under reduced pressure to 30 ml.
of isopropanol and recrystallized from boiling isopropanol:MeOH. Yield = 1.315 g (3.9 mmol, 80% yield),
Melting point 261°-262°C (decomposed), predicted analytical value C 42.7
%, H 4.18% N 12.5%; Experimental value C 42.7%,
H 4.16%, N 12.4%. The structure of
Confirmed by NMR.

Example This example demonstrates the preparation of protected amino-derivatized nucleoside phosphoramidites.

5′-trifluoroacetamide-5′-deoxy-
3'-N,N-diisopropylphosphoramidothymidine (): The syringes and capillary tubes used in this reaction, all made of glass, were kept in a dry open for 1 hour.
It was fired in the evening. Dimethylformamide (DMF) is
It was stored in 4 ml Molecule Sheep (Linde). Diisopropylethylamine (DIPEA) is
It was distilled from potassium hydroxide and then from calcium hydroxide and stored in 4 ml molecular sheep.

63 mg (0.19 mmol) of Compound V was added to a dry three-necked 50 ml round bottom flask equipped with a stir bar.
Rubber plugs were placed on each of the three necks, and needles were inserted into these plugs. The flask was vacuumed in a desiccator of dry CaCl ₂ for several hours. Keep the flask under a gentle stream of dry nitrogen gas to dry 2 ml.
DMF was added via syringe. 60μ DIPEA (0.34 mmol) was added in a dry 100μ capillary. 40μ of chloro-N,N-diisopropylaminomethoxyphosphine (American Bionuclea, Emerpil, Calif.) was added (dry).
by 100μ capillary). The reaction was stirred gently until all starting material was dissolved and then allowed to stand at room temperature (always under _N2 ). After 1 hour, HCCl ₃ :EtOH:
TLC on a silica gel F-254 plate at Et ₃ N=88:10:2 showed a single spot of the product which was significantly more mobile than starting material V. Attempts to purify this product in a manner similar to that described for nucleoside phosphoramidite (4) were unsuccessful due to product degradation. Therefore,
The crude reaction mixture was used directly for coupling to synthetic oligonucleotides on solid support.
The structure of is deduced from the reactivity of this product upon addition to oligonucleotides and the expected product of the reaction based on literature results.

Example This example shows that the 5′-
Figure 2 shows the preparation of an oligonucleotide attached at the 5' end to the 3'-hydroxyl of amino-5'-deoxythymidine.

Addition to the 5'-end of the oligonucleotide: the sequence 5'OH- attached to the solid phase at the 3' end
AGC ACT TTT AGA GT 3' base-protected synthetic oligonucleotides were synthesized into dimethoxytrityl nucleoside phosphor on solid supports, published by Applied Biosystems, Inc., Foster City, California. It was prepared by the method described in detail in the paper entitled "Synthesis of deoxyoligonucleotides using amidites". The differences in the reaction with oligonucleotides are (a) Steps 4.22 and
Mix 1 ml of the freshly prepared reaction mixture containing instead of 4.23 with 1 ml of 0.5 M tetrazole in acetonitrile and add this to the reaction vessel; (b) wash twice with acetonitrile for 30 seconds after step 4.33. and (c) omitting capping step 5 (allowing analysis of unreacted hydroxyl groups in subsequent additions).

The efficiency of the coupling reaction is easily monitored by coupling with the "normal" phosphoramidite and cleavage of the dimethoxytrityl group, allowing colorimetric analysis. If the 5'-hydroxyl group of the oligonucleotide reacts with the first bond, it is no longer available for reaction in the subsequent bond, and DCA after the second bond
Almost no color develops during processing. In this example,
An OD ₄₅₀ =1.12 (after dilution) for the DMT group was generated from the oligonucleotide before reacting with.
The OD ₄₅₀ for DMT = 0.026 (after dilution) was generated from the G residue added after reaction with. Therefore, 98% of the 5'-OH groups were protected by reaction with. This oligonucleotide is deprotected and
and cleaved from the solid phase by conventional treatment with thiophenol and concentrated NH ₄ OH, and
_NH4OH was removed under reduced pressure. The oligonucleotide was dissolved in 1 ml of distilled water. The OD ₂₈₀ is 128 for this solution, which means that the concentration of DNA is 4.5
Indicates mg/ml. Dilute 50 μ of this solution to 1 ml with water and add several hundred (100-200) mg of
AG50W-X4 (sodium type) ion exchange resin
Mixed for 15 minutes to exchange DNA from ammonium salt to sodium salt (ammonium ions inhibit subsequent ninhydrin analysis). The resin was removed by centrifugation, and the supernatant was dried in a Savant type rotary concentrator. Quantitative ninhydrin analysis [Sarin, VK et al., Analytical Biochemistry, Vol. 117, No. 147-
157 (1981)] gave about 1 mole of amino groups per mole of oligonucleotide (the molar concentration of oligonucleotide was determined based on the nucleotide composition from the calculated extinction coefficient E ₂₆₀ =1.55×10 ⁵ ). Ninhydrin analysis on the same molar amount of the unconjugated comparison oligonucleotide showed no amino-positive reaction.

Example 5 Coupling with dye: To the above solution of 100 μ of amino oligonucleotide, 200 μ of H ₂ O and 50 μ of 1
molar carbonate/bicarbonate buffer (PH 9.0) and 25μ of freshly prepared 10mg/ml fluorescein isothiocyanate (FITC) (Molecular Blobs, Inc., Yankee City, Oregon). A solution in dimethyl sulfoxide was added. The mixture was left at room temperature for several hours and then washed with Cephadex G in H ₂ O.
It was purified by chromatography on a column (1 cm x 9 cm) of -25 medium. A yellow product eluted in the elution volume and was separated cleanly from unreacted dye. A comparative reaction with an unreacted oligonucleotide gave almost no coloration to the elution volume of the column, indicating that the dye had indeed reacted with the added amino groups. Dye-
Oligonucleotide conjugate has OD ₂₆₀ = 2.3, OD ₄₉₃ =
It had a value of 0.54. Based on E ₄₉₅ =7×10 ⁴ for FITC, this gives 7.7 μmoles of dye solution. _E260
= 1.55 x ¹⁰⁵ , DNA is 12.8 μmol.
Therefore, approximately 60% (~60%) of the DNA molecules were labeled with the dye. This is a rough estimate and does not take into account variations in the absorption of bound versus unbound fluorescein, nor does it take into account variations in shorter contaminating and unreacted oligonucleotides. An aliquot of this colored DNA was electrophoresed in a 20% polyacrylamide gel and was clearly visible as a single colored (and fluorescent) band of appropriate mobility for an oligonucleotide of this length.

Dye-conjugated oligonucleotides are phase-reversed using high-performance liquid chromatography (HPLC).
It was easily purified on a _C18 column (Waters) using an acetonitrile:0.1M triethylammonium acetate (PH7.0) gradient for elution.

Many uses are possible for the novel amino-derivatized oligonucleotides described above. Aliphatic amino groups react easily and specifically with many functional groups. This means that virtually any desired molecule can be attached to the oligonucleotides created as described above. This includes enzymes, other proteins, fluorescent labels, bioluminescent labels, chromophores, and the like. Oligonucleotides are widely used in many fields, often in combination with radioactive labels. Non-radioactive sample molecules can also be used in place of radioactive labels. This makes the method of using oligonucleotides cheaper and easier to use, and more suitable for clinical use. The novel amino-derivatized oligonucleotides can also be radiolabeled, eg with I ¹²⁵ , although radiolabeling is less preferred. Only three specific examples of applications of novel amino-derivatized oligonucleotides are illustrated: 1. Automated DNA sequencing; 2. Detection of genetic diseases by DNA hybridization; 3. General use of fluorescence for detection of hybridization. .

Detection of genetic abnormalities: Oligonucleotides can be used to determine an individual's genotype. This is done on DNA samples obtained from fetuses by aminocentesis. This information is constant for genetic counseling of couples at risk for various genetic diseases. Genotyping of adults will also be determined to allow effective diagnosis and early treatment. One notable example of this technique is the detection of sickle cell anemia (Connor et al., Proceedings National Academy of Sciences, USA Vol. 80, p. 278 (1983)). A synthetic oligonucleotide of 19 base pairs in length was synthesized, one containing the normal human β-globin gene (β ^A ).
and one is complementary to sickle cell β-
It is complementary to the globin gene (β ^S ). These molecules were radiolabeled and used as samples in DNA hybridization. Under appropriate hybridization conditions, these samples can be used to distinguish the β ^A gene from the β ^S allele. This allows diagnosis of sickle cell anemia. More generally, as pointed out in Connor et al.
"The allele-specific hybridization properties of oligonucleotides provide a general diagnostic method for any genetic disease involving a point mutation in the DNA sequence of a single-copy gene." The present invention can be used directly in this technology. An oligonucleotide sample having an amino group is prepared and labeled with a fluorescent label. Fluorescent sample molecules are infinitely stable compared to the short lifetime of radioactive samples and do not require special precautions in use or handling. This makes the method highly suitable for clinical use, and this is in fact the main area of use.

Oligonucleotide samples are widely used in research as well as clinical applications. These are "libraries"
i.e. cloned into a plasmid or phage vector to contain the entire genome (or expression
It is commonly used to detect DNA pieces and desired sequences in collections of DNA fragments with sequences containing RNA). Furthermore, these
It is also used to hybridize to a given sequence of DNA in a "blot" of a restriction digest of a specific piece of DNA. In all of these and other examples, the oligonucleotide is generally labeled with ^P32 at the 5' end, and the molecule is detected by autoradiography. The present invention discloses the labeling of oligonucleotides with fluorescent dyes. Fluorescence can therefore be used to detect molecules with any technique in which radioactivity is conventionally used.
This has many advantages over radioactivity, such as sample stability, cost and ease of use, and disposal.

Although the present invention has been described above, the present invention is not limited thereto.

[Brief explanation of drawings]

FIG. 1 is a partial structure of an oligonucleotide. Figure 2 shows 5'-trifluoroacetamide
5'-deoxy-3'-N,N-diisopropylphosphoramidothymidine. FIG. 3 is a synthetic route for compound VI (5'-trifluoroacetamido-5'-deoxy-3'-N,N-diisopropylphosphoramidothymidine) from commercially available compound I (thymidine).

Claims (1)

[Claims] 1. A single-stranded oligonucleotide containing an amino group at the 5' carbon atom of the 5' terminal nucleoside. 2. The compound according to claim 1, wherein the oligonucleotide contains a 5'-terminal 5'-amino-5'-deoxythymidine nucleoside. 3. In a method for producing a single-stranded oligonucleotide containing an amino group at the 5' carbon atom of the 5'-terminal nucleoside, an aminonucleoside phosphoramidite is reacted in the final coupling step of oligonucleotide synthesis on a solid support. A production method characterized in that, in particular, an amino group is introduced. 4 Protected 5'-amino-5'-deoxy-3'-
4. The method according to claim 3, wherein an amino group is introduced by reacting phosphoramidothymidine. 5 The phosphoramidite compound has the following general formula: wherein B is a nucleoside base or nucleoside base analog, and R ₁ and R ₂ are lower alkyl. 6. The method of claim 5, wherein the oligonucleotide is then cleaved from the support.