KR20060136482A - Oligonucleotide for detecting target dna or rna - Google Patents

Abstract

Oligonucleotides that can be used to detect the presence of target DNA or RNA in a sample, methods of detecting target DNA or RNA using the oligonucleotides, and kits comprising the oligonucleotides are provided. The oligonucleotide comprises a fluorescently labeled nucleoside and at least one neighboring nucleoside positioned next to the fluorescently labeled nucleoside, the base of the neighboring nucleoside being thymine or It is cytosine.

Description

Oligonucleotide for detection of target DNA or RNA {OLIGONUCLEOTIDE FOR DETECTING TARGET DNA OR RNA}

The present invention relates to oligonucleotides for detecting target DNA or RNA, comprising a nucleoside labeled with a fluorescent substance and at least one specific nucleoside positioned next to the nucleoside labeled with the fluorescent substance.

Sequence selective DNA detection is a powerful tool for observing many biological changes in the field of biotechnology. A new group of oligonucleotide probes, often called molecular beacons, have been developed to facilitate the detection of specific nucleic acid target sequences (Piatek et al., 1998, Nature Biotechnol. 16: 359-363; and Tyagi and Kramer). , 1996, Nature Biotechnol. 14: 303-308). The molecular label is a nucleic acid sequence having a fluorophore and a quencher at the 5 'and 3' ends, respectively. The molecular label forms a stem-loop structure, and the fluorescence emitted from the fluorescent material is absorbed by the fluorescent attenuator when it receives light capable of exciting the fluorescent material.

The molecular label is designed to have a nucleotide sequence complementary to that of the target DNA or RNA of interest. When the molecular label encounters a target sequence having a sequence complementary to its own sequence, hybridization between the sequences takes place to form a double helix, and the resulting torsional force indicates that the stem region of the molecular label is released. Induce. As a result, the fluorescent material is pulled away from the fluorescent attenuating material and eliminates the role of the fluorescent attenuating material.

However, general molecular labels have the following disadvantages. First, the molecular label can detect only target DNA or RNA having a sequence that is completely complementary to its sequence, and second, because the fluorescent and fluorescent attenuators occupy the ends of the molecular label, for example, There is no space to attach any useful functional group that can be used to fix the label, and third, a complex and expensive method must be used to attach the fluorescent attenuator.

Therefore, the inventors have made diligent efforts to develop a new oligonucleotide probe system that solves the above problems.

Accordingly, an object of the present invention comprises (i) a fluorescently labeled nucleoside and (ii) at least one nucleoside located next to the fluorescently labeled nucleoside and having a thymine or cytosine base. To provide an oligonucleotide for detecting target DNA or RNA.

Another object of the present invention is to provide a method for detecting the presence of target DNA or RNA using the oligonucleotide.

Still another object of the present invention is to provide a kit for detecting a target DNA or RNA, comprising the oligonucleotide.

The present invention provides a target DNA comprising (i) a fluorescently labeled nucleoside and (ii) at least one nucleoside located next to the fluorescently labeled nucleoside and having a thymine or cytosine base. Or it relates to oligonucleotides for RNA detection.

Oligonucleotide of the present invention is characterized in that it contains a fluorescent material without a fluorescent attenuator.

In the present invention, nucleosides labeled with a fluorescent substance such as 2'-dioxyuridine labeled with a fluorescent substance can be prepared by methods known in the art. The fluorescent material may be selected from the group consisting of fluorene, pyrene, fluorescein, rhodamine and coumarin, preferably fluorene. .

Oligonucleotides of the present invention comprise a nucleoside labeled with a fluorescent substance and at least one nucleoside located next to the nucleoside labeled with the fluorescent substance and having a thymine or cytosine base. In preparing the oligonucleotides according to the present invention, any method known in the art for synthesizing oligonucleotides can be used. Preferably, an automated DNA synthesizer is used.

The phosphor may be present at any position within the oligonucleotide, but is preferably located at the center of the oligonucleotide.

Oligonucleotides of the present invention also prevent the use of fluorescent attenuators because thymine or cytosine based nucleosides located next to the fluorescently labeled nucleosides play an important role in attenuating the fluorescence emitted from the fluorescent material. It is characterized by the needlessness.

When the oligonucleotides of the present invention hybridize with a target DNA or RNA having a perfectly matched sequence, the fluorescence intensity increases rapidly compared to the fluorescence intensity of the oligonucleotide in the free state. On the other hand, when the sample tested contains DNA or RNA having a base that does not match only one sequence of the oligonucleotide of the present invention, the fluorescence intensity is markedly reduced compared to the fluorescence intensity of the oligonucleotide in the free state. Thus, oligonucleotides according to the present invention can be advantageously used for the detection of the presence of target DNA or RNA with fully matched or single base mismatched sequences in a sample.

One example of an oligonucleotide according to the present invention has one of the nucleotide sequences of SEQ ID NOs: 1 and 6 (see FIGS. 2 and 4).

Oligonucleotides of the present invention may form a stem-loop structure like a typical molecular label, but are not limited to the class of oligonucleotides that form the stem-loop structure.

Oligonucleotides of the present invention can be usefully used to detect the presence of a target DNA or RNA having a nucleotide sequence that is completely identical or has a single base mismatch with the oligonucleotide. Specifically, the oligonucleotides of the present invention may hybridize with DNA or RNA in a sample and then measure the fluorescence intensity to check whether the fluorescence intensity increases or decreases relative to the fluorescence intensity of the free oligonucleotide. When the sample comprises DNA or RNA having a nucleotide sequence complementary to the oligonucleotide of the present invention, the fluorescence intensity increases at a rate of 2 or more compared to the fluorescence intensity of the free form oligonucleotide, while the single base mismatched base When DNA or RNA having a sequence is present in the sample, the fluorescence intensity decreases to a size of 0.1 to 0.3 times as compared to the value of the oligonucleotide in the free state. Thus, oligonucleotides of the present invention can detect DNA or RNA with sequences that are completely identical or have single base mismatches. Thus, the oligonucleotides of the present invention can be used as an efficient fluorescence ON / OFF system for detecting DNA or RNA with fully identical or single base mismatched sequences.

The oligonucleotides of the present invention do not contain any fluorescent attenuator at their ends, making the manufacturing process simple and allowing the introduction of any functional group that can be used for a wide range of applications at the free ends.

The present invention also relates to (i) reacting an oligonucleotide of the invention with a sample so that any possible hybridization occurs and (ii) measuring the intensity of fluorescence emitted from the hybridization mixture so that (iii) the target DNA or RNA is A method of detecting the presence of a target DNA or RNA in a sample, comprising determining whether it is present in the sample.

The present invention further provides a kit for detecting a target DNA or RNA comprising the oligonucleotide of the present invention. The kit may further include those known in the art, such as general buffers, additives used for hybridization.

The above and other objects and features of the present invention will become apparent from the following description of the invention in conjunction with the accompanying drawings:

1 is a schematic diagram for preparing 2'-dioxyuridine labeled with a fluorescent material,

2 is an example of an oligonucleotide (SEQ ID NO: 1) according to the present invention;

3 is a fluorescence spectrum observing a fully matched nucleotide (SEQ ID NOs: 1 and 5) and a single base mismatched nucleotide (SEQ ID NOs: 1 and 2, SEQ ID NOs: 1 and 3, SEQ ID NOs: 1 and 4),

4 is the stem-loop structure of SEQ ID NO: 6,

5 is a fluorescence spectrum observing a fully matched nucleotide (SEQ ID NOs: 6 and 7) and a single base mismatched nucleotide (SEQ ID NOs: 6 and 8).

The following examples are only for illustrating the present invention, but the present invention is not limited thereto.

¹ H, ¹³ C and ³¹ P NMR spectra were analyzed on Bruker NMR spectrometers (Aspect 300 MHz), FAB mass spectra and JEOL four sector tandem mass spectrometers (JMS-HX / HX110A). Obtained using. Furthermore, the percentages in the solid / solid mixtures, liquid / liquid mixtures and solid / liquid mixtures presented below represent weight / weight, volume / volume and weight / volume, respectively. Unless otherwise noted, all reactions were carried out at room temperature.

Example 1 Preparation of 2′-Deoxyuridine Labeled with Fluorescent Materials

(1-1) 5'- O - [bis (4-methoxyphenyl) phenylmethyl] -2'-deoxy-5- (fluorenyl ethynyl 2) Preparation of uridine

5-iodo-5'-dimethoxytrityl-2'-dioxyuridine (497 mg, 0.757 mmol) (see Compound 1 in Figure 1) and 2-ethynylfluorene (231 mg, 1.21 mmol) To a solution obtained by dissolving in Et ₃ N (4 mL) and THF (12 mL), (PPh ₃ ) ₂ PdCl ₂ (53 mg, 0.076 mmol) and CuI (14 mg, 0.074 mmol) were added. Argon was bubbled into the mixture for 2 minutes and the mixture was pumped / purged 10 times. The mixture was then stirred at 45-50 ° C. for 2 hours and the solvent was evaporated under reduced pressure. As a result, the residue was subjected to column chromatography (Merck 60 silica gel, 230-400 mesh) using hexane / EtOAc (1: 5) as the elution solution to obtain the title compound (434 mg, 80%), which was CHCl Recrystallized from ₃ / MeOH (1: 1) (see compound 2 in FIG. 1).

M.p. 160-161 ° C.

[a] ¹⁴ D = + 40 ° ( c = 1.05, CHCl ₃ ).

IR (film): ν 3425, 3185, 3013, 2932, 2836, 2261, 1700, 1608, 1508, 1453, 1251, 1177, 1094, 1034, 829, 767, 701, 668 cm ^-1 .

¹ H NMR (300 MHz, CDCl ₃ ): δ 10.26 (br s, 1H; NH), 8.32 (s, 1H; H-6), 7.73 (d, J = 7.4 Hz, 1H: fluorene-H), 7.58-7.51 (m, 4H; fluorene-H), 7.43-7.26 (m, 2H + 6H; fluorene-H and DMT-H), 7.15 (d, J = 7.5 Hz, 2H; DMT-H), 7.04 ( br s, 1H; DMT-H), 6.83 and 6.81 (2d, J = 8.5 Hz, 4H; DMT-H), 6.48 (t, J = 5.9 Hz, 1H; H-1 ′), 4.63 (br s, 1H; H-3 '), 4.26 (br s, 1H; H-4'), 3.99 (br s, 1H; OH), 3.71 (s, 2H; ArCH ₂ ), 3.64 and 3.63 (2s, 6H; OCH ₃ ), 3.50 (br d, J = 9.2 Hz, 1H; H-5 '), 3.33 (br d, J = 8.1 Hz, 1H; H-5'), 2.68 (br s, 1H; H-2 ' ), 2.39 (br s, 1 H; H-2 ').

¹³ C NMR (75 MHz, CDCl ₃ ): δ 162.0, 158.3, 149.6, 144.4, 143.4, 142.5, 142.0, 141.5, 140.9, 135.5, 135.4, 130.2, 129.9, 129.8, 128.1, 127.9, 127.8, 126.9, 126.7, 124.9, 120.3, 120.0, 119.2, 113.2, 100.7, 94.5, 86.8, 85.9, 79.9, 77.2, 72.3, 63.5, 55.0, 41.5, 36.5.

HRMS-FAB ( m / z ): [M + Na] ⁺ calcd for C ₄₅ H ₃₈ N ₂ O ₇ Na, 741.2578; found, 741.2577.

(1-2) 5'- O- [bis (4-methoxyphenyl) phenylmethyl] -2'-dioxy-5- (2-ethynylfluorenyl) -3 '-[2-cyanoethyl Preparation of Bis (1-methylethyl) phosphoramidyl] uridine

In the solution obtained by dissolving the compound prepared in Example (1-1) (301 mg, 0.44 mmol) and 4-methylmorpholine (140 μL, 1.27 mmol) in CH ₂ Cl ₂ (12 mL) at room temperature. 2-cyanoethyldiisopropyl chlorophosphoramidate (120 μL, 0.537 mmol) was added dropwise. After the reaction was completed (about 30 minutes), the mixture was concentrated under reduced pressure and purified by column chromatography (Merck 60 silica gel, 230-400 mesh) using hexanes / EtOAc (1: 1) to give the title compound ( 285 mg, 74%) (see compound 3 in FIG. 1).

M.p. 98-100 ° C.

[α] ¹⁴ D = + 35 ° ( c = 0.995, CHCl ₃ ).

IR (film): ν 3186, 3016, 2967, 2837, 2254, 1699, 1608, 1581, 1508, 1455, 1364, 1304, 1251, 1178, 1155, 1035, 880, 830, 771, 667 cm ^-1 .

¹ H NMR (300 MHz, CDCl ₃ ): δ 8.33 and 8.28 (2s, 1H; NH), 7.74 and 7.71 (2s, 1H; H-6), 7.57-7.48 (m, 4H; fluorene-H), 7.40 -7.26 (m, 3H + 6H; fluorene-H + DMT-H), 7.17-7.05 (m, 2H; DMT-H), 6.99 and 6.95 (2s, 1H; DMT-H), 6.80 (d, J = 8.7 Hz, 4H; DMT-H), 6.39 (dd, J = 12.7, 5.6 Hz, 1H; H-1 '), 4.63 (br s, 1H; H-3'), 4.26 and 4.21 (2br s, 1H ; H-4 '), 3.88-3.75 (m, 1H; PCH ₂ ), 3.71 and 3.70 (2s, 2H; ArCH ₂ ), 3.67 and 3.65 (2s, 6H; OCH ₃ ), 3.64-3.50 (m, 4H NCH, PCH ₂ , H-5 '), 3.32-3.28 (m, 1H; H-5'), 2.66-2.58 (m, 2H; CH ₂ CN, H-2 '), 2.46-2.35 (m, 2H; CH ₂ CN, H-2 '), 1.18 and 1.07 (2d, J = 6.7 Hz, 12H; NCHC H ₃ ).

¹³ C NMR (75 MHz, CDCl ₃ ): δ 161.4, 158.5, 149.2, 1443, 144.3, 143.5, 142.5, 141.8, 141.6, 141.0, 135.4, 130.3, 129.9, 128.2, 128.0, 127.0, 126.8, 125.0, 120.4, 120.1, 119.2, 117.6, 117.4, 113.2, 100.8, 94.5, 87.0, 86.2, 85.7, 79.8, 63.2, 63.0, 58.2, 58.0, 55.1, 43.3, 43.2, 43.1, 43.0, 40.8, 36.5, 24.6, 24.5, 20.4, 20.3, 20.2, 20.1, 19.2.

³¹ P NMR (121 MHz, CDCl ₃ ): δ 151.6, 151.1.

HRMS-FAB ( m / z ): [M + Na] ⁺ calcd for C ₅₄ H ₅₅ N ₄ O ₈ PNa, 941.3658; found, 941.3654.

Example 2: Synthesis of Oligonucleotides According to the Invention

The compound obtained in Example (1-2) was subjected to an automated DNA synthesizer (PerSeptive Biosystems 8909 Expedite ^™ Nucleic Acid on a Controlled Pore Glass 9CPG solid support as a building block for preparing fluorescence oligonucleotides of SEQ ID NOs: 1-6). Synthesis System) was introduced as the phosphoramidite approach. The prepared oligonucleotides were analyzed by MALDI-TOF mass spectrometer as follows:

Oligonucleotide of SEQ ID NO: 1, calcd m / z 4717, found 4723; And

Oligonucleotide of SEQ ID NO: 6, calcd m / z 5903, found 5903.

Furthermore, oligonucleotides of SEQ ID NOs: 2 to 5, 7 and 8, which are target DNA candidates, were prepared using the same automated DNA synthesizer.

As shown in Table 1, the oligonucleotides of SEQ ID NOs: 5 and 7 have base sequences complementary to SEQ ID NOs: 1 and 6, respectively. On the other hand, the oligonucleotides of SEQ ID NOs: 2 to 4 and the oligonucleotides of SEQ ID NO: 8 have base sequences that do not match only one base with SEQ ID NOs: 1 and 6, respectively.

The synthesized oligonucleotides are cleaved from the solid support by treating 30% aqueous NH ₄ OH (1.0 mL) at 55 ° C. for 10 hours. The crude product obtained by automated oligonucleotide synthesis was lyophilized and diluted with distilled water (1 mL). The oligo nucleotide was purified by ^{HPLC (Merck LichoSPHER ® 100 RP-} 18 endcapped column, 10 × 250 mm, 5 μm). The HPLC mobile phase was maintained isocratically with 5% acetonitrile / 0.1 M triethylammonium acetate (TEAA) (pH 7.0) at a flow rate of 2.5 mL / min for 10 minutes. The concentration gradient was then linearly increased for at least 10 minutes from 5% acetonitrile / 0.1 M TEAA to 50% acetonitrile / 0.1 M TEAA at the same flow rate. Fractions containing purified oligonucleotides were collected and lyophilized. 80% aqueous AcOH was added to the oligonucleotides. After 30 minutes at ambient temperature the solvent was evaporated under reduced pressure. The residue was diluted with distilled water (1 mL) and then purified by HPLC under the same conditions as described above. For characterization, matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry data of the oligonucleotides were analyzed using Voyager RP (PerSeptive Biosystems, Framingham, MA, USA) time-of-flight (TOF). ) was collected by a dual-stage reflector mass spectrometer. The instrument used a nitrogen laser at 337 nm to desorb / ionize the sample. The acceleration voltage was 20 kV and the flight distance was 1.1 m.

Example 3: Measurement of Fluorescence Intensity

In terms of whether the fluorescing oligonucleotides (SEQ ID NOs: 1 and 6) of the present invention can be used to detect targets with fully identical or single base mismatched sequences, are examined as follows:

The oligonucleotides of SEQ ID NO: 1 are molar ratios of 1: 1 in each oligonucleotides of SEQ ID NOs: 2-5 and buffer (100 mM NaCl, 20 mM MgCl ₂ and 10 mM Tris-HCl buffer, pH 7.2). MD-5020 PTI model using a 0.5 × 2 cm quartz cuvette with hybridization, and its steady-state fluorescence (FL) spectrum with a bandwidth of 15 nm and a light transmission distance of 1 cm Obtained by a microscope photometer. The cell support was insulated with circulating water controlled by PolyScience digital temperature controller 9110. Fluorescence measurements were performed under the same buffer as used in the hybridization. Fluorescence emission spectra are shown in FIG. 3. Fluorescence intensities measured at the maximum wavelength (λ max) of 425 nm are listed in Table 2.

Thus, for duplexes where all base pairs between oligonucleotides were completely identical (oligonucleotide duplexes of SEQ ID NOs: 1 and 5), the fluorescence intensity increased markedly. On the other hand, in the case of duplexes with one base pair mismatch (SEQ ID NOS: 1 and 2, 1 and 3, and oligonucleotide duplexes of 1 and 4), the fluorescence intensity decreased drastically.

In addition, the above experiment was repeated for fluorescence oligonucleotides of SEQ ID NO: 6 except for using oligonucleotides of SEQ ID NOs: 7 and 8 instead of oligonucleotides of SEQ ID NOs: 2-5. The observed fluorescence emission spectra are shown in FIG. 5 and the fluorescence intensities measured at 425 nm are listed in Table 3.

Thus, when all base pairs between oligonucleotides are completely matched (oligonucleotide duplexes of SEQ ID NOs: 6 and 7), the fluorescence intensity has increased significantly, whereas if one base pair mismatches between oligonucleotides (Oligonucleotide duplexes of SEQ ID NOs: 6 and 8), fluorescence intensity markedly decreased.

While the invention has been described in connection with the specific embodiments described above, it should be understood that those skilled in the art may variously modify and change the invention within the scope of the invention as defined by the appended claims.

It can be seen that the oligonucleotides of the present invention can be advantageously used to detect DNA or RNA with nucleotide sequences that are completely identical or single base mismatched by measuring changes in fluorescence intensity.

SEQUENCE LISTING <110> POSTECH FOUNDATION <120> OLIGONUCLEOTIDE FOR DETECTING TARGET DNA OR RNA <130> PCA40323 / PSC <150> US 60 / 561,146 <151> 2004-04-12 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> An exemplary oligonucleotide of the present invention <220> <221> misc_structure <222> (8) N is fluorophore-labeled 2'-deoxyuridine <400> 1 tggactcnct caatg 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide having single base noncomplementary to SEQ NO: 1 <400> 2 cattgagtga gtcca 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide having single base noncomplementary to SEQ NO: 1 <400> 3 cattgaggga gtcca 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide having single base noncomplementary to SEQ NO: 1 <400> 4 cattgagcga gtcca 15 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide having complementary to SEQ NO: 1 <400> 5 cattgagaga gtcca 15 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> An exemplary oligonucleotide of the present invention <220> <221> modified_base <222> (10) N is fluorophore-labeled 2'-deoxyuridine <400> 6 ttctgactcn ctttcagaa 19 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide having complementary to SEQ NO: 6 <400> 7 ttctgaaaga gagtcagaa 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide having single base noncomplementary to SEQ NO: 6 <400> 8 ttctgaaagc gagtcagaa 19  

Claims (10)

Target DNA or RNA detection comprising (i) a fluorescently labeled nucleoside and (ii) the fluorescent material is located next to the labeled nucleoside and has at least one nucleoside having a thymine or cytosine base Oligonucleotides. The method of claim 1, Oligonucleotide, characterized in that the fluorescently labeled nucleoside is 2'-dioxyuridine. The method of claim 1, Oligonucleotide, characterized in that the fluorescent material is located in the center of the oligonucleotide. The method of claim 1, Oligonucleotide, characterized in that the fluorescent material is selected from the group consisting of fluorene (pyrene), pyrene (pyrene), fluorescein (fluorescein), rhodamine (rhodamine) and coumarin (coumarin). The method of claim 4, wherein Oligonucleotide, characterized in that the fluorescent material is fluorene. The method according to any one of claims 1 to 5, Oligonucleotide having a nucleotide sequence of SEQ ID NO: 1. The method according to any one of claims 1 to 5, Oligonucleotides that form a stem-loop structure. The method of claim 7, wherein Oligonucleotide having a nucleotide sequence of SEQ ID NO: 6. (i) reacting the oligonucleotide according to any one of claims 1 to 5 with a sample so that any possible hybridization occurs; (ii) measuring the intensity of fluorescence emitted from the hybridization mixture; (iii) determining whether a target DNA or RNA is present in the sample, A method for detecting the presence of target DNA or RNA in a sample. A target DNA or RNA detection kit comprising the oligonucleotide according to any one of claims 1 to 5.

Images