KR20030094716A - Novel linker for oligonucleotides - Google Patents

Abstract

PURPOSE: A novel linker for oligonucleotides is provided, which connects fluorescence material or a solid support as a reporter or a terminator to oligonucleotides, so that oligonucleotides can be easily purified and assayed. CONSTITUTION: A novel linker for oligonucleotides is characterized by having the structure of the formula 1, wherein HCA is 5- or 6-membered heterocyclic amine; n is 0 to 10; X is connected to nitrogen of the heterocyclic amine and is one ring selected from C1-C10 single bond carbohydrate ring, C1-C10 single bond carbohydrate in which 1 to 5 hetero atoms are inserted into its carbon site, or C1-C10 single bond carbohydrate ring in which one bond selected from amide, ester, ether, amine, sulfonyl or a combination thereof is added; R1 is hydrogen or a hydroxy protecting group; R2 is hydrogen, phosphoramidite or H-phosphonate, or a solid support; and R3 is a reporter molecule, a terminator molecule, an amino protecting group, an amino acid or a peptide.

Description

Novel Linker for Oligonucleotides

The present invention relates to linkers of oligonucleotides, and more particularly, to novel linkers for linking fluorescent materials or solid supports as reporters or destructors to oligonucleotides and methods for their preparation.

In recent years, automated DNA synthesizers have revolutionized the field of molecular biology and biochemistry. As a result, linear DNA oligonucleotides with specific sequences have become commercially available. Synthesis of oligonucleotides most commonly used at present is a phosphite-triester method for synthesizing oligonucleotides from a solid support using nucleoside phosphoramidite. In general, the process consists of deblocking, coupling, oxidation and capping processes.

Oligonucleotides can be applied to a variety of fields. For example, DNA oligonucleotides can be used as primers for cDNA synthesis, primers for PCR, templates for RNA transcription, linkers for plasmid construction, and hybridization probes for research and diagnostics.

Modifications of oligonucleotides are required for various applications of oligonucleotides. Various methods can be applied to the modification of the oligonucleotides, but the simplest is to label the oligos through the oligo synthesizer using the phosphoramidite method.

Usually oligonucleotides are synthesized and labeled to facilitate tracking and quantitative analysis of oligonucleotide-target complexes, and specific labeling of oligonucleotides is an important technique for synthesizing DNA-probes (Goodchild, J. Bioconjugate Chem. , 1: 165 (1990)).

Since the labeling of oligonucleotides using conventional radioisotopes is dangerous and difficult to handle, the replacement of the oligonucleotides using fluorescent materials instead of the conventional radioisotopes has been accelerated. Smith, LM et al., Nature , 321: 674-679 (1986); and Agrawal, S. et al., Nucleic Acid Res. , 14: 6227-6245 (1986). Accordingly, several methods have been developed for the synthesis of oligonucleotide derivatives having amino or thiol groups to label oligonucleotides with fluorescent materials (Ono, K. et al., Bioconjugate Chem. , 4: 499-508 (1993). And Douglas, ME et al., BioMed. Chem. Lett. 4 (8): 995-1000 (1994)).

The reporter may be attached to the synthesized oligonucleotide using a chemical method or an enzyme, and a reporter or a destructor may be introduced using a compound that can be used for the phosphoramidite method in the oligonucleotide synthesis process (Kempe , T. et al., Nucleic Acid Res. 13: 45-57 (1985); WO 91/17169; and Gibson, KJ et al., Nucleic Acid Res. 15: 6455-6467 (1987)).

Currently, various types of linkers have been developed for introducing such reporters into oligonucleotides. 1992, Nelson et al. Nucleic Acid Res. 20: 6253-6259 (1992) is the first skeleton based on 2- (4-aminobutyl) -1 and 3-propanediol to which three functional compounds can be attached through covalent bonds. Following the development of a linker, Mullah B. et al. Published a linker incorporating an amide functional group in the stomach skeleton ( Nucleic Acid Res. 26: 1026-1031 (1998)).

However, since these linkers exist as racemates to generate two diastereomers when combined with oligonucleotides, there is a problem that the purification or analysis using the synthetic oligonucleotides is complicated.

Throughout this specification, numerous patent documents and articles are referenced and their citations are indicated. The disclosures of cited patent documents and articles are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors have completed the present invention by preparing a novel linker that is easy to purify and analyze oligonucleotides as a linker of oligonucleotides.

Accordingly, it is an object of the present invention to provide modifications, including oligonucleotide labeling, or novel linkers for use in the synthesis of oligonucleotides.

Fig. 1A shows the sequence of the 35S promoter of four kinds of cauliflower mosaic virus.

Fig. 1B is a diagram showing the nucleotide sequence of the 35S promoter of four different cauliflower mosaic viruses;

2 is a graph showing the change in intensity of fluorescence measured according to the present invention; And

3 is a graph showing a standard curve obtained according to the present invention and quantifying an unknown sample through it.

According to one aspect of the invention, the invention provides a novel linker having the structure of formula

In the above formula, HCA is a heterocyclic amine consisting of 5 atoms or 6 atoms; n is 0-10; X is connected to the nitrogen atom of the heterocyclic amine, C ₁ -C ₁₀ single bond hydrocarbon chain, C ₁ -C ₁₀ hydrocarbon chain having 1-5 hetero molecules at the carbon position of the single bond hydrocarbon chain, or C _{One of the} hydrocarbon chains to which the bond selected from the group consisting of amides, esters, ethers, amines, sulfonyls, or combinations thereof is added to the _1- C ₁₀ single bond hydrocarbon chain; R 1 is hydrogen or a hydroxy protecting group; R2 is hydrogen, phosphamidite group or H-phosphonate, or a solid support; And R3 is one of a reporter molecule, a destructor molecule, an amino protecting group, an amino acid or a peptide.

The heterocyclic amine located in the central portion of the linker of the present invention is a non-aromatic heterocyclic amine, which is a heterocyclic amine consisting of five atoms, such as pyrrolidine, or a heterocyclic amine consisting of six atoms, such as Piperidine. In addition, the heterocyclic amine may comprise one or more nitrogens (eg piperazine) and may contain one or more hetero atoms other than nitrogen (eg morpholine).

Thus, according to a preferred embodiment of the invention (when the central heterocyclic amine is pyrrolidine), the linker of the invention is represented by the following formula (2):

In the above formula, n, X, R1, R2 and R3 are the same as in the above formula (1).

According to another preferred embodiment of the invention (when the central heterocyclic amine is piperidine), the linker of the invention is represented by the formula:

In the above formula, n, X, R1, R2 and R3 are the same as in the above formula (1).

The linker of the present invention is capable of binding to any nucleoside constituting an oligonucleotide with or without a reporter. As used herein, an "oligonucleotide" is a substance in which nucleotides are repeatedly connected, and one or more nucleotides may be present as a derivative, and linkers may be attached with appropriate intervals. Wherein nucleotides include ribonucleotides and deoxyribonucleotides, which may have natural bases adenine, cytosine, thymine, guanine, uracil and derivatives thereof.

Substances introduced into the oligonucleotides by the linker are reporter molecules, ?? molecules, amino protecting groups, amino acids, peptides and the like.

The reporter is a group that enables light sensing, binding, quantification, etc. of oligonucleotides, and includes oligonucleotides having reporter molecules. Preferably, the reporter is a fluorescent reporter molecule. The reporter may include a spacer so as not to affect the action of the amine group when combined with the amine group.

The fluorescent reporter molecule is 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2 ', 4', 1,4-tetrachlorofluoro Tetrachlorofluroscein (TET), 2 ', 4', 5 ', 7', 1,4-hexachlorofluorescein (HEX) and 2 ', 7'-dimethoxy-4'5 '-Dichloro-6-carboxyrhodamine (JoE) and the like are preferred.

On the other hand, the destructor is located in close proximity to the reporter to temporarily cancel the reporter's function, when the distance from the reporter is separated due to separation, such as a group for regenerating the function of the reporter, including an oligonucleotide having a destructor molecule do.

Preferably the destructor is a fluorescent destructor molecule. The destructor may include a spacer so as not to affect the action of the amine group when combined with the amine group.

The fluorescent destructor molecules are tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), cyanine, androquinone, nitrothiazole and nitro. Amidazole and the like are preferred.

On the other hand, in each step of the oligonucleotide synthesis process, a large number of functional groups must be protected such as various functional groups such as four base functional groups (e.g., amino groups), phosphoric acid groups and 5'-hydroxy groups so that only desired chemical reactions occur.

First, the amino group present in the base should be protected in order to prevent the acetylation reaction and phosphorylation reaction from occurring in the amino group during oligonucleotide synthesis. Generally protecting groups are used which are stable to acids and easy to remove from alkalis.

The amino protecting group used in the linker of the present invention is preferably a benzyloxycarbonyl group.

The 5'-hydroxy group must be protected during condensation, oxidation and capping reactions and the protecting group must be removed by a weak acid such as trichloroacetic acid (TCA) just before the next nucleotide is added.

The hydroxy protecting group is 4,4-dimethoxytrityl (DMT), 9-phenylxanthen-9-yl (yl) (pixyl) and 9-fluorenylmethoxycarbonyl (Fmoc) And the like, most preferably DMT.

According to a preferred embodiment of the invention, n is 0-10. More preferably, n is 0-1 and smaller is n, that is, the shorter the length of the single-bond hydrocarbon chain connected to the heterocyclic amine in the central portion of the linker and the less steric hindrance, the more linker having simpler structure do.

According to a preferred embodiment of the present invention, X is a C ₄ -C ₈ hydrocarbon chain having an amide group, more preferably X is a C ₆ hydrocarbon chain having one amide group. Amide groups are advantageous in that they are easy to manufacture, stable under oligonucleotide synthesis conditions, and are easy to analyze. When a hetero atom is included in X, preferably the hetero atom is oxygen or nitrogen.

The linker of the present invention has a variety of uses, but is typical of the introduction of fluorescent materials into oligonucleotides and their use in the synthesis of oligonucleotides.

When the linker of the present invention is used to introduce a fluorescent material into the oligonucleotide, one specific embodiment of the present invention is represented by the following formula (4).

In the above formula, iPr represents an isopropyl group.

In the linker represented by Chemical Formula 4, a reporter molecule or a destructor molecule is bonded instead of Fmoc, an amino protecting group.

When the linker of the present invention is used for the synthesis of oligonucleotides, typically phosphoramidite DNA synthesis, the linker of the present invention may be attached to the 3'-end of the nucleotide or oligonucleotide and added to the DNA synthesizer. It can be used instead of the protective deoxynucleoside phosphoramidite.

In addition, instead of the protected deoxynucleoside derivatized long chain alkylamine-controlled pore glass (lcaa-CPG) column commonly used in phosphoramidite DNA synthesis, the linker of the present invention is linked to a solid resin. Can be used.

That is, one specific embodiment of the linker of the present invention is an immobilized linker represented by the following formula (5):

In the above formula, n, X and R 1 are the same as those of Formula 1 above, and Y is a single bond, nucleotide or oligonucleotide; FS is a functional spacer; SSM is a solid support; And R 3 is an amino protecting group, reporter or destructor molecule.

In Formula 5, non-limiting examples of SSM include long-chain alkylamine CPG (lcaa-CPG), 3-amino-propyl silica gel, aminomethyl polystyrene resin, TengaGel ^TM (polyethylene glycol-TENTAcles grafted on low cross-linked gelatinous polystyrene matrix) and the like.

The SSM is separated from the finally synthesized oligonucleotide and has a functional spacer between the central portion of the linker. Functional spacers include, but are not limited to, succinyl groups, glutaryl groups, and the like, and any spacer used for chemical synthesis of oligonucleotides may be used.

According to the most preferred embodiment of the present invention, in the linker of Formula 5, n is 0 or 1, X is a C ₆ hydrocarbon chain having one amide group, Y is a single bond, FS is a succinyl spacer, SSM is CPG, R3 is a reporter or destructor, and R1 is a DMT.

One specific embodiment of the linker of Chemical Formula 5 is represented by the following Chemical Formula 6:

Synthesis of oligonucleotides using the linker of the present invention can be carried out according to conventional methods, which methods are described in Caruthers, MH Science , 230: 281-285 (1985), Itakura, K. et al., Ann. Rev. Biochem. 53: 323-356 (1984), and Hunkapiller, M. et al., Nature , 310: 105-111 (1984), which is incorporated herein by reference.

On the other hand, oligonucleotides comprising the linker of the present invention can be used, for example, for real-time monitoring of DNA amplification processes, and can typically be used for real-time PCR. Such a method of real-time PCR is disclosed in US Pat. No. 5,538,848, which is incorporated herein by reference.

The chemical formula of the probe sequence used when performing real-time PCR using an oligonucleotide including a linker of the present invention is as follows.

As described above, the oligonucleotide of the present invention may be usefully used to introduce a fluorescent material or to bind a solid resin.

In addition, since the linker of the present invention is made of a compound having a pure enantiomer structure, the linker of the present invention has an advantage of eliminating the difficulty of purification and analysis caused by the racemate linker. Such pure enantiomeric structures are prepared using pure natural chiral compounds (eg, proline with certain stereoisomerism). The preparation of the linkers of the invention with specific enantiomer structures is illustrated in detail in the examples below.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self-evident.

Example

Example 1 Synthesis of Linker Linked Phosphoramidite

Scheme 1 is a diagram illustrating a method for preparing a linker having a phosphoramidite according to an embodiment of the present invention. According to Scheme 1, a linker capable of attaching a fluorescent substance to an oligonucleotide can be synthesized, and the linker can be attached to the 3'-end or the middle of the oligonucleotide. Referring to Scheme 1, one embodiment of a method for preparing a linker with phosphoramidite according to the present invention will be described in detail.

Step 1: Synthesis of trans-4-hydroxy-L-proline methyl ester hydrochloride (Compound 2)

Trans-4-hydroxy-L-proline (Compound 1) (10 g, 76.3 mmol) was dissolved in 200 mL of methanol and acetyl chloride (7.6 mL, 106.8 mmol) was added dropwise and stirred to reflux overnight. Thereafter, 500 ml of ether was added thereto, and the resulting white solid was filtered and dried in vacuo to give Compound 2 (12.7 g, 87.6%): ¹ H NMR (CDCl ₃ ) δ2.07-2.30 (m, 1H, C3-H ), 2.33-2.50 (m, 1H, C3-H), 3.29-3.34 (m, 2H, C2, C5-H), 3.45-3.50 (m, 1H, C5-H), 3.84 (s, 3H, CO ₂ CH ₃ ), 4.43-4.60 (br, 1H, NH), 4.62-4.72 (m, 1H, C4-H); Mass EI m / z 146.80, 87.00, 68.75.

Step 2: Synthesis of 1-benzyl-4-hydroxy-pyrrolidine-2-carboxylic acid methyl ester (compound 3)

CH ₃ CN (100 mL) dried Compound 2 (12.7 g, 66.6 mmol), K ₂ CO ₃ (26.4 g, 199.8 mmol), BnCl (6.5 mL, 56.6 mmol) and tBu ₄ NI (cat.) ), And refluxed under stirring overnight. It was cooled to room temperature, diluted with 200 mL of water, extracted with AcOEt (50 mL × 3), and washed with water. The organic layer was dried over MgSO ₄ and concentrated to give an oily residue. The residue was separated by column chromatography (MC: MeOH = 20: 1/15: 1) to give a colorless oily compound 3 (10.57 g, 67.5%): ¹ H NMR (CDCl ₃ ) δ2.03- 2.11 (m, 1H, C3-H), 2.20-2.29 (m, 1H, C3-H), 2.46 (dd, 1H, J = 3.82, 10.14 Hz, C5-H), 3.32 (dd, 1H, J = 5.62, 10.14 Hz, C5-H), 3.60 (m, 1H, C2-H), 3.65 (s, 3H, CO ₂ CH ₃ ), 3.66 (d, 1H, J = 12.87 Hz, CH ₂ Ph), 3.89 (d, 1H, J = 12.87 Hz, CH ₂ Ph), 4.44 (m, 1H, C 4 -H), 7.24-7.32 (m, 5H, Ph); Mass EI m / z 235.20, 176.10, 91.15.

Step 3: Synthesis of 1-benzyl-5-hydroxymethyl-pyrrolidin-3-ol (Compound 4)

To a THF (150 mL) suspension of LAH (2.16 g, 57.12 mmol) was added Compound 3 (11.2 g, 47.6 mmol) dropwise at 0 ° C. under a nitrogen stream for 20 minutes and stirred at room temperature for 3 hours. Excess LAH was treated with acetone and water. The resulting gray precipitate was removed using celite and the filtrate was concentrated. The remaining small amount of water was removed using an azeotropic point with toluene and then dried in vacuo to afford crude compound 4 (9.54 g, 97.0%). This compound was used in the following reaction without further purification: ¹ H NMR (CDCl ₃ ) δ 1.77-1.90 (m, 1H, C3-H), 2.07-2.22 (m, 1H, C3-H), 2.38 (dd, 1H, J = 4.88, 10.17 Hz, C5-H), 3.04-3.13 (m, 1H, CH ₂ OH), 3.24 (dd, 1H, J = 5.70, 10.17 Hz, C5-H), 3.41 (dd, 1H, J = 2.03, 11.39 Hz, C2-H), 3.48 (d, J = 13.02 Hz, CH ₂ Ph), 3.67 (dd, 1H, J = 3.26, 10.99 Hz, CH ₂ OH), 3.99 (d, 1H, J = 13.02 Hz, CH ₂ Ph), 4.28-4.80 (m, 1H, C4-H), 7.25-7.33 (m, 5H, Ph); Mass EI m / z 207.35, 176.15, 90.85.

Step 4: Synthesis of 5-hydroxymethyl-pyrrolidin-3-ol (Compound 5)

Compound 4 (9.54 g, 46.0 mmol) was dissolved in dry ethanol (50 mL), 10% Pd-C (1.0 g) was added, and stirred for 5 hours under a hydrogen gas stream at 25 ° C. and 40 psi. The remaining amount of Pd-C was filtered off and concentrated under reduced pressure to give crude compound 5 (4.8 g, 88.9%): ¹ H NMR (D ₂ O) δ 1.93-2.14 (m, 1H, C3-H), 2.15 -2.25 (m, 1H, C3-H), 3.34-3.40 (m, 1H, C5-H), 3.46-3.54 (m, 1H, C5-H), 3.71-3.81 (m, 1H, CH ₂ OH) , 3.94-4.02 (m, 1H, CH ₂ OH), 4.05-4.10 (m, 1H, C2-H), 4.69-4.78 (m, 1H, C4-H).

Step 5: Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5-hydroxymethyl-pyrrolidin-3-ol (Compound 6)

Compound 5 (0.3 g, 2.5 mmol), 6-N-Fmoc-ε-aminocaproic acid (0.88 g, 2.5 mmol), 1-hydroxy-7-azabenzotriazole (HOAT) (0.34 g, 2.5 mmol) and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyl uronium hexafluorophosphate (HBTU) (0.95 g, 2.5 mmol) were added to DMF (1 ML), and diisopropylethylamine (DIPEA) (0.44 mL, 2.5 mmol) was added dropwise under 25 DEG C, atmospheric nitrogen stream and stirred for 2 hours 30 minutes. The reaction mixture was concentrated under reduced pressure, dissolved in chloroform (100 mL) and washed successively with 5% hydrochloric acid solution (50 mL), water (50 mL), and brine (50 mL). The organic layer was dried over MgSO ₄ , concentrated under reduced pressure, and ethanol (2 mL) was added to the oily residue, which was left in the freezer for one day. The resulting needle-like precipitate was dried to give compound 6 (0.98 g, 86.7%): ¹ H NMR (CDCl ₃ ) δ 1.27-1.35 (m, 2H, CH ₂ -CH ₂ NHFmoc), 1.41-1.50 (m , 2H, CH ₂ -CH ₂ NHFmoc), 1.55-1.64 (m, 4H, CH ₂ -CH ₂ NHFmoc), 2.10 (dd, 1H, J = 7.50, 13.59 Hz, C3-H), 2.13-2.32 (m , 2H, C3, C5-H), 3.08-3.16 (m, 2H, CH ₂ -CH ₂ NHFmoc), 3.40-3.49 (m, 3H, C2-H, CH ₂ ODMT), 3.59-3.65 (m, 1H , C5-H), 4.14 (dd, 1H, J = 6.77, 13.53 Hz, C4-H), 4.25-4.33 (m, 2H, NHCO ₂ CH ₂ ), 4.93 (s, 1H, NH), 5.34 (d , 1H, J = 8.21 Hz, Fmoc), 7.19-7.26 (m, 2H, Fmoc), 7.30-7.35 (m, 2H, Fmoc), 7.50-7.53 (m, 2H, Fmoc), 7.67-7.70 (m, 2H, Fmoc); Mass FAB ⁺ cal. 452.2311 found 453.2390 (+ H).

Step 6: Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5-O-DMT Methyl-pyrrolidin-3-ol (Compound 7)

Compound 6 (0.3 g, 0.66 mmol) and DMTCl (0.22 g, 0.66 mmol) and catalytic amount of DMAP were dissolved in dry CH ₂ Cl ₂ (2 mL), and TEA (0.18 mL, 1.32 m) at -20 ° C under nitrogen stream Mol) was added and stirred for 4 hours. After treating with methanol, the mixture was diluted with CH ₂ Cl ₂ (10 mL), washed with brine (10 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was separated by column chromatography (50: 1 = MC: MeOH, with TEA 5%) to give compound 7 (0.32 g, 64.2%): ¹ H NMR (CDCl ₃ ) δ1.12-1.17 (m, 2H , CH ₂ -CH ₂ NHFmoc), 1.20-1.33 (m, 2H, CH ₂ -CH ₂ NHFmoc), 1.34-1.48 (m, 2H, CH ₂ -CH ₂ NHFmoc), 1.49-1.60 (m, 2H, CH ₂ -CH ₂ NHFmoc), 1.83-1.91 (m, 1H, C3-H), 2.09-2.33 (m, 2H, C3, C5-H), 2.99-3.08 (m, 2H, CH ₂ -CH ₂ NHFmoc) , 3.32-3.36 (m, 1H, C5-H), 3.55-3.60 (m, 1H, C2-H), 3.65-3.66 (brs, 8H, CH ₂ ODMT, PhO CH ₃ ), 4.01-4.12 (m, 1H, C4-H), 4.26-4.31 (m, 2H, NHCO ₂ CH ₂ ), 5.16 (s, 1H, Fmoc), 5.20-5.22 (br, 1H, NH), 6.67-6.73 (m, 4H, DMT ), 7.05-7.17 (m, 9H, DMT), 7.22-7.29 (m, 4H, Fmoc), 7.48-7.51 (m, 2H, Fmoc), 7.62-7.66 (m, 2H, Fmoc); Mass FAB ⁺ cal. 754.3618 found 777.3514 (+ Na).

Step 7: Synthesis of 1-N-Fmoc-5-O-DMT Methyl-pyrrolidine-3-O-2-cyanoethyl-N, N-diisopropyl phosphoramidite (Compound 8)

Compound 7 (0.10 g, 0.13 mmol) was dissolved in CH ₂ Cl ₂ (1 mL) and diisopropylethylamine (DIPEA) (67 μl, 0.39 mmol) and 2-cyanoethyl-N, N at 0 ° C. -Diisopropylchlorophosphoamidite (43 μl, 0.20 mmol) was added dropwise and stirred for 1 hour, followed by another 2 hours at room temperature. The reaction mixture was treated with methanol and concentrated under reduced pressure. The residue was dissolved in AcOEt, washed with NaHCO ₃ aqueous solution and brine, dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was separated by column chromatography (Hex: AcOEt = 3: 2, containing 2% TEA) to give compound 8 (0.076 g, 58.8%): ¹ H NMR (CDCl ₃ ) δ1.10-1.15 (m, 2H , CH ₂ -CH ₂ NHFmoc), 1.16 (d, 6H, J = 0.9 Hz, iPr), 1.18 (d, 6H, J = 1.0 Hz, iPr), 1.21-1.31 (m, 2H, CH ₂ -CH ₂ NHFmoc), 1.30-1.46 (m, 2H, CH ₂ -CH ₂ NHFmoc), 1.46-1.57 (m, 2H, CH ₂ -CH ₂ NHFmoc), 1.80-1.90 (m, 1H, C3-H), 2.07- 2.32 (m, 2H, C3, C5-H), 2.64 (t, 2H, J = 5.9 Hz, O CH ₂ CH ₂ CN), 2.95 (m, 1H, iPr), 2.96-3.07 (m, 2H, CH ₂ -CH ₂ NHFmoc), 3.12 (m, 1H, iPr), 3.30-3.34 (m, 1H, C5-H), 3.54-3.59 (m, 1H, C2-H), 3.65-3.66 (brs, 8H, CH ₂ ODMT, PhO CH ₃ ), 3.75 (m, 2H, OCH ₂ CH ₂ CN), 4.00-4.11 (m, 1H, C4-H), 4.24-4.30 (m, 2H, NHCO ₂ CH ₂ ), 5.15 (s, 1H, Fmoc), 5.20-5.22 (br, 1H, NH), 6.66-6.73 (m, 4H, DMT), 7.05-7.17 (m, 9H, DMT), 7.22-7.29 (m, 4H, Fmoc ), 7.48-7.51 (m, 2H, Fmoc), 7.62-7.66 (m, 2H, Fmoc); Mass FAB ⁺ cal. 968.4853 found 991.4754 (+ Na).

Example 2: Synthesis of Linker with CPG

Scheme 2 is FIG. 4 is a view illustrating a method of manufacturing a linker having a controlled pore glass (CPG) according to an embodiment of the present invention. FIG. According to Scheme 2, a linker to which a solid support is attached can be synthesized. With reference to Scheme 2 will be described in detail one embodiment of a method for producing a CPG attached linker according to the present invention.

Step 1: Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5-O-DMT-pyrrolidine-3-succinate (Compound 9)

Compound 7 (0.10 g, 0.13 mmol), succinic anhydride (17 mg, 0.17 mmol) and catalytic amount of DMAP were dissolved in dry CH ₂ Cl ₂ (0.5 mL) and TEA (18 μL, 0.13 mmol) was added dropwise overnight. Stirred. The reaction mixture was diluted with CH ₂ Cl ₂ (5 mL) and washed successively with 5% aqueous citric acid solution (5 mL) and brine (5 mL). The organic layer was dried over MgSO ₄ , concentrated under reduced pressure, and the residue was separated by column chromatography (CH ₂ Cl ₂ : MeOH = 50: 1/20: 1, containing 5% TEA) to thereby give Compound 9 (0.079 g, 71.8%). Obtained: ¹ H NMR (CDCl ₃ ) δ 1.38-1.48 (m, 4H, CH ₂ -CH ₂ NHFmoc), 1.55-1.66 (m, 4H, CH ₂ -CH ₂ NHFmoc), 1.97-2.04 (m, 1H , C3-H), 2.09-2.24 (m, 2H, C3, C5-H), 2.46-2.48 (m, 4H, CH ₂ CH ₂ CO ₂ H), 3.04-3.11 (m, 2H, CH ₂ -CH ₂ NHFmoc), 3.38-3.48 (m, 1H, C5-H), 3.54-3.60 (m, 1H, C2-H), 3.69 (brs, 8H, CH ₂ ODMT, PhO CH ₃ ), 4.13-4.15 (m , 1H, C4-H), 4.28-4.33 (m, 2H, NHCO ₂ CH ₂ ), 5.18-5.23 (m, 1H, Fmoc), 5.32 (br, 1H, NH), 6.70-6.75 (m, 4H, DMT), 7.10-7.23 (m, 9H, DMT), 7.26-7.30 (m, 4H, Fmoc), 7.50-7.53 (m, 2H, Fmoc), 7.66-7.69 (m, 2H, Fmoc); Mass FAB ⁺ cal. 854.3778 found 877.3679 (+ Na).

Step 2: CPG Adhesion (Compound 10)

Compound 9 (0.025 g, 0.029 mmol), aminopropyl CPG (164 mg, 89.2 μmol / g amine loading, 14.6 μmol), 1-hydroxybenzotriazole (HOBT) (3.9 mg, 29 μmol) and 2 -(1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (11 mg, 29 μmol) was dissolved in dry DMF (4 mL) Diisopropylethylamine (DIPEA) (8.4 μl, 49.3 μmol) was added dropwise under normal temperature, nitrogen stream and stirred for 3 days on a rotary stirrer. The solid support was washed successively with DMF (5 mL × 3) and CH ₃ CN (2 mL × 2) and vacuum dried overnight to afford compound 10 (158 mg, 71.6 μmol / g).

Step 3: Removal of Fmoc Protectors (Compound 11)

Compound 10 (158 mg, 71.6 μmol / g) was capped by vibration stirring in acetic anhydride / lutidine-THF (10% solution, 3 mL) for 1 h and then washed with CH ₃ CN (3 mL × 3). The residue was treated four times for 10 minutes with 20% piperidine / DMF (4 mL) to remove the Fmoc protecting group. The removal status of the Fmoc protecting group was measured continuously at UV 302 nm. The residue was washed with DMF (4 mL x 3) and dried in vacuo to afford compound 11 (150 mg, 71.6 μmol / g, 10.7 μmol).

Step 4: Synthesis of Compound 12

Compound 11 (150 mg, 71.6 μmol / g, 10.7 μmol), TAMRA N-hydroxysuccinimide (NHS) ester (30 μl, 57 μmol) and TEA (15 μl, 171 μmol) were dried with DMF ( 4 ml) and shaken for 3 days. The reaction mixture was washed with DMF (1 mL × 3) and CH ₃ CN (1 mL × 2). The residue was capped by vibration stirring in acetic anhydride / lutidine-THF (10% solution, 2 mL) for 1 h, then washed with CH ₃ CN (5 mL × 3) and vacuum dried for 3 days to give compound 12 (145 mg , 71.6 mol / g, 10.4 mol).

In order to verify the effect of the oligonucleotide as a probe using a linker according to the present invention, the following experiment was performed in which the probe was applied to quantitative detection of genetically modified plants. The genetically modified plant is genetically modified using the 35S promoter of cauliflower mosaic virus.

Experimental Example 1: Plant DNA Extraction

Non-transformed native waxy corn, transgenic maize (BT-11 corn, fluca), untransformed conventional soybean, Roundup Ready Soybean (fluka) or genetically modified to detect genetically modified organisms DNA of each plant of unknown magnitude is reported in Edwards K., et al. According to the method ( Nucleic Acids Research , 19: 1349 (1991)), extraction was carried out as follows: First, 200 μl of the extraction buffer consisting of 200 mM Tris HCl, pH 7.5, 250 mM NaCl, 25 mM EDTA and 0.5% SDS was added. Tissue samples were crushed using a pestle after addition to the tissue of the crop to be investigated. Then, an equal amount of extraction buffer was further added and then spun for 10 minutes. Then, 300 μl of crushed liquid was transferred to a new tube, and then 300 μl of chloroform: isoamyl alcohol (24: 1) was added, followed by inverting and centrifugation at 13,000 rpm for 5 minutes. The supernatant was then transferred to a fresh Eppendorf tube, then 300 μl of isopropanol was added and mixed. The precipitated DNA sample was then washed twice with 70% ethanol and redissolved with 50 μl ddH ₂ O. DNA of genetically modified soybean or corn and DNA of untransformed soybean or corn were prepared by diluting at a concentration of 50 ng / μl, respectively. This is for use as a positive control in the real-time PCR described in Experimental Example 4 below.

On the other hand, known DNA samples with known genetically modified plant content should be used for highly purified products, because if the purity of the DNA sample is low, it will cause a false standard curve. Therefore, in the present invention, a DNA sample having a purity of 99.99% was used as a positive control.

Genetically modified plant content (%) DNA of genetically modified plant (50 ng / μl) DNA of non-genetically modified plant (50 ng / μl) Final volume One 10 μl 990 μl 1000 μl 3 30 μl 970 μl 1000 μl 5 50 μl 950 μl 1000 μl 10 100 μl 900 μl 1000 μl 50 500 μl 500 μl 1000 μl

Experimental Example 2: Preparation of Primer

In order to construct a primer to be used for PCR for detecting a transgenic plant having the 35S promoter of Cauliflower mosaic virus, first, the optimal site to be amplified in the 35S promoter sequence is known (GenBank accession No. AJ251014, X84105, AF044029). And X04879). First, the above known sequences were aligned, the consensus sequence was examined, and conserved sequences were found (see FIGS. 1A and 1B). 1A and 1B, TBI251014, Astdnabv, Af044029, and Arcamvpr each represent GenBank accessionNo. For AJ251014, X84105, AF044029 and X04879, the shaded portion of the region adjacent to the 3'-end is the region showing the conserved sequence. The base sequence of the site having the conserved sequence is shown in the attached SEQ ID NO: 1. In consideration of the fact that it is performed on various plant samples, the site to be amplified by the real-time PCR of the present invention was determined as the site having the above conserved sequence. Then, a forward primer (see SEQ ID NO: 2) and a reverse primer (see SEQ ID NO: 3) to be used for amplification of the selected site were synthesized. In addition to the primers described above, a number of primers such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 which can be hybridized with the above conserved sequence sites or adjacent sites thereof were synthesized, and these primers were also prepared in real time as described in Experimental Example 4 below. PCR showed excellent primer properties.

Experimental Example 3: Preparation of a real-time PCR probe using a linker of the present invention with CPG

In order to prepare a probe added during the real-time PCR of the present invention, first, the site where the probe is to be located on the template DNA was selected. The probe was positioned between the forward primer and the reverse primer prepared in Experimental Example 2, but was prepared to be closer to the forward primer to quickly confirm the result of the amplification.

All probes were synthesized using Expedite 8909 DNA / PNA Synthesizer (PerSeptive Biosystems) in trityl-on at 1 μmol scale according to the instructions for use of the synthesizer. Compound 12 of Example 2 was used for the introduction of a 3′-terminated TAMRA destructor. Introduction of the FAM at the 5'-terminus position includes 5-fluorescein phosphoramidite ([(3 ', 6'-dipivaloylfluoresynyl) -6-carboxamidohexyl] -1-O- ( 2-cyanoethyl)-(N, N-diisopropyl) -phosphoamidite (Glen Research) was used and each base was dA ^bz , dC ^bz , dG ^dmf , T phosphoramidite (ABI) was used For the condensation of FAM at the 5 'terminal position, the condensation time was 3 minutes (typical condensation time is 1 minute) The solid support and protecting group were t-butylamine: methanol: distilled water (1: The solution was lyophilized after being separated by heating at 65 ° C. for 3 hours using a mixture of 1: 2), and the residue was a solution of 100 mM triethylammoonium acetate (hereinafter referred to as “TEAA”) at pH 7. Dissolve in 1 ml and reverse-phase high-quality chromatography (Hamilton PRP-1, 300 mm x 7 mm, 18-38% acetonitrile / 100 mM TEAA, monitored by UV at pH 7, 260 nm and 560 nm) The desired fractions were lyophilized and dissolved by addition of 1 ml of distilled water twice and lyophilized again to completely remove the remaining TEAA salts, after which 1 ml of distilled water was added to the residue and the solution was dried at 70 ° C. The absorbance coefficients of the natural nucleotides for the concentration calculations are as follows: dAMP, 15200: dCMP, 7700: TMP, 8830: dGMP, 11500. FAM, 20958: TAMRA, 31980.

The composition of the oligomer was followed by HPLC (Hewlett Packard, ODS hypersil, C-18; 20 mM K ₂ HPO ₄ , pH 5.6 (A), MeOH (B), after enzymatic digestion (alkaline phosphatase and snack vernom phosphodiesterase)). 100% A: 40% B, 20 minutes) and laser desorption mass spectrometry. The finally synthesized oligonucleotide is 5 'FAM-aag gaa agg cca tcg ttgaag atg c-TAMRA-3' (see SEQ ID NO: 8).

Experimental Example 4: Real-time PCR experiment using the probe of Experimental Example 3

Three real-time PCR reaction solutions were prepared per unknown sample and standard sample for standard curve. The total volume of one real-time PCR reaction was 50 μl and the composition was 39.25 μl of reaction cocktail, AmpliTaq Gold ^TM DNA polymerase (5 U / μl, Perkin-Elmer), 0.25 μl, AmpErase uracil N-glycosylase (UNG) (1 U / μl, Perkin-Elmer) 0.5 μl, DNA template 10 μl (unknown sample and soy or corn positive standard). The composition of the reaction cocktail is as follows: PCR reaction buffer (10X, Roche), MgCl ₂ (3.5 mM), dATP (200 μM), dCTP (200 μM), dGTP (200 μM), dUTP (400 μM), Forward primer (300 nM), reverse primer (300 nM) and probe (200 nM). The composition of the PCR reaction buffer (10X) is 100 mM Tris / HCl, 400 mM KCl, 15 mM MgCl ₂ , 10 mM DTT and 5 μg / ml BSA.

Immediately before starting the PCR reaction, 0.25 μl of AmpliTaq Gold ^™ DNA polymerase and 0.5 μl of AmpErase uracil N-glycosylase (UNG) were added and mixed to 39.25 μl of the PCR cocktail. Subsequently, the PCR cocktail and enzyme mixture were dispensed into each PCR tube, and then a DNA template was added and mixed quickly. The sample used as the DNA template is a positive control obtained in Experiment 1, that is, 10 μl of DNA solution of 1%, 3%, 5%, 10% and 50% transgenic maize or transgenic soybean (50 ng / μl each) , 10 μl of distilled water as a negative control and untransformed soybean or corn DNA solution (50 ng / μl each), 10 μl (50 ng / μl) each of DNA Sample A and Sample B of unknown genetic modification.

Then, pre-denaturation reaction (modified AmpErase uracil N-glycosylase) for 2 minutes at 50 ℃, then activated AmpliTaq Gold ^TM DNA polymerase for 10 minutes at 95 ℃, 15 seconds at 95 ℃ A total of 40 cycles consisting of a denaturation reaction, an annealing for 1 minute at 64 ° C., and an elongation reaction were carried out. Real-time PCR was performed using the Rotor Gene 2000 instrument of Corbett, the degree of amplification of template DNA in real time was measured by fluorescence, and the analysis of PCR results was also obtained by the program in the apparatus described above.

The results of the real time PCR are shown in FIGS. 2 and 3. As can be seen in Figure 2, as the number of PCR cycles increases the intensity of fluorescence in the sigmoid (sigmoid) pattern, it can be seen that the primers and probes produced by the present invention has its own function.

3 shows standard curves obtained from the positive and negative controls. As can be seen in Figure 3, the correlation coefficient of the standard curve (correlation coefficient) is 0.999, very close to the outlier 1, it can be seen that the genetically modified plant content of the unknown sample accurately calculated, unknown sample A is 0.35%, sample It can be seen that B has 0% genetically modified plant. Table 2 summarizes the results of this experiment.

No. sample Sample type Content of given genetically modified plant (%) % Of genetically modified plant measured C _V C _t C _t standard deviation One H ₂ O Negative control 0 0 - - - 2 1% soybean Positive control 1.00 1.00 0.06% 31.45 0.01 3 1% soybean Positive control 1.00 1.05 5.49% 31.38 0.01 4 3% beans Positive control 3.00 2.79 6.88% 30.09 0.11 5 3% beans Positive control 3.00 2.86 4.74% 30.06 0.11 6 5% beans Positive control 5.00 5.23 4.56% 29.26 0.03 7 5% beans Positive control 5.00 5.19 3.77% 29.27 0.03 8 10% beans Positive control 10.00 9.71 2.91% 28.44 0.12 9 50% beans Positive control 50.00 50.34 0.69% 26.26 0.09 10 50% beans Positive control 50.00 50.34 0.69% 26.26 0.09 11 Sample A Unknown sample - 0.37 - 32.78 - 12 Sample A Unknown sample - 0.33 - 32.92 - 13 Sample B Unknown sample - 0 - 38.95 - 14 Sample B Unknown sample - 0.01 - 37.94 -

In Table 1, the C _t value represents a threshold cycle number and represents the number of PCR cycles showing fluorescence that is clearly distinguished from the background fluorescence value.

Experimental Example 5: Comparison of effects of Taqman probe and probe of the present invention

Real time PCR was performed in the same manner as in Example 4 using the probe of the present invention and Taqman probe (ABI) prepared in Example 3, and the effects were compared.

The results are shown in Table 3 below.

sample C _t Taqman probe Probe of the invention 5% beans 31.5 31.33 10% beans 30.67 30.41 50% beans 28.14 27.87

As shown in Table 3, the probe of the present invention showed a slightly faster C _t than Taqman probe, showing excellent sensitivity. Therefore, it can be seen that the probe of the present invention can be usefully used as a probe of real-time PCR.

The invention, which is easy to purify and analyze oligonucleotides, provides a novel linker for use in the modification, including the labeling of oligonucleotides, or for the synthesis of oligonucleotides. The linker of the present invention can be used to introduce a substance such as a reporter into the oligonucleotide, and to facilitate the purification and analysis of the oligonucleotide. In addition, it can be used as a material for the synthesis of oligonucleotides by attaching a phosphoramidite or a solid support to the linker of the present invention.

Claims (9)

Linkers of novel oligonucleotides having the structure Formula 1 In the above formula, HCA is a heterocyclic amine consisting of 5 atoms or 6 atoms; n is 0-10; X is linked to the nitrogen atom of the heterocyclic amine, a C ₁ -C ₁₀ single bond hydrocarbon chain, a hydrocarbon chain having 1-5 hetero molecules at a carbon position of the C ₁ -C ₁₀ single bond hydrocarbon chain, or _{One of the} hydrocarbon chains to which the bond selected from the group consisting of amides, esters, ethers, amines, sulfonyl or combinations thereof is added to the C ₁ -C ₁₀ single bond hydrocarbon chain; R 1 is hydrogen or a hydroxy protecting group; R2 is hydrogen, phosphamidite group or H-phosphonate, or a solid support; And R3 is one of a reporter molecule, a destructor molecule, an amino protecting group, an amino acid or a peptide. The linker of an oligonucleotide according to claim 1, wherein the novel linker represented by Chemical Formula 1 is represented by the following Chemical Formula 2 or Chemical Formula 3: Formula 2 Formula 3 In the above formula, n is 0-10; X is linked to the nitrogen atom of the heterocyclic amine, a C ₁ -C ₁₀ single bond hydrocarbon chain, a hydrocarbon chain having 1-5 hetero molecules at a carbon position of the C ₁ -C ₁₀ single bond hydrocarbon chain, or _{One of the} hydrocarbon chains added to the C ₁ -C ₁₀ single bond hydrocarbon chain a bond selected from the group consisting of amides, esters, amides, esters, ethers, amines, sulfonyls or combinations thereof; R 1 is hydrogen or a hydroxy protecting group; R2 is hydrogen, phosphamidite group or H-phosphonate, or a solid support; And R3 is one of a reporter molecule, a destructor molecule, an amino protecting group, an amino acid or a peptide. The linker of oligonucleotide according to claim 1, wherein n is 0-1. The linker of oligonucleotide according to claim 1, wherein X is a C ₄ -C ₈ hydrocarbon chain having an amide group. The linker of oligonucleotide according to claim 4, wherein X is a C ₆ hydrocarbon chain having one amide group. The linker of oligonucleotide according to claim 1, wherein R1 is a hydroxy protecting group. The linker of oligonucleotide according to claim 6, wherein R1 is a dimethoxytrityl group. The linker of oligonucleotide according to claim 2, wherein the linker is represented by the following Chemical Formula 4: Formula 4 In the above formula, iPr represents an isopropyl group. The linker of oligonucleotide according to claim 2, wherein the linker is represented by the following Chemical Formula 5: Formula 5 In the above formula, n is 0-10; X is connected to the nitrogen atom of the heterocyclic amine, a C ₁ -C ₁₀ single bond hydrocarbon chain, a hydrocarbon chain having 1-5 hetero molecules at a carbon position of the C ₁ -C ₁₀ single bond hydrocarbon chain, or _{One of the} hydrocarbon chains added to the C ₁ -C ₁₀ single bond hydrocarbon chain a bond selected from the group consisting of amides, esters, amides, esters, ethers, amines, sulfonyls or combinations thereof; R 1 is hydrogen or a hydroxy protecting group; Y is a single bond, nucleotide or oligonucleotide; FS is a functional spacer; SSM is a solid support; And R 3 is an amino protecting group, reporter or destructor molecule.