Single-stranded oligonucleotides labeled with fluorescent and quenching substances
By introducing non-complementary base pairs and binding quenching substances into the hairpin structure of single-stranded oligonucleotides, the problem of low synthesis efficiency is solved, and the effect of efficient synthesis of labeled oligonucleotides is achieved.
Patent Information
- Application Number
- CN202380046720.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-06-20
- Filing Date
- 2023-03-30
- Publication Date
- 2025-02-28
AI Technical Summary
Synthesis efficiency is low when synthesizing single-stranded oligonucleotides labeled with fluorescent substances and quenching substances.
By introducing non-complementary base pairs into the hairpin structure of a single-strand oligonucleotide and combining the quenching substance on the bases of the nucleotide residues, a three-layer chain structure that can be cut off by the flap endonuclease is formed, so that the fluorescent substance and the quenching substance are free and luminous.
The synthesis efficiency of single-stranded oligonucleotides labeled with fluorescent substances and quenching substances is significantly improved, and high yields of target oligonucleotides can be produced in solid phase synthesis.
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Abstract
Description
Technical Field
[0001] The present invention relates to a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance. This application claims priority based on Japanese Patent Application No. 2022-099034 filed in Japan on June 20, 2022, the contents of which are incorporated herein by reference. Background Art
[0002] As a method for detecting a trace amount of an analyte molecule, a method utilizing FRET (Fluorescence resonance energy transfer) is known.
[0003] For example, in genetic diagnosis, there are many methods for accurately and rapidly detecting and quantifying target nucleic acids, among which Invasive Cleavage Assay (ICA) is excellent in operability and reaction stability (see, for example, Non-Patent Document 1).
[0004] Here, refer to Figure 1 ICA will be described. Figure 1 is a schematic diagram for explaining an example of ICA. Figure 1 In the example of , the presence of T (thymine) base 101 in the target nucleic acid 100 is detected. First, a flap probe 110 and an invader probe 120 complementary to the target nucleic acid 100 are hybridized. As a result, the invader probe 120 hybridizes to a portion of the target nucleic acid 100 adjacent to the position to which the flap probe 110 hybridizes. Furthermore, at least one base at the 3' end of the invader probe 120 invades the position of the 5' end of the region 141 where the flap probe 110 hybridizes with the target nucleic acid 100, forming a first triple-stranded structure 130.
[0005] Next, when flap endonuclease is reacted with the first triplex structure 130, the flap site 140 of the first triplex structure 130 is cut to generate the nucleic acid fragment 140. Next, the nucleic acid fragment 140 hybridizes with the nucleic acid fragment 150 to form the second triplex structure 160.
[0006] exist Figure 1 In the example of , the 5' end of the nucleic acid fragment 150 is bound to a fluorescent substance F, and a quenching substance Q is bound to the 3' side several bases away from the 5' end of the nucleic acid fragment 150. The fluorescent substance F and the quenching substance Q are located in close proximity in space. Therefore, the fluorescence emitted by the fluorescent substance F can be quenched by the quenching substance Q.
[0007] Next, when the flap endonuclease is reacted with the second three-layer chain structure 160, the flap site 170 of the second three-layer chain structure 160 is cut, generating a nucleic acid fragment 170. As a result, the fluorescent substance F is released from the quenching substance Q, and emits fluorescence under the irradiation of the excitation light. By detecting this fluorescence, the presence of the T (thymine) base 101 in the target nucleic acid 100 can be detected.
[0008] Prior art literature
[0009] Patent Literature
[0010] Patent Document 1: Japanese Patent Application Publication No. 2004-309405
[0011] Non-patent literature
[0012] Non-patent document 1: Eis P Set al, An invasive cleavage assay for direct quantitation of specific RNAs, Nature Biotechnology, 19(7), 673-676, 2001. Summary of the invention
[0013] Problems to be solved by the invention
[0014] The inventors have found that when synthesizing a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, the synthesis efficiency is sometimes low. The present invention aims to provide a technique for improving the synthesis efficiency of a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance.
[0015] Means for solving problems
[0016] The present invention includes the following aspects.
[0017] [1] A single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, wherein the single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-stranded structure is formed at the 5' terminal portion, and the fluorescent substance and the quenching substance are freed by being cut by a flap nuclease that recognizes the triple-stranded structure, and emit fluorescence under the irradiation of excitation light; wherein the hairpin structure contains non-complementary base pairs inside, and the quenching substance is bound to the base of the nucleotide residue.
[0018] [2] The single-stranded oligonucleotide according to claim 1, wherein the fluorescent substance is labeled on the nucleotide residue at the 5' end, and the quenching substance is labeled on the nucleotide residue located more on the 5' side among the nucleotide residues constituting the non-complementary base pair.
[0019] [3] The single-stranded oligonucleotide according to [1] or [2], wherein the base of the nucleotide residue at the 5′ terminal is a pyrimidine base.
[0020] [4] The single-stranded oligonucleotide according to any one of [1] to [3], wherein the quencher is labeled at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.
[0021] [5] A method for improving the synthesis efficiency of a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, wherein as the single-stranded oligonucleotide, the following single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance is synthesized:
[0022] A hairpin structure is formed on the 5' side by self-hybridization. When a single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-chain structure is formed at the 5' end portion. The triple-chain structure is cut by the flap nuclease that recognizes the flap nuclease, and the fluorescent substance and the quenching substance are freed to emit fluorescence under the irradiation of excitation light; wherein, non-complementary base pairs are contained inside the hairpin structure.
[0023] [6] The method according to [5], wherein the single-stranded oligonucleotide is synthesized as follows: the quencher substance is bound to the base of the nucleotide residue.
[0024] Effects of the Invention
[0025] According to the present invention, it is possible to provide a technique for improving the synthesis efficiency of a single-stranded oligonucleotide labeled with a fluorescent substance and a quencher substance. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a schematic diagram for explaining an example of invasion lysis assay (ICA). DETAILED DESCRIPTION
[0027] [Single-stranded oligonucleotide]
[0028] In one embodiment, the present invention provides a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, which is the following single-stranded oligonucleotide: a hairpin structure is formed on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed and emit fluorescence under the irradiation of excitation light by the flap nuclease that recognizes the triple-chain structure; wherein the interior of the hairpin structure contains non-complementary base pairs, and the quenching substance is bound to the base of the nucleotide residue.
[0029] The single-stranded oligonucleotide of this embodiment is a fluorescent substrate for ICA, and can also be said to be a substrate for flap endonuclease. Examples of flap endonucleases include flap endonuclease 1 (NCBI accession number: WP_011012561.1, Holliday junction 5′flap endonuclease (GEN1)) (NCBI accession number: NP_001123481.3), excision repair protein (NCBI accession number: AAC37533.1), and the like.
[0030] Here, the single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization. When a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-stranded structure formed at the 5' terminal portion corresponds to Figure 1 The second triple strand structure 160 is shown. That is, the single-stranded oligonucleotide having a base sequence complementary to the 3′ side corresponds to the nucleic acid fragment 140 derived from the flap site 140 .
[0031] As described later in the Examples, by including a non-complementary base pair inside the hairpin structure, the synthesis efficiency of a single-stranded oligonucleotide labeled with a fluorescent substance and a quencher substance can be significantly improved compared to a case where the hairpin structure does not include a non-complementary base pair inside.
[0032] The single-stranded oligonucleotide of the present embodiment has a self-complementary sequence, so if the synthesis is advanced, a ring structure is formed. Therefore, it is believed that the reaction point volume of the elongation of chemical synthesis becomes larger, and the elongation of nucleotides is hindered due to steric hindrance. Therefore, it is believed that by introducing 1 to several mismatched bases in the self-complementary sequence portion (inside of the hairpin structure), steric hindrance can be relaxed, and the obstruction of chemical synthesis elongation can be suppressed. As a result, it is believed that the synthesis efficiency of oligonucleotides can be improved.
[0033] In the single-stranded oligonucleotide of the present embodiment, the number of non-complementary base pairs may be 1 to 3, 1 or 2, or even 1.
[0034] In the single-stranded oligonucleotide of the present embodiment, the quenching substance is bound to the base of the nucleotide residue. As described later in the examples, by binding the quenching substance to the base of the nucleotide residue, the synthesis efficiency of the single-stranded oligonucleotide labeled with the fluorescent substance and the quenching substance can be significantly improved compared with the case where the quenching substance is bound to a portion other than the base of the nucleotide residue. The inventors speculate that by binding the quenching substance to the base portion, steric hindrance is alleviated and the synthesis efficiency is improved.
[0035] The single-stranded oligonucleotide of this embodiment preferably has a length of about 20 to 150 bases. In addition, the number of base pairs forming the hairpin structure is preferably about 5 to 50.
[0036] (Fluorescent substance)
[0037] The fluorescent substance is not particularly limited, and examples thereof include fluorescein, ATTO425, ATTO488, Alexa488, ATTO542, Yakima Y, Redmond R, ATTO643, Alexa647, Alexa680, Alexa568, FAM, ATTO633, Cy5, HiLyte Fluor 647, and ATTO663.
[0038] The single-stranded oligonucleotide of the present embodiment preferably has a fluorescent substance labeled on the nucleotide residue at the 5' end, and a quencher substance labeled on the nucleotide residue located further to the 5' side among the nucleotide residues constituting the non-complementary base pair.
[0039] As described later in the examples, such fluorescent substrates can be synthesized with high synthesis efficiency. In this specification, high synthesis efficiency can also be said to be high yield, for example, in the case of solid phase synthesis based on the phosphoramidite method using a 1 mol column, it means that more than 90 pmol of the target single-stranded oligonucleotide can be synthesized.
[0040] (Quenching substance)
[0041] The quenching substance is not particularly limited as long as it is a substance that can quench the fluorescence of the fluorescent substance used, and examples thereof include Black Hole Quencher (BHQ) (registered trademark)-1, BHQ (registered trademark)-2, BHQ (registered trademark)-3, Fluorescence Quencher 1 (Tide Quencher 1, TQ1), Fluorescence Quencher 2 (Tide Quencher 2, TQ2), Fluorescence Quencher 2WS (Tide Quencher 2WS, TQ2WS), Fluorescence Quencher 3 (Tide Quencher 3, TQ3), Fluorescence Quencher 3WS (Tide Quencher 3WS, TQ3WS), Fluorescence Quencher 4 (Tide Quencher 4, TQ4), Fluorescence Quencher 4WS (Tide Quencher 4WS, TQ4WS), Fluorescence Quencher 5 (Tide Quencher 5, TQ5), Fluorescence Quencher 5WS (Tide Quencher 5WS, TQ6), Fluorescence Quencher 6WS (Tide Quencher 6WS, TQ7), Fluorescence Quencher 7WS (Tide Quencher 7WS, TQ8), Fluorescence Quencher 8WS (Tide Quencher 8WS, TQ9), Fluorescence Quencher 9WS (Tide Quencher 9WS, TQ10), Fluorescence Quencher 10WS (Tide Quencher 11WS, TQ12), Fluorescence Quencher 12WS (Tide Quencher 13WS, TQ14), Fluorescence Quencher Quencher5WS, TQ5WS), Fluorescence Quencher 6WS (Tide Quencher 6WS, TQ6WS), Fluorescence Quencher 7WS (Tide Quencher 7WS, TQ7WS), QSY35, QSY7, QSY9, QSY21, Iowa Black FQ, Iowa Black RQ, etc. As the quenching substance, a substance that can quench the fluorescence of the fluorescent substance used is selected.
[0042] In the case where the fluorescent substance is located near the space of the quenching substance, the fluorescence from the fluorescent substance is quenched by the quenching substance. "Quenched fluorescence" means the following. In the absence of a quenching substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as A. In addition, in the case where the quenching substance is located near the space of the fluorescent substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as B. Here, "quenched fluorescence" means that the value of the above B / A is less than 40%.
[0043] The distance between the fluorescent substance and the quenching substance when the fluorescent substance is located near the space of the quenching substance is not particularly limited as long as the fluorescence emission from the fluorescent substance is suppressed by the quenching substance, but is preferably 10 nm or less, more preferably 5 nm or less, and further preferably 2 nm or less.
[0044] In the single-stranded oligonucleotide of the present embodiment, the quenching substance is bound to the base of the nucleotide residue. As a more specific example of the quenching substance bound to the base of the nucleotide residue, for example, the quenching substance shown in the following formula (1) can be cited. The quenching substance in the following formula (1) is BHQ (registered trademark)-1.
[0045] [Chemical formula 1]
[0046]
[0047] (Oligonucleotide)
[0048] The single-stranded oligonucleotide constituting the fluorescent substrate of the present embodiment is not particularly limited as long as it can exert the effects of the present invention, and may be a natural nucleic acid or a synthetic nucleic acid.
[0049] As natural nucleic acid, for example, genomic DNA, mRNA, rRNA, hnRNA, miRNA, tRNA, etc. can be enumerated. Natural nucleic acid can be nucleic acid recovered from an organism, or nucleic acid recovered from water, organic matter, etc. in contact with an organism. As a method for recovering natural nucleic acid, known methods such as the phenol / chloroform method can be enumerated.
[0050] Examples of synthetic nucleic acids include synthetic DNA, synthetic RNA, cDNA, bridged nucleic acid (BNA), locked nucleic acid (LNA), and the like.
[0051] The synthesis method of nucleic acid is not particularly limited, and known chemical synthesis methods such as solid phase synthesis, known nucleic acid amplification methods, reverse transcription reaction, etc. can be enumerated. As nucleic acid amplification methods, for example, PCR method, LAMP method, SMAP method, NASBA method, RCA method, etc. can be enumerated. Wherein, the single-stranded oligonucleotide of the present embodiment is preferably synthesized by solid phase synthesis.
[0052] Solid phase synthesis can be performed by repeatedly performing a first step and a second step, wherein the first step removes the protective group of a nucleotide derivative having a protective group or an oligonucleotide derivative having a protective group bound to the solid phase to generate a nucleotide or an oligonucleotide, and the second step binds the nucleotide derivative having a protective group to the above-mentioned nucleotide or oligonucleotide.
[0053] As the protecting group, there is no particular limitation, and examples thereof include dimethoxytrityl (DMT), acetyl (Ac); benzoyl (Bz); ether protecting groups such as trityl (Tr), monomethoxytrityl (MMT), and trimethoxytrityl (TMT); acetal protecting groups such as β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), and tetrahydropyranyl (THP); silyl ether groups such as tert-butyldimethylsilyl (TBS), etc. These protecting groups are used when the protected functional group is a hydroxyl group. When the protected functional group is an amino group, etc., a suitable protecting group may be appropriately selected and used.
[0054] The protecting group may be used alone or in combination of two or more. For example, a nucleotide derivative having a protecting group having a different chemical structure may be used each time a nucleotide derivative is bound.
[0055] Nucleotide derivatives with protecting groups can be used in common nucleic acid synthesis methods. As nucleic acid synthesis methods, as an example, the phosphoramidite method can be cited, and as nucleotide derivatives, phosphoramidite-modified nucleotide derivatives can be used. In addition, as the protecting group of the nucleotide derivative, the above-mentioned protecting group can be exemplified.
[0056] As the solid phase, for example, beads can be used. Examples of the material of the solid phase include silicone, glass, quartz, soda-lime glass, and polyamide resin.
[0057] As the functional group protected by the protecting group, a hydroxyl group bonded to the carbon at the 5-position of ribose or deoxyribose can be cited, but it is not limited thereto. As nucleotide derivatives, for example, DMT-dA phosphoramidite, DMT-dT phosphoramidite, DMT-dG phosphoramidite, DMT-dC phosphoramidite, etc. can be cited, but it is not limited thereto. As the nucleotide derivatives, commercially available nucleotide derivatives for nucleic acid synthesis can be used. In addition, the nucleotides from which the nucleotide derivatives are derived can be RNA, or artificial nucleic acids such as BNA (bridged nucleic acids) or PNA (peptide nucleic acid).
[0058] By using a nucleotide derivative to which a fluorescent substance is bound or a nucleotide derivative to which a quenching substance is bound as a nucleotide derivative, a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance can be synthesized.
[0059] As described above, solid phase synthesis can be performed by repeatedly performing a first step and a second step, wherein the first step causes the protective group of a nucleotide derivative having a protective group or an oligonucleotide derivative having a protective group bound to the solid phase to be released to generate a nucleotide or an oligonucleotide, and the second step causes the nucleotide derivative having a protective group to be bound to the above-mentioned nucleotide or oligonucleotide.
[0060] The nucleotide derivative having a protecting group or the oligonucleotide derivative having a protecting group refers to an oligonucleotide in the middle of synthesis, which has a protecting group at the terminal. The protecting group is the same as described above, for example, it can be an acid-decomposable protecting group that is deprotected by the action of an acid.
[0061] In the first step, for example, an acid is allowed to act on an oligonucleotide derivative having a protecting group to remove the protecting group, thereby generating a hydroxyl group at the 5' end of the oligonucleotide. For example, when the protecting group is a DMT group, the removed protecting group is a DMT cation.
[0062] In the second step, a nucleotide derivative having a protecting group is bonded to the deprotected oligonucleotide, the nucleotide chain is extended, and an oligonucleotide derivative having a protecting group at the terminal is formed again.
[0063] The second step may further include the following steps: capping the unreacted functional groups by acetylation, etc., so that they do not participate in the subsequent cycle; oxidizing the binding part between the oligonucleotide and the terminal nucleotide derivative, and converting the trivalent phosphorus to a pentavalent phosphate. By repeating the first step and the second step, the oligonucleotide can be elongated.
[0064] The single-stranded oligonucleotide of this embodiment preferably has a fluorescent substance disposed near the nucleotide residue at the 5' end, and the base of the nucleotide residue at the 5' end is a pyrimidine base. Purine bases tend to quench the fluorescence of the fluorescent substance. Therefore, if the base of the nucleotide residue at the 5' end is a pyrimidine base, the quenching of the fluorescence of the fluorescent substance can be suppressed, and therefore it is preferred.
[0065] [Method for improving the synthesis efficiency of single-stranded oligonucleotides labeled with fluorescent substances and quenching substances]
[0066] In one embodiment, the present invention provides a method for improving the synthesis efficiency of a single-stranded oligonucleotide labeled with a fluorescent substance and a quencher substance, wherein a single-stranded oligonucleotide having a predetermined structure is synthesized as the single-stranded oligonucleotide.
[0067] In the method of this embodiment, the single-stranded oligonucleotide forms a hairpin structure on the 5' side through self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-chain structure is formed at the 5' end portion, which is cut by the flap nuclease that recognizes the triple-chain structure, and the fluorescent substance and the quenching substance are freed and emit fluorescence under the irradiation of excitation light; wherein, the interior of the hairpin structure contains non-complementary base pairs, and the quenching substance is bound to the base of the nucleotide residue.
[0068] As described later in Examples, the inventors found that the synthesis efficiency can be improved by synthesizing an oligonucleotide having the above structure as a single-stranded oligonucleotide.
[0069] In the method of this embodiment, the fluorescent substance, the quenching substance, the oligonucleotide, the method for synthesizing the oligonucleotide, etc. are the same as those described above.
[0070] The single-stranded oligonucleotide preferably has a fluorescent substance labeled on the nucleotide residue at the 5' end, and a quenching substance labeled on the nucleotide residue located further to the 5' side among the nucleotide residues constituting the non-complementary base pair.
[0071] It is preferred that the fluorescent substance is disposed on the nucleotide residue at the 5' terminal of the single-stranded oligonucleotide, and the base of the nucleotide residue at the 5' terminal is a pyrimidine base.
[0072] The single-stranded oligonucleotide is preferably labeled with a quencher substance at a position closer to the 3' side than the fluorescent substance, so that when cleaved by flap endonuclease, an oligonucleotide fragment containing the fluorescent substance is released.
[0073] Example
[0074] [Experimental Example 1]
[0075] (Study on fluorescent substrates)
[0076] The single-stranded oligonucleotides shown in the following Table 1 were synthesized by solid phase synthesis based on the phosphoramidite method, and the yields were compared.
[0077] [Table 1]
[0078] name Serial Number Base sequence (5'→3') Fluorescent Substrate 1 1 F-TCT-T(Q)-AGCCGGTTTTCCGGCTAAGACCTCGGCGCG Fluorescent Substrate 2 2 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG
[0079] The fluorescent substrate 1 (sequence number 1) shown in Table 1 above is a fluorescent substrate that does not contain a non-complementary base pair inside the hairpin structure, and specifically has a structure as shown in the following formula (2). F in the fluorescent substrate 1 represents ATTO488, and Q represents BHQ (registered trademark)-1. As shown in the following formula (2), the fluorescent substrate 1 does not contain a non-complementary base pair inside the hairpin structure. In addition, in the fluorescent substrate 1, the quenching substance is bound to the base of the nucleotide residue.
[0080] [Chemical formula 2]
[0081]
[0082] The fluorescent substrate 2 (sequence number 2) shown in Table 1 above is a fluorescent substrate containing non-complementary base pairs inside the hairpin structure, and specifically has a structure as shown in the following formula (3). F in fluorescent substrate 2 represents ATTO488, and Q represents BHQ (registered trademark)-1. As shown in the following formula (3), fluorescent substrate 2 contains non-complementary base pairs inside the hairpin structure. In the following formula (3), the T (thymine) residue and the G (guanine) residue surrounded by the box are not complementary to each other. In addition, in fluorescent substrate 2, the quenching substance is bound to the base of the nucleotide residue.
[0083] [Chemical formula 3]
[0084]
[0085] A 1 mol column was used for the solid phase synthesis. Table 2 below shows the yield of each single-stranded oligonucleotide.
[0086] [Table 2]
[0087] name Serial Number Yield (pmol) Fluorescent Substrate 1 1 71 Fluorescent Substrate 2 2 93
[0088] The results showed that the fluorescent substrate 2, which contained a non-complementary base pair inside the hairpin structure and to which the quencher substance was bound to the base of the nucleotide residue, had a significantly higher yield than the fluorescent substrate 1.
[0089] Industrial Applicability
[0090] According to the present invention, it is possible to provide a technique for improving the synthesis efficiency of a single-stranded oligonucleotide labeled with a fluorescent substance and a quencher substance.
[0091] Description of Reference Numerals
[0092] 100 Target nucleic acid
[0093] 101 bases
[0094] 110 flap probe
[0095] 120 Intrusion Probe
[0096] 130 The first three-layer chain structure
[0097] 140, 170 flap part (nucleic acid fragment)
[0098] 141 Area
[0099] 150 Nucleic Acid Fragments
[0100] 151 Mismatch site
[0101] 160 The second third-layer chain structure
[0102] F Fluorescent material
[0103] Q Quenching substance
Claims
1. A single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, wherein the single-stranded oligonucleotide forms a hairpin structure at the 5' side by self-hybridization, forms a triple-stranded structure at the 5' end portion when hybridized with a single-stranded oligonucleotide having a base sequence complementary to the 3' side, and is cut by a flap endonuclease that recognizes the triple-stranded structure, so that the fluorescent substance and the quenching substance are freed and emit fluorescence under the irradiation of excitation light; in, The hairpin structure contains non-complementary base pairs in its interior, The quenching substance binds to the base of the nucleotide residue.
2. The single-stranded oligonucleotide according to claim 1, wherein The fluorescent substance is labeled on the nucleotide residue at the 5' end. The quenching substance is labeled on a nucleotide residue located further to the 5′ side among the nucleotide residues constituting the non-complementary base pair.
3. The single-stranded oligonucleotide according to claim 1 or 2, wherein The base of the nucleotide residue at the 5' end is a pyrimidine base.
4. The single-stranded oligonucleotide according to claim 1 or 2, wherein The quencher is labeled at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.
5. A method for improving the synthesis efficiency of a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, wherein the single-stranded oligonucleotide is synthesized as follows: A hairpin structure is formed on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-stranded structure is formed at the 5' end portion, and the triple-stranded structure is cut by the flap endonuclease that recognizes the triple-stranded structure, and the fluorescent substance and the quenching substance are freed and emit fluorescence under the irradiation of the excitation light; in, The interior of the hairpin structure contains non-complementary base pairs.
6. The method according to claim 5, wherein: As the single-stranded oligonucleotide, the following single-stranded oligonucleotide was synthesized: The quenching substance binds to the base of the nucleotide residue.
Citation Information
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Micro-chamber used for detecting unimolecular enzyme activity
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