New method for detecting and quantifying RNA

Abstract

To provide nucleic acid aptamer molecules that can be effectively used for real-time labeling of RNA / DNA in living cells.SOLUTION: The present disclosure relates to a nucleic acid aptamer molecule, a complex containing the aptamer and fluorophore small molecules, a method for detecting intracellular or extracellular RNA, DNA or other target molecules by means of the aptamer molecule, as well as a kit containing the aptamer. The aptamer of the present disclosure can specifically bind to one type of fluorophore small molecules, and can significantly improve their fluorescence intensity under light excitation at suitable wavelength.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a nucleic acid aptamer molecule (aptamer nucleic acid molecule), a method using the nucleic acid aptamer molecule for detecting intracellular or extracellular RNA, DNA or other target molecules, and a kit containing the aptamer. The aptamer of the present invention can specifically bind to a type of fluorophore small molecule and significantly increase the fluorescence intensity when excited with light of an appropriate wavelength. [Background technology]

[0002] RNA is one of the most important biomolecules, and there are many kinds of RNA, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), long non-coding RNA (lncRNA), etc. In recent years, scientists have been discovering the crucial functions that RNA plays in multiple biological activities, including many RNA-protein complexes, such as telomerase, splicing enzymes, ribozymes and riboswitches. In addition, there are many non-coding RNAs in cells, such as short interfering RNA (siRNA), microRNA (microRNA), and long non-coding RNA (lncRNA), which play an irreplaceable role in controlling gene expression at the post-transcriptional level. Therefore, real-time monitoring of RNA transport and metabolic processes in cells is extremely important for research into the relationship between RNA localization, gene expression, and cell control processes.

[0003] Currently, methods for labeling RNA include in situ hybridization, RNA binding protein-fluorescent protein technology, and RNA aptamer-fluorescent dye technology. Fluorescent in situ hybridization is a technology for directly labeling RNA with fluorophore-modified oligonucleotides, and its basic principle is that the fluorophore-modified oligonucleotides have a partial sequence that is complementary to RNA, and realize the labeling of the target RNA through base pairing (Raj et al. Nature Methods 2018.5:877-879). Its advantage is that the target RNA itself does not need to be modified or genetically modified, but is directly labeled. Its disadvantages are that oligonucleotides are difficult to enter cells, require auxiliary procedures for introducing into cells, are prone to damage to cells, have relatively high background fluorescence, require complicated elution procedures, can only be used for the study of fixed cells, and cannot be used for real-time monitoring of the dynamic change process of RNA in living cells. Molecular beacon technology is based on an oligonucleotide probe with a hairpin structure. After binding to a target RNA, the quenching effect of the quenching group labeled at one end on the fluorescent group labeled at the other end is eliminated, and the fluorescent group emits fluorescence, or the FRET of the fluorescent groups at both ends disappears (Chen et al. Nucleic Acids Res 2010.38:e148). The advantage is that molecular beacons have a higher signal-to-noise ratio than other oligonucleotide probes because they have no background fluorescence themselves. The disadvantages are that they are difficult to enter cells, tend to gather in the cell nucleus and emit nonspecific fluorescence, are easily affected by RNA secondary structures, and require oligonucleotide probes, etc. to be specially prepared for each RNA (You et al. Ann. Rev. Biophys 2015.44:187-206), which limits the application of the technology.

[0004] RNA-binding protein-fluorescent protein technology is currently the conventional method for labeling live cell RNA. Conventional RNA-binding protein systems include the MCP / MS2 phage system (Bertrand et al. Molecular Cell 1998.2:437-445), λ N These include the / boxB phage system (Daigle et al. Nature Methods 2007.4:633-636) and the dCas9-sgRNA system (Nelles et al. Cell 2016.165:488-496). The basic principle is to fuse a fluorescent protein with one RNA-binding protein that can bind to a specific RNA sequence to obtain an RNA-binding protein-fluorescent protein fusion protein, and then fuse this specific RNA sequence with a target RNA to achieve the goal of labeling the target RNA with a fluorescent protein through the binding of the RNA-binding protein to the specific RNA sequence. This method is widely used to label RNA in live cells, but the unbound RNA-binding protein-fluorescent protein fusion protein leads to relatively high imaging background fluorescence. Scientists have improved the signal-to-noise ratio of imaging by linking multiple RNA sequences identified by the RNA-binding protein in series to bind multiple fluorescent proteins to one target RNA, but the carrying of a relatively large complex may affect the normal physiological function of RNA.

[0005] Fluorescent RNA technology using RNA aptamer-fluorescent dye is a relatively desirable RNA labeling technology. Its basic principle is that the fluorescent dye has a relatively low fluorescence before binding to the RNA aptamer, and the fluorescence intensity increases after binding to the RNA aptamer. The advantage of this technology is that the system is simple, only the RNA aptamer needs to be fused with the target RNA, and furthermore, it has gene coding properties and modularity, and can be widely used for different RNAs. Early fluorescent RNAs were developed based on thiazole orange and its analogs, or fluorophore-quenching groups (Dolgosheina et al. ACS chemical biology 2014.9:2412-2420; Sunbul et al. Angewandte Chemie 2013.52:13401-13404), and these fluorescent dyes themselves have disadvantages such as high background fluorescence, high cytotoxicity, or difficulty in entering cells, making them unsuitable for labeling live cell RNA, especially mammalian cells. In 2011, S. Jaffrey's research group screened and obtained an RNA aptamer called Spinach based on fluorescent protein chromophore analogue (DFHBI) (Paige et al. Science 2011.333:642-646). Compared with previous fluorescent RNAs, DFHBI has the advantages of low background fluorescence, easy cell entry, and no cytotoxicity. After that, the research group further developed Spinach2, Broccoli and Corn fluorescent RNAs (Song et al. Nature Chemical Biology 2017.13:1187-1194; Filonov et al. Journal of the American Chemical Society 2014.136:16299-16308; Strack et al. Nature methods 2013.10:1219-1224). Although these fluorescent RNAs can be used to label and image multiple highly abundant RNAs in mammalian cells, they still suffer from unstable fluorescence, low brightness, and Mg sensitivity. 2+and dependency on assembly properties, which limits their widespread use. In 2019, Chen et al. developed a fluorescent RNA called Pepper, which has advantages such as high brightness and high stability and can be used to label and image multiple types of RNA in living cells (Chen et al. Nature Biotechnology 2019.37:1287-1293). However, in living cells, multiple types of RNA need to be simultaneously involved in the execution of many cellular functions, so it is necessary to develop multiple types of fluorescent RNA with excellent properties and bioorthogonal capabilities to realize labeling and imaging of multiple types of RNA in cells and explore their biological functions. Summary of the Invention

[0006] The present invention provides nucleic acid aptamer molecules, DNA molecules encoding the nucleic acid aptamer molecules, conjugates between nucleic acid aptamer molecules and fluorophore molecules, and uses of the conjugates.

[0007] The present invention provides the following configurations.

[0008] 1. The present invention relates to a nucleic acid aptamer molecule comprising a nucleotide sequence (a), a nucleotide sequence (b), or a nucleotide sequence (c), The nucleotide sequence (a) is 1 AGAUUGUAAACAN 14 -N 15 -N 16 GACACUN 23 (General formula: Carrot structure) 1 , N 14 , N 15 , N 16 and N 23 represents a fragment equal to or greater than one nucleotide in length, and N 1 and N 23 At least one pair of bases in the nucleotide sequence forms a complementary base pair, 14 and N 16At least one pair of bases in the nucleotide sequence forms a complementary base pair; The nucleotide sequence (b) has at least 70% identity with the nucleotide sequence (a); The nucleotide sequence (c) is a nucleotide sequence (a) containing N 1 , N 14 , N 15 , N 16 and N 23 and has aptamer function and is derived from nucleotide sequence (a).

[0009] 2. In some embodiments of the present application, the nucleic acid aptamer molecule is a sequence having at least 72%, 77%, 83%, 88%, 94% or 100% identity to the Carrot structure nucleotide sequence of nucleotide sequence (a).

[0010] 3. In some embodiments of the present application, the nucleotide sequence (c) is a nucleotide sequence having a N in the Carrot structure nucleotide sequence defined in the nucleotide sequence (a). 1 , N 14 , N 15 , N 16 and N 23 A nucleic acid aptamer molecule in which 5, 4, 3, 2 or 1 nucleotides have been substituted, deleted and / or inserted at positions not containing:

[0011] 4. In some embodiments of the present application, the nucleotide sequence (c) is a nucleotide sequence (a) having N 1 , N 14 , N 15 , N 16 and N 23 The nucleic acid aptamer molecule is a nucleic acid aptamer molecule in which 4, 3, 2 or 1 nucleotide is substituted at positions that do not contain:

[0012] 5. In some embodiments of the present application, N in the nucleotide sequence (a) 1 and N 23 If forms a complementary base pair, then N1 The nucleotide sequence is 5'-3' oriented, 23 The nucleotide sequence is oriented 3'-5', 14 and N 16 If forms a complementary base pair, then N 14 The nucleotide sequence is 5'-3' oriented, 16 The orientation of the nucleotide sequence is 3'-5'.

[0013] 6. In some embodiments of the present application, N 1 and N 23 If at least one fragment in is equal to or greater than five nucleotide bases in length, 1 and N 23 At least two pairs of bases in the nucleotide sequence form complementary base pairs, 14 and N 16 If at least one fragment in is equal to or greater than five nucleotide bases in length, 14 and N 16 At least two pairs of bases in the nucleotide sequence form complementary base pairs.

[0014] 7. The nucleic acid aptamer molecule has nucleotide substitutions in the general Carrot structure of A4U, A4G, A4C, U5A, U5G, U5C, G7C, G7A, G7U, U8C, A10U, A10G, A10C, A11U, A11G, A11C, C12G, C12A, C12U, A13U, A13G, A13C, G17 A, C19A, C19U, A20C, A4C / U5A, A4C / U5C, A4C / A11G, A4C / C12A, A4C / A13C, U5A / A11G, U 5A / C12A, U5A / A13C, U5G / A13C, U5C / G7U, U5C / A11G, U5C / C12G, U5C / C12A, U5C / C12U, U 5C / A13C, G7U / A13C, A11G / A13C, C12G / A13C, C12A / A13C, C12U / A13C, A4C / U5C / A13C, U5C / G7U / A13C, U5C / C12G / A13C, U5C / C12U / A13C, U5C / A11G / A13C, U5C / C12A / A13C, A4 C / U5C / A11G / A13C, A4C / U5C / C12A / A13C, A4C / U5C / G7U / A11G / A13C, A4C / U5C / G7U / C12A / A13C, A4C / U5C / A11G / C12A / A13C, and A4C / U5C / A11G / C12A / A13C.

[0015] 8. In some embodiments of the present application, the nucleotide substitutions into the general Carrot structure are A4C, U5A, U5G, U5C, G7U, A11G, C12G, C12A, C12U, A13C, A4C / U5A, A4C / U5C, A4C / A11G, A4C / C12A, A4C / A13C, U5A / A11G, U5A / C12A, U5A / A13C, U5G / A13C, U5C / G7U, U5C / A11G, U5C / C12G, U5C / C12A, U5 It is one selected from the group consisting of C / C12U, U5C / A13C, G7U / A13C, A11G / A13C, C12G / A13C, C12A / A13C, C12U / A13C, A4C / U5C / A13C, U5C / G7U / A13C, U5C / C12G / A13C, U5C / C12U / A13C, U5C / A11G / A13C, U5C / C12A / A13C, A4C / U5C / A11G / A13C, and A4C / U5C / C12A / A13C.

[0016] 9. In some embodiments of the present application, the nucleotide substitution into the general Carrot structure is one selected from the group consisting of A4C, U5A, U5G, U5C, G7U, A11G, C12G, C12A, C12U, A13C, A4C / U5C, A4C / A13C, U5A / A13C, U5C / A11G, U5C / C12A, U5C / A13C, A11G / A13C, C12A / A13C, A4C / U5C / A13C, U5C / A11G / A13C, U5C / C12A / A13C.

[0017] 10. The nucleic acid aptamer molecule is a nucleotide sequence (a) of N 1 and N 23 The nucleotide sequence at this point is F30 or a tRNA scaffold RNA sequence.

[0018] 11. In some embodiments of the present application, the aptamer molecule is an RNA molecule or a base-modified RNA molecule.

[0019] 12. In some embodiments of the present application, the aptamer molecule is a DNA-RNA hybrid molecule or a base-modified DNA-RNA molecule.

[0020] 13. The nucleic acid aptamer molecule is N 14 -N 15 -N 16 However, it contains one nucleotide sequence that can distinguish the target molecule.

[0021] 14. In some embodiments of the present application, the target molecule includes, but is not limited to, a protein, a nucleic acid, a lipid molecule, a carbohydrate, a hormone, a cytokine, a chemokine, a metabolite, and a metal ion.

[0022] 15. In some embodiments of the present application, N in the nucleic acid aptamer molecule 14 -N 15 -N 16 is a nucleotide sequence capable of distinguishing between S-adenosylmethionine and adenosine molecules.

[0023] 16. In some embodiments of the present application, the aptamer function is that the nucleic acid aptamer can increase the fluorescence intensity of the fluorophore molecule with excitation light of an appropriate wavelength by at least 2-fold, at least 5-10-fold, at least 20-50-fold, at least 100-200-fold, or at least 200-fold or more.

[0024] 17. In some embodiments of the present application, the nucleic acid aptamer molecule further comprises a concatemer that can bind to multiple fluorophore molecules, the concatemers are linked by a spacer sequence of appropriate length, and the number can be 2, 3, 4, 5, 6, 7, 8 or more. The nucleotides of the concatemers can be selected from, but are not limited to, sequences SEQ ID No: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0025] 18. The nucleic acid aptamer molecule has the sequence SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30 or 31.

[0026] 19. A complex of a nucleic acid aptamer molecule and a fluorophore molecule, wherein the nucleic acid aptamer molecule is the above-mentioned nucleic acid aptamer molecule, and the fluorophore molecule has a structure represented by the following formula (I):

[0027] [ka]

[0028] In the formula: Ar 1、 Ar 2 are independently a 6-membered aryl group, a 6-membered heteroaryl group, and D- is HO- or N(X 1 )(X 2 )- and X 1 , X 2 are each independently selected from hydrogen, an alkyl group, and a modified alkyl group; 1 , X 2 may be bonded to each other to form an aliphatic heterocycle together with the N atom, and D- is N(X 1 )(X 2 )-Ar 1 When is a phenyl group, X 1 , X 2 independently form a saturated or unsaturated ester heterocycle with a benzene ring, D- is HO-, and Ar 1 is a phenyl group, at least one hydrogen atom adjacent to HO- is replaced with a halogen, Y is O, S, and R 1 is hydrogen, an alkyl group, or a modified alkyl group; R 2 is a hydrogen atom, a halogen atom, -OH, or -CN, However, each of the "alkyl groups" is independently C 1 -C 10 A straight or branched chain alkyl group, C 1 -C 7 may be a linear or branched alkyl group; 1 -C 5It may be a straight-chain or branched-chain alkyl group, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, an s-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, an isopentyl group, a 1-ethylpropyl group, a neopentyl group, an n-hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 3-methylpentyl group, an isohexyl group, and a 1,1-dimethylbutyl group. a 2,2-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, a 2-ethylbutyl group, a n-heptyl group, a 2-methylhexyl group, a 3-methylhexyl group, a 2,2-dimethylpentyl group, a 3,3-dimethylpentyl group, a 2,3-dimethylpentyl group, a 2,4-dimethylpentyl group, a 3-ethylpentyl group or a 2,2,3-trimethylbutyl group; 20. In some embodiments of the present application, the "modified alkyl group" is independently selected from the group consisting of a halogen atom, -OH, -CO-, -O-, -CN, -SO 3 H, a primary amino group, a secondary amino group, a tertiary amino group, wherein the modified alkyl group has 1 to 10 carbon atoms, and each carbon-carbon single bond may be independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond; The carbon atom being replaced means that the carbon atom, or the carbon atom together with the hydrogen atom thereon, is replaced with the corresponding group; The "modified alkylene group" is C 1 -C 10 (Preferably, C 1 -C 6 any carbon atom of the alkylene group is replaced by a group selected from -O-, -OH, -CO-, -CS-, and -(S=O)-; Preferably, the "modified alkyl group" is a group selected from the group consisting of -OH, -O-, ethylene glycol unit (-(CH 2 CH 2 O) n -), monosaccharide units, -O-CO-, -NH-CO-, -SO 2-O-, -SO-, Me 2 N-, Et 2 Contains one or more groups selected from N-, -SS-, -CH=CH-, F, Cl, Br, I, and a cyano group; Preferably, Ar 1 is a structure selected from the following formulas (II-1) to (II-15),

[0029] [ka]

[0030] Ar 2 is a structure selected from the following formulae (III-1) to (III-25).

[0031] [ka]

[0032] The compound of formula (I) is selected from the following compounds:

[0033] [ka] [ka]

[0034] 21. In some embodiments of the present application, the aptamer molecule in the complex comprises the nucleotide sequence SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30 or 31. 22. The complex as described above for the detection or labelling of a target nucleic acid molecule in vitro or in vivo. 23. The conjugate as described above for detecting or labelling an extracellular or intracellular target molecule. 24. The complex for imaging genomic DNA. 25. The complex for detecting the interaction of a protein with RNA. 26. A DNA molecule that transcribes the nucleic acid aptamer molecule. 27. An expression vector comprising the DNA molecule. 28. A host cell containing the expression vector. 29. A kit comprising said nucleic acid aptamer molecule and / or said expression vector and / or said host cell and / or said complex.

[0035] 30. a) adding said complex to a solution containing a target molecule; b) exciting the complex with light of an appropriate wavelength; c) measuring the fluorescence of the complex; A method for detecting a target molecule comprising the steps of:

[0036] 31. A method for detecting genomic DNA, comprising imaging the genomic DNA with the complex. 32. A method for purifying and purifying RNA, comprising purifying and purifying RNA using the complex.

[0037] The present inventors have designed a novel nucleic acid aptamer molecule, synthesized a novel fluorophore molecule, and constructed a novel fluorophore-nucleic acid aptamer complex, which can improve the fluorescence intensity of the fluorophore molecule under the excitation light of an appropriate wavelength after the aptamer molecule is bound to the fluorophore molecule, thereby eliminating the shortcomings of the previous fluorophore-nucleic acid aptamer complex, and can be effectively used for real-time labeling of RNA / DNA in living cells. The nucleic acid aptamer of the present invention has a relatively strong affinity for the fluorophore molecule and exhibits a different fluorescence spectrum. These nucleic acid aptamer-fluorophore molecule complexes can be used for real-time labeling and imaging of RNA / DNA in prokaryotic and eukaryotic cells, detecting RNA-protein interactions, or as tags for RNA purification and purification. [Brief description of the drawings]

[0038] [Figure 1]Secondary structure prediction of the general structure of the Carrot nucleic acid aptamer molecule. [Diagram 2] Secondary structure prediction of Carrot-1 and Carrot-2 nucleic acid aptamer molecules. [Diagram 3] Secondary structure prediction of F30-Carrot-2. [Figure 4] Secondary structure prediction of tRNA-Carrot-2. [Diagram 5] Characterization of the Carrot-IV-39 complex. (a) Fluorescence excitation and emission spectra of the Carrot-1-IV-39 complex. (b) Measurement of the dissociation constant when Carrot-1 binds to IV-39. (c) Measurement of the Mg2+ dependency of the Carrot-1-IV-39 complex. [Figure 6] Activation effect of Carrot with different base modifications on IV-39. (a) is a schematic diagram of Carrot with different base modifications. In the case of Carrot-3, the dark base in the structural diagram is a deoxyribose nucleotide base, and in the case of Carrot-4, the dark base in the structural diagram is a base modified with 2'-F. (b) is the fluorescence activation effect of Carrot-3 and Carrot-4 on IV-39 fluorophore molecules. In the "control", Carrot-3 or Carrot-4 aptamer RNA was replaced with buffer. [Figure 7] Activation effect of different Carrot concatemers on IV-39. (a) is a schematic diagram of serially linked form 1, (b) is a schematic diagram of serially linked form 2, (c) is a schematic diagram of serially linked form 3, (d) is the fluorescence activation effect of Carrot concatemers in serially linked form 1 on the IV-39 fluorophore molecule, (e) is the fluorescence activation effect of Carrot concatemers in serially linked form 2 on the IV-39 fluorophore molecule, and (f) is the fluorescence activation effect of Carrot concatemers in serially linked form 3 on the IV-39 fluorophore molecule. [Figure 8]Effect of using F30-Carrot-2-IV-4 complex to label RNA in bacteria. [Figure 9] Effect of using F30-Carrot-2-IV-4 complex for labeling RNA in yeast cells. [Figure 10] The effect of using Carrot and IV-39 and its analogs for labeling RNA in mammalian cells. (a) The effect of using tRNA-Carrot-2-IV-39 complex for labeling RNA in mammalian cells, (b) The statistical result of using tRNA-Carrot-2-IV-39 complex for labeling RNA in mammalian cells, (c) The effect of using F30-4Carrot-2 and IV-39 analogs for labeling RNA in mammalian cells. [Figure 11] 1. Probe construction using Carrot-1. (a) Schematic diagram of probe construction, (b) Fluorescence activation effect on IV-39 fluorophore molecules in the absence of probe targets. [Figure 12] 4Carrot-2 traces the location of RNA in cells. (a) 4Carrot-2 was used to identify the location of GAPDH mRNA in cells, and (b) 4Carrot-2 was used to identify the location of ACTB mRNA in cells. [Figure 13] Imaging results using Carrot for detection of genomic DNA. (a) Schematic diagram of different interdigitated sgRNAs. (b) Imaging results of different interdigitated sgRNAs at gene sites in living cells. [Figure 14] These are the results of using Carrot to detect RNA-protein interactions. [Figure 15] This is the result of using Carrot for RNA purification and purification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention will now be described in more detail with reference to the following definitions and examples. The disclosures of all patents and publications referred to herein, including all sequences disclosed in such patents and publications, are expressly incorporated herein by reference. Hereinafter, the terms "nucleotide" and "nucleotide base" are used interchangeably and have the same meaning.

[0040] nucleic acid aptamer molecule The "nucleic acid aptamer molecule" according to the present invention is also called an "aptamer molecule." The nucleic acid aptamer molecule comprises: (a) N 1 AGAUUGUAAACAN 14 -N 15 -N 16 GACACUN 23 (corresponding to the general Carrot structure in FIG. 1 ), or (b) a sequence having at least 70% identity to said nucleotide sequence (a), with the proviso that 1 and N 23 At least one pair of bases in the nucleotide sequence forms a reverse complementary base pair, i.e., N 1 The nucleotide sequence is 5'-3' oriented, 23 The orientation of a nucleotide sequence includes nucleotide sequences that are 3'-5'. 1 and N 23 If at least one of the nucleotide bases is 4 or less in length, at least one pair of bases must form a complementary base pair, and N 1 and N 23 If at least one of the nucleotide bases is 5 or more in length, at least two pairs of bases must form complementary base pairs. 14 and N 16 At least one pair of bases in the nucleotide sequence forms a reverse complementary base pair, i.e., N 14 The nucleotide sequence is 5'-3' oriented, 16 The orientation of the nucleotide sequence is 3'-5'. 14 and N 16If at least one of the nucleotide bases is 4 or less in length, at least one pair of bases must form a complementary base pair, and N 14 and N 16 If at least one of the nucleotide bases is 5 or more in length, at least two pairs of bases must form complementary base pairs. 15 is a nucleotide sequence of any length and configuration, or (c) a nucleotide sequence having from 1 to 5 nucleotide substitutions, deletions and / or insertions at any position of the nucleotide sequence (a) above.

[0041] The nucleic acid aptamer molecules include nucleotide substitutions into the general Carrot structure, the substitutions being A4U, A4G, A4C, U5A, U5G, U5C, G7C, G7A, G7U, U8C, A10U, A10G, A10C, A11U, A11G, A11C, C12G, C12A, C12U, A13U, A13G, A13C, G17A, C19A, C19U, A20C, A4C / U5A, A4C / U5C, A4C / A11G, A4C / C12A, A4C / A13C, U5A / A11G, U5A / C12 A, U5A / A13C, U5G / A13C, U5C / G7U, U5C / A11G, U5C / C12G, U5C / C12A, U5C / C12U, U5C / A13C, G 7U / A13C, A11G / A13C, C12G / A13C, C12A / A13C, C12U / A13C, A4C / U5C / A13C, U5C / G7U / A13C , U5C / C12G / A13C, U5C / C12U / A13C, U5C / A11G / A13C, U5C / C12A / A13C, A4C / U5C / A11G / A13C , A4C / U5C / C12A / A13C, A4C / U5C / G7U / A11G / A13C, A4C / U5C / G7U / C12A / A13C, A4C / U5A / A11G / C12A / A13C, A4C / U5C / A11G / C12A / A13C (i.e., the aptamer molecular structure in Table 1). These mutants can specifically bind to fluorophore molecules, and after binding, can significantly improve the fluorescence intensity of the fluorophore molecules with excitation light of an appropriate wavelength. Among them, the position sequence of nucleotides corresponds to the positions in Figure 1.

[0042] The above mutations represent nucleotide substitutions at the corresponding sites of the aptamer nucleotide sequence of the general Carrot structure, for example, A4U represents Carrot in which the 4th adenine nucleotide A is replaced with a uracil nucleotide U, i.e., Carrot(A4U) in Table 1. U5C / A13C represents Carrot in which the 5th U is replaced with a C and the 13th A is replaced with a C, i.e., Carrot(U5C / A13C) in Table 1.

[0043] [Table 1(1)] [Table 1(2)]

[0044] Aptamer molecules are single-stranded nucleic acid molecules with a secondary structure consisting of one or more base-paired regions (stalks) and one or more non-base-paired regions (loops) (Figure 1). The nucleic acid aptamer molecule of the present invention contains one secondary structure as predicted in Figure 1. The 5' or 3' end of said structure can be bound to any target RNA molecule and used for detecting extracellular or intracellular target RNA molecules. In one preferred embodiment of the present invention, the 5' end of the nucleic acid aptamer molecule binds to ACTB RNA sequence (Genebank: BC016045). In another preferred embodiment of the present invention, the 5' end of the nucleic acid aptamer molecule binds to GAPDH RNA sequence (Genebank: BC009081).

[0045] The stem-ring structure in Fig. 1 (N 14 -N 15 -N 16 ) serves to stabilize the overall structure of the nucleic acid aptamer molecule and can be replaced with any nucleotide sequence of any length and configuration that can form other stem-ring structures. The aptamer molecules of the present invention further include N 14 -N 15 -N 16The inserted nucleotide sequence may include another nucleotide sequence inserted at position 1 of the stem-ring structure (N 14 -N 15 -N 16 ) to replace the nucleotide sequence. The nucleotide sequence can specifically identify / bind to the target molecule. In the absence of the target molecule, the aptamer molecule has a weak binding ability with the fluorophore molecule, so that the fluorophore molecule exhibits weak fluorescence, but in the presence of the target molecule, the binding between the target molecule and the aptamer promotes the binding between the aptamer and the fluorophore molecule, and significantly enhances the fluorescence intensity of the fluorophore molecule under an excitation light of an appropriate wavelength. The target molecule can be a small molecule, a signal molecule on the cell surface, etc. These nucleic acid aptamers bind to specific target molecules in a non-covalent manner, and such non-covalent binding is mainly due to intermolecular ionic forces, dipole forces, hydrogen bonding forces, van der Waals forces, electron-positron interactions, stacking effects, or a combination of multiple forces mentioned above. The stem-ring structure (N 14 -N 15 -N 16 ) can be replaced with an RNA sequence that identifies the target molecule and used to detect the target molecule extracellularly or intracellularly.

[0046] In a preferred embodiment of the invention, the nucleic acid aptamer molecule is preferably SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30 or 31, or a variant thereof that is capable of binding to a fluorophore molecule to significantly enhance the fluorescence of the fluorophore molecule upon excitation with light of an appropriate wavelength.

[0047] The nucleic acid aptamer molecule of the present invention may further include a nucleotide sequence that improves its stability. In one preferred embodiment of the present invention, F30 scaffold RNA (sequence 3) is used, and the binding form with the nucleic acid aptamer molecule is as shown in Figure 3. In another preferred embodiment of the present invention, tRNA scaffold RNA (sequence 4) is used, and the binding form with the nucleic acid aptamer molecule is as shown in Figure 4.

[0048] identity "Identity" is used herein to describe the relatedness between two nucleotide sequences. In calculating the identity of two aptamer nucleotide sequences of the present invention, (a) the number of Ns in the sequence 1 , N 14 , N 15 , N 16 , N 23 For the present invention, the degree of identity between two nucleotide sequences is determined, for example, using the Needle program of the EMBOSS software suite (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277), preferably using version 3.0.0 or higher, implementing the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453). Optional parameters used are a gap penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). The output labeled "longest identity" in Needle (obtained using the -nobrief option) was used to calculate the percentage identity as follows:

[0049] (Identical residues × 100) / (comparison length − total number of deletions in comparison).

[0050] As shown in Table 1 of the present invention, the sequences of Carrot-1 and Carrot-1(U5C) are 1 AGAUUGUAAACAN 14 -N 15 -N 16 GACACUN 23 and N 1 AGACUGUAAACAN 14 -N 15 -N16 GACACUN 23 and when comparing their identities, according to the definition of the present invention, N 1 , N 14 -N 15 -N 16 and N 23 Since the sequence should not contain any nucleotide bases, the identity comparison of those sequences results in 94.4% (one nucleotide difference).

[0051] Fluorophore molecules The "fluorophore molecule" according to the present invention is also referred to as "fluorophore" or "fluorescent molecule". "Fluorophore molecule" is a fluorophore molecule that is activated according to a certain condition in the present invention. They show a relatively low quantum yield in the absence of a nucleic acid aptamer. In a specific embodiment, when not bound to a specific aptamer, the quantum yield of the fluorophore is less than 0.1, more preferably less than 0.01, and most preferably less than 0.001. After the fluorophore is bound to a specific aptamer, the quantum yield of the fluorophore is improved by more than 2 times, more preferably more than 10 times, and most preferably more than 100 times. The fluorophore molecule is preferably water-soluble, non-toxic to cells, and easily permeable through the cell membrane. The fluorophore of the present invention is preferably capable of entering the cytoplasm or periplasm through the cell membrane or cell wall by active transport or passive diffusion. In an embodiment of the invention, the fluorophore permeates the outer and inner membranes of gram-negative bacteria, the cell walls and membranes of plant cells, the cell walls and membranes of fungi, the membranes of animal cells, and the GI and endothelial cell membranes of living animals.

[0052] The nucleic acid aptamer molecule of the present invention can specifically bind to a certain fluorophore and significantly increase the fluorescence value when excited at a specific wavelength. The fluorophore molecule is selected from the structure (I).

[0053] [ka]

[0054] (Ι) In the formula: Ar 1、 Ar 2 are independently a 6-membered aryl group, a 6-membered heteroaryl group, and D- is HO- or N(X 1 )(X 2 )- and X 1 , X 2 are each independently selected from hydrogen, an alkyl group, and a modified alkyl group; 1 , X 2 may be bonded to each other to form an aliphatic heterocycle together with the N atom, and D- is N(X 1 )(X 2 )-Ar 1 When is a phenyl group, X 1 , X 2 independently form a saturated or unsaturated ester heterocycle with a benzene ring, D- is HO-, and Ar 1 is a phenyl group, at least one hydrogen atom adjacent to HO is replaced by a halogen, Y is O, S, and R 1 is hydrogen, an alkyl group, or a modified alkyl group; R 2 is a hydrogen atom, a halogen atom, -OH, or -CN, wherein each of the "alkyl groups" is independently selected from C 1 -C 10 A straight or branched chain alkyl group, C 1 -C 7 may be a linear or branched alkyl group; 1 -C 5It may be a straight-chain or branched-chain alkyl group, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, an s-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, an isopentyl group, a 1-ethylpropyl group, a neopentyl group, an n-hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 3-methylpentyl group, an isohexyl group, and a 1,1-dimethylbutyl group. a 2,2-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, a 2-ethylbutyl group, a n-heptyl group, a 2-methylhexyl group, a 3-methylhexyl group, a 2,2-dimethylpentyl group, a 3,3-dimethylpentyl group, a 2,3-dimethylpentyl group, a 2,4-dimethylpentyl group, a 3-ethylpentyl group or a 2,2,3-trimethylbutyl group; In the formula: The "modified alkyl group" is independently selected from any carbon atom of the alkyl group, and is selected from the group consisting of a halogen atom, -OH, -CO-, -O-, -CN, -SO 3 H, a primary amino group, a secondary amino group, a tertiary amino group, wherein the modified alkyl group has 1 to 10 carbon atoms, and each carbon-carbon single bond may be independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond; In the formula: the carbon atom is replaced means that the carbon atom, or the carbon atom together with the hydrogen atom thereon, is replaced with the corresponding group, and the "modified alkylene group" is C 1 -C 10 (Preferably, C 1 -C 6 any carbon atom of the alkylene group is replaced by a group selected from -O-, -OH, -CO-, -CS-, and -(S=O)-; Preferably, the "modified alkyl group" is a group selected from the group consisting of -OH, -O-, ethylene glycol unit (-(CH 2 CH 2 O) n -), monosaccharide units, -O-CO-, -NH-CO-, -SO 2 -O-, -SO-, Me 2 N-, Et2 Contains one or more groups selected from N-, -SS-, -CH=CH-, F, Cl, Br, I, and a cyano group; Preferably, Ar 1 is a structure selected from the following formulas (II-1) to (II-15),

[0055] [ka]

[0056] Ar 2 is a structure selected from the following formulae (III-1) to (III-25).

[0057] [ka]

[0058] Preferably, the compound of formula (I) is selected from the following compounds:

[0059] [ka] [ka]

[0060] In a preferred embodiment of the invention, the fluorophore molecule comprises IV-1, IV-2, IV-3, IV-4, IV-5, IV-6, IV-7, IV-8, IV-9, IV-10, IV-11, IV-12, IV-13, IV-14, IV-15, IV-16, IV-17, IV-18, IV-19, IV-20, IV-21, IV-22, IV-23, IV-24, IV-25, IV-26, IV-27, IV-28, IV-29, IV-30, IV-31, IV-32, IV-33, IV-34, IV-35, IV-36, IV-37, IV-38, IV-39, IV-40. In the present invention, "improvement of the fluorescent signal", "increase in fluorescence", "improvement of the fluorescent intensity", and "increase in the fluorescent intensity" refer to an improvement in the quantum yield of the fluorophore when irradiated with excitation light of an appropriate wavelength, or a shift in the maximum emission peak of the fluorescent signal (relative to the emission peak of the fluorophore itself in ethanol or aqueous solution), or an increase in the molar extinction coefficient, or two or more of these. In one preferred embodiment of the present invention, the increase in quantum yield is at least 2-fold, in another preferred embodiment of the present invention, the increase in quantum yield is at least 5-10-fold, in another more preferred embodiment of the present invention, the increase in quantum yield is at least 20-50-fold, in another more preferred embodiment of the present invention, the increase in quantum yield is at least 100-200-fold, and in another more preferred embodiment of the present invention, the increase in quantum yield is at least 200-fold or more. The light source that excites the fluorophore to produce a fluorescent signal may be any suitable light emitting device, including, for example, LED lights, incandescent lamps, fluorescent lamps, and lasers, and the excitation light may be emitted directly from these devices or indirectly via another fluorophore, for example a donor fluorophore in fluorescence resonance energy transfer (FRET) or a donor luminophore in luminescence resonance energy transfer (BRET).

[0061] target molecule A target molecule according to the present invention may be any biomaterial or small molecule, including but not limited to proteins, nucleic acids (RNA or DNA), lipid molecules, carbohydrates, hormones, cytokines, chemokines, metabolites, metal ions, etc. A target molecule may also be a molecule involved in a disease or pathogenic infection.

[0062] In the aptamer molecule of the present invention, for example, in the structure shown in FIG. 1, the inserted nucleotide sequence is N 14 -N 15 -N 16 By replacing the stem-ring structure of the aptamer molecule with the nucleotide sequence, the aptamer molecule can specifically identify / bind to the target molecule. In the absence of the target molecule, the aptamer molecule does not bind to the fluorophore molecule or has a weak binding ability, and cannot significantly enhance the fluorescence of the fluorophore molecule under the excitation light of an appropriate wavelength, but in the presence of the target molecule, the binding between the target molecule and the nucleotide sequence promotes the binding between the aptamer molecule and the fluorophore molecule, and significantly enhances the fluorescence of the fluorophore molecule under the excitation light of an appropriate wavelength, thereby realizing the detection, imaging and quantitative analysis of the target molecule.

[0063] The target molecule may also be a molecule expressed throughout the cell or throughout the cell surface. Exemplary cells include, but are not limited to, cancer cells, bacterial cells, fungal cells, and normal animal cells. The target molecule may also be a virus particle. Currently, many aptamers for the above target molecules have been identified, and they can be integrated into the multiple nucleic acid aptamers of the present invention. Currently, RNA aptamers that can bind to target molecules that have been reported include, but are not limited to, T4 RNA polymerase aptamer, HIV reverse transcriptase aptamer, and phage R17 capsid protein aptamer.

[0064] target nucleic acid molecule "Target nucleic acid molecule" is also called "target nucleic acid molecule", and refers to a nucleic acid molecule to be detected, which may be intracellular or extracellular, including target RNA molecule and target DNA molecule. The present invention links the target nucleic acid molecule with the nucleic acid aptamer molecule, and binds the fluorophore molecule with the nucleic acid aptamer molecule, thereby significantly improving the fluorescence value of the fluorophore molecule under the excitation light of an appropriate wavelength, and further achieving the purpose of measuring the content and distribution of the target nucleic acid molecule.

[0065] In the present invention, the term "target RNA molecule" includes any RNA molecule, including, but not limited to, pre-mRNA, mRNA encoding a cell's own or exogenous expression product, pre-rRNA, rRNA, tRNA, hnRNA, snRNA, miRNA, siRNA, shRNA, sgRNA, crRNA, lncRNA, phage capsid protein MCP recognition binding sequence MS2RNA, phage capsid protein PCP recognition binding sequence PP7RNA, lambda phage transcription termination protein N recognition binding sequence boxB RNA, etc. The target RNA is the 5'-end or 3'-end or N-end of the RNA aptamer molecule of the present invention. 14 -N 15 -N 16 It can be fused to the position.

[0066] In the present invention, "sgRNA" refers to a single guide RNA (sgRNA) formed after modifying tracrRNA and crRNA using the CRISPR / Cas9 system, and a sequence of about 20 nt at its 5' end targets a DNA site by complementary base pairing, promoting the Cas9 protein to induce breaks in the DNA double strand at that site.

[0067] Concatamers of nucleic acid aptamers The nucleic acid aptamer molecule of the present invention may further include a concatemer capable of linking a plurality of fluorophore molecules. The concatemer may be linked by a spacer sequence of an appropriate length, and the number of Carrot structures linked in series may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The concatemer may have various forms, and in a preferred embodiment of the present invention, the form of the tandem linkage is "tandem linkage 1", and as shown in FIG. 7a, the preferred nucleotide sequence is SEQ ID NO: 7, 8, 9, 10 or 11, in which 2Carrot-2 represents concatemer 1 having two Carrot-2 structures. In another preferred embodiment of the present invention, the form of the tandem linkage is "tandem linkage 2", and as shown in FIG. 7b, the preferred nucleotide sequence is SEQ ID NO: 12, 13, 14, 15 or 16, in which 2×Carrot-2 represents concatemer 2 having two Carrot-2 structures. In another preferred embodiment of the present invention, the form of tandem linkage is "tandem linkage 3", and the preferred nucleotide sequence is SEQ ID NO: 17, 18, 19 or 20, as shown in Figure 7c, in which 2x2 Carrot-2 represents concatemer 3 having 4 Carrot-2 structures. In any form, the spacer sequence between the concatemers is replaceable.

[0068] The monomeric aptamer according to the present invention is an aptamer containing only one Carrot structure, that is, containing only one aptamer shown in FIG.

[0069] The multimeric aptamer includes, but is not limited to, an aptamer that contains one or more aptamers with a Carrot structure and is configured in a tandem connection form as shown in FIG.

[0070] Aptamer-fluorophore conjugates The aptamer-fluorophore complex of the present invention comprises one nucleic acid aptamer molecule and one or more fluorophore molecules. In a specific embodiment of the present invention, the molecular complex comprising one nucleic acid molecule and one fluorophore molecule is Carrot-1-IV-1, Carrot-1-IV-2, Carrot-1-IV-3, Carrot-1-IV-4, Carrot-1-IV-5, Carrot-1-IV-6, Carrot-1-IV-7, Carrot-1-IV-8, Carrot-1-IV-9, Carrot-1-IV-10, Carrot-1-IV-11, Carrot-1-IV-12, Carrot-1-IV-13, Carrot-1-IV-14, Carrot-1-IV-15, Carrot-1-IV-16, Carrot-1-IV-17, Carrot-1-IV-18, Carrot-1-IV -19, Carrot-1-IV-20, Carrot-1-IV-21, Carrot-1-IV-22, Carrot-1-IV-23, Carrot-1-IV-24, C arrot-1-IV-25, Carrot-1-IV-26, Carrot-1-IV-27, Carrot-1-IV-28, Carrot-1-IV-29, Carrot- 1-IV-30, Carrot-1-IV-31, Carrot-1-IV-32, Carrot-1-IV-33, Carrot-1-IV-34, Carrot-1-IV- 35, Carrot-1-IV-36, Carrot-1-IV-37, Carrot-1-IV-38, Carrot-1-IV-39, Carrot-1-IV-40.

[0071] In another specific embodiment of the present invention, a complex is formed between a concatemeric nucleic acid molecule and a plurality of fluorophore molecules, for example, complexes F30-4Carrot-2 containing four aptamer units formed in the form of "serial connection 1" and four fluorophore molecules are formed, such as F30-4Carrot-2-4×(IV-1), F30-4Carrot-2-4×(IV-2), F30-4Carrot-2-4×(IV-3), F30-4Carrot-2-4×(IV-4), F30-4Carrot-2-4×(IV-5), F30-4Carrot-2-4×(IV-6), F30-4Carrot-2-4×(IV-7), F30-4Carrot-2-4×(IV-8), F30-4Carrot-2-4×(IV-9), F30-4Carrot-2-4×(IV-10), F30-4Carrot-2-4×(IV-11), F30-4Carrot-2-4×(IV-12), F30-4Carrot-2-4×(IV-13), F30-4Carrot-2-4×(IV-14), F30-4Carrot-2-4×(IV-15), F30-4Carrot-2-4×(IV-16), F30-4Carrot-2-4×(IV-17), F30-4Carrot-2-4×(IV-18), F30-4Carrot-2-4×(IV-19), F30-4Carrot-2-4×(IV-20), F30-4Carrot-2-4×(IV-21), F30-4Carrot-2-4×(IV-22), F30-4Carrot-2-4×(IV-23), F30-4Carrot-2-4×(IV-24), F30-4Carrot-2-4× 0-4Carrot-2-4x (IV-5), F30-4Carrot-2-4x (IV-6), F30-4Carrot-2-4x (IV-37), F30-4Carrot-2-4x (IV-17), F30-4Carrot-2-4x (IV-18), F30-4Carrot-2-4x (IV-19), F30-4Carrot-2-4x (IV-20), F30-4Carrot-2-4x (IV-21), and F30-4Carrot-2-4x (IV-22). The molecular complex may exist as two independent solutions in vitro, or may exist in the same solution, or may exist in a cell.

[0072] Nucleic acid aptamer function The aptamer function of the present invention is that it can significantly improve the fluorescence intensity of the fluorophore molecule when excited by light of an appropriate wavelength, and the aptamer can be measured using the conventional experimental method (5) Functional detection of nucleic acid aptamers described in the specific examples. In one preferred embodiment of the present invention, the increase in fluorescence intensity is at least 2-fold (fluorescence intensity is detected according to experimental method (5)), in another preferred embodiment of the present invention, the increase in fluorescence intensity is at least 5-10-fold, in another more preferred embodiment of the present invention, the increase in fluorescence intensity is at least 20-50-fold, in another more preferred embodiment of the present invention, the increase in fluorescence intensity is at least 100-200-fold, and in another more preferred embodiment of the present invention, the increase in fluorescence intensity is at least 200-fold or more.

[0073] Nucleic acid aptamer secondary structure The secondary structure of the nucleic acid aptamer in the present invention was obtained by simulation prediction using mFold online analysis software (http: / / unafold.rna.albany.edu / ?q=mfold). The stem structure in the secondary structure is a local double-stranded structure formed by complementary base pairs formed by hydrogen bonding forces in a partial region within a single strand of a nucleic acid aptamer molecule. In general, the formation of a double-stranded structure does not require all nucleotides in this partial region to form complementary base pairs. In general, N 1 and N 23 , and N 14 and N 16 If at least 50% of the nucleotides in a portion of the sequence of N form complementary base pairs with another fragment, the stem structure can be formed. 1 and N 23 If is a single nucleotide, then N 1 and N 23 If the complements are not perfect, they cannot form a stem structure (as shown in Figure 1).

[0074] DNA molecules expressing nucleic acid aptamers The DNA molecule comprises a DNA sequence capable of encoding a nucleic acid aptamer molecule of the present invention. 1 AGATTGTAAACAR 14 -R 15 -R 16 GACACTR 23 and nucleotide sequences having at least 70% identity thereto. 1 In the general Carrot structure, N 1 Code and R 14 In the general Carrot structure, N 14 Code and R 15 In the general Carrot structure, N 15 Code and R 16 In the general Carrot structure, N 16 Code and R 23 In the general Carrot structure, N 23The DNA molecule may further comprise a promoter for controlling DNA transcription, and the promoter is operably linked to the DNA sequence encoding the nucleic acid aptamer. In a specific embodiment of the present invention, the DNA molecule comprises a U6 promoter, and in another specific embodiment of the present invention, the DNA molecule comprises a CMV promoter. In addition to the DNA molecule, the DNA molecule may further comprise a DNA sequence encoding any target nucleic acid molecule. In a specific embodiment of the present invention, the DNA molecule encoding the target RNA comprises a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin (the sequences of the chimeric RNAs are SEQ ID No: 26 and 27, respectively).

[0075] promoter In the present invention, the term "promoter" includes eukaryotic and prokaryotic promoters. The promoter sequence of eukaryotic cells is completely different from that of prokaryotic cells. In general, eukaryotic promoters cannot be recognized by RNA polymerase in prokaryotic cells to mediate RNA transcription. For the same reason, prokaryotic promoters cannot be recognized by RNA polymerase in eukaryotic cells to mediate RNA transcription. Different promoters have a large difference in strength (strength refers to the ability to mediate transcription). Depending on the actual application, a strong promoter can be used to achieve a high level of transcription. For example, when used for labeling, a high level of expression is preferred, but when evaluating transcription behavior, a relatively low level of transcription can allow for timely adjustment of the transcription process of the cell. Depending on the host cell, one or more suitable promoters can be selected and used. For example, when used in E. coli cells, there are T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, PR and PL promoters in λ phage, and other promoters, including but not limited to lacUV5 promoter, ompF promoter, bla promoter, lpp promoter, etc. In addition, a hybrid trp-lacUV5 promoter (tac promoter) or other E. coli promoters obtained by DNA recombination or synthesis techniques can both be used to transcribe the RNA aptamer of the present invention. Some of the bacteria's own operator sequences can be combined with promoter sequences to form inducible promoters, in which case the transcription of the DNA molecule cannot be induced unless a specific inducer is added. For example, the lac operator requires the addition of lactose or a lactose analog (IPTG) to induce its expression, and other operators include trp, pro, etc.

[0076] As mentioned above, the regulatory sequence at the 5' end of the sequence coding for the DNA molecule is a promoter. Whether obtaining RNA aptamers by in vitro transcription or expressing aptamers in cultured cells or tissues, it is necessary to select an appropriate promoter according to its strength. Since aptamers expressed in vivo can be genetically engineered, another type of promoter is an inducible promoter that induces DNA transcription in response to a specific environment, such as being expressed in a specific tissue, at a specific time, at a specific developmental stage, etc. These different promoters can be distinguished by RNA polymerase I, II, or III.

[0077] In eukaryotic cells, a suitable promoter is also required for the initiation of transcription, including, but not limited to, β-globulin promoter, CAG promoter, GAPDH promoter, β-actin promoter, actin promoter, Cstf2t promoter, SV40 promoter, PGK promoter, MMTV promoter, adenovirus Ela promoter, CMV promoter, etc. In eukaryotic cells, the termination of transcription depends on a specific cleavage site in the RNA sequence. Similarly, there are large differences in the transcription terminators depending on the gene transcribed by RNA polymerase. However, screening for a suitable 3' transcription terminator region can be achieved by those skilled in the art using basic experimental techniques.

[0078] Expression system The "expression system" of the present invention is also called an "expression vector" and contains an integrated DNA molecule that expresses a nucleic acid aptamer. The expression system of the present invention may be a plasmid or a viral particle.

[0079] "Expression vector" Recombinant viruses can be obtained by transfecting a plasmid into a cell infected with the virus. Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems gt11, gt WES.tB, Charon 4. Plasmid vectors include pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG399, pR290, pKC37, pKC101, pBluescript II SK+ / - or KS+ / - (see Stratagene cloning systems), pET28 series, pACYCDuet1, pCDFDuet1, pRSET series, pBAD series, pQE, pIH821, pGEX, pIIIEx426RPR, etc.

[0080] Many host expression systems can be used to express the DNA molecules of the present invention. Mainly, the vector system must be compatible with the host cell used, and host-vector systems include, but are not limited to, bacteria transformed with phage DNA, or plasmid DNA, or cosmid DNA, yeast with yeast vectors, mammalian cells infected with viruses (e.g., adenovirus, adeno-associated virus, reverse transcription virus), mammalian cells transfected with plasmids, insect cells infected with viruses (e.g., baculovirus), plant cells infected with bacteria or transformed by particle bombardment. The strength and properties of the transcription elements in the vectors vary greatly. Depending on the host-vector system used, any one or more suitable transcription elements are selected and used.

[0081] Once the constructed DNA molecules have been cloned into a vector system, they can be easily transferred into host cells. Depending on the vector or host cell system, methods include, but are not limited to, transformation, transduction, conjugation, fixation, electroporation, etc.

[0082] In a specific embodiment of the present invention, expression plasmids pET28a-T7-F30-Carrot-2 and pYES2.1-F30-Carrot-2 are provided, which contain a DNA molecule encoding F30-Carrot-2 RNA. In another specific embodiment of the present invention, expression plasmid pU6-tRNA-Carrot-2 is provided, which contains a DNA molecule encoding tRNA-Carrot-2 RNA. In another specific embodiment of the present invention, expression plasmid pU6-F30-4Carrot-2 is provided, which contains a DNA molecule encoding F30-4Carrot-2 RNA. In another specific embodiment of the present invention, expression plasmids pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 and pCDNA3.1 hygro(+)-ACTB-4Carrot-2 are provided, which contain a DNA molecule encoding GAPDH-4Carrot-2 and ACTB-4Carrot-2. In another specific embodiment of the present invention, expression plasmids psgRNA-Carrot-2-1, psgRNA-Carrot-2-2 and psgRNA-Carrot-2-3 are provided, which contain DNA molecules encoding sgRNA-Carrot-2-1, sgRNA-Carrot-2-2 and sgRNA-Carrot-2-3. In another specific embodiment of the present invention, expression plasmid pU6-Carrot-1-MS2 is provided, which contains DNA molecules encoding Carrot-1-MS2.

[0083] The present invention also provides an expression vector that integrates a DNA molecule encoding a nucleic acid aptamer, but lacks a target RNA molecule coding DNA sequence. Due to the lack of a target RNA molecule coding DNA sequence, users can select the DNA sequence of the target RNA molecule they want to detect, for example, the coding DNA sequence corresponding to GAPDH mRNA, and insert the DNA sequence into such an expression vector of the present invention by standard recombinant DNA technology, and then introduce (transfect, transform, infect, etc.) the resulting expression vector into a host cell to detect the content and distribution of the target RNA.

[0084] host cell In the present invention, the term "host cell" includes, but is not limited to, bacteria, yeast, mammalian cells, insect cells, plant cells, zebrafish cells, Drosophila cells, and nematode cells. The host cell is more preferably a cultured in vitro cell or a whole in vivo living tissue. The host cell in the present invention includes, but is not limited to, mammalian cells including 297T, COS-7, BHK, CHO, HEK293, HeLa, H1299, fertilized egg stem cells, induced totipotent stem cells, primary cells directly isolated from mammalian tissues, and the like, including, but not limited to, E. coli cells including, but not limited to, BL21(DE3), BL21(DE3,Star), TOP10, Mach1, and DH5α, and including, but not limited to, yeast cells including, but not limited to, BY4741, BY4742, and AH109.

[0085] Detection Array The detection array according to the present invention comprises one or more nucleic acid aptamer molecules according to the present invention, among which the nucleic acid aptamer molecules are anchored at separate positions on the array surface, which is composed of a solid support, including but not limited to glass, metal, ceramics, etc. The anchoring of the nucleic acid aptamer molecule according to the present invention to the array surface can be achieved by, but not limited to, the following methods: (1) The 5' or 3' end of the nucleic acid aptamer molecule is labeled with biotin, streptavidin is coated on the array surface, and the nucleic acid aptamer molecule is anchored by the specific binding between biotin and streptavidin. (2) The phage capsid protein MCP recognition binding sequence MS2, the phage capsid protein PCP recognition binding sequence PP7, or the λ phage transcription termination protein N recognition binding sequence boxB RNA sequence is fused to the 5', 3', or stem ring structure of the nucleic acid aptamer molecule, and the recognition and binding proteins MCP, PP7, or λ are fused to the 5', 3', or stem ring structure of the nucleic acid aptamer molecule. N Proteins were coated onto the surface of the array, and MS2 and MCP proteins, PP7 and PCP proteins, or boxB RNA and λ NThe nucleic acid aptamer molecule is anchored by specific interaction with the protein. (3) A part of RNA or DNA sequence is fused to the 5' or 3' end of the nucleic acid aptamer molecule, and an RNA sequence complementary to the RNA sequence or a DNA sequence complementary to the DNA sequence is anchored to the array surface, and the nucleic acid aptamer molecule is anchored to the array surface by the principle of molecular hybridization. The detection array can be used to detect the presence or absence and the concentration of a target molecule, so that only when a target molecule is present, the nucleic acid aptamer molecule binds to a fluorophore molecule, significantly increasing the fluorescence intensity at the appropriate excitation light wavelength, and within a certain range, the higher the concentration of the target molecule, the higher the fluorescence intensity.

[0086] kit The kit of the present invention comprises a nucleic acid aptamer molecule and / or a fluorophore molecule according to the present invention and corresponding instructions, or comprises an expression system for expressing the nucleic acid aptamer molecule and / or a fluorophore molecule and corresponding instructions, or comprises a host cell for expressing the nucleic acid aptamer molecule expression system and / or a fluorophore molecule and corresponding instructions. The nucleic acid aptamer molecule and the fluorophore molecule in the kit are each present in separate solutions, or the nucleic acid aptamer molecule and the fluorophore molecule are present in the same solution.

[0087] Working Example The present invention will be further described below with reference to examples. These examples are merely illustrative and do not limit the technical scope of the present invention. The examples mainly use conventional gene process molecular biology cloning methods, which are familiar to those skilled in the art, such as the "Molecular Biology Laboratory Reference Manual" by Jane Roskams et al. (Chinese translation version) and the relevant chapters and sections of "Molecular Cloning Laboratory Manual" by J. Sambrook, DW Russell, translated by Huang Pedang et al. (Third Edition, August 2002, Science Press, Beijing). It is not difficult for those skilled in the art to successfully carry out the present invention by following the following examples and making some modifications and changes according to the actual situation.

[0088] The pCDNA3.1 hygro(+) plasmid vector used in the examples was purchased from Invitrogen, the pLKO.1-puro plasmid vector was purchased from Sigma, the pET28a plasmid vector was purchased from Novagen, and the pYES2.1 TOPO TA plasmid vector was purchased from Invitrogen. All primers used in PCR were synthesized, purified, and identified as accurate by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. All expression plasmids constructed in the examples were sequenced, and the sequence analysis was completed by Jierui Quantum Co., Ltd. The Taq DNA polymerase used in each example was purchased from Shanghai Yishengsheng Biological Technology Co., Ltd., the PrimeSTAR DNA polymerase was purchased from TaKaRa, and all three types of polymerases were accompanied by the corresponding polymerase buffer and dNTPs when purchased. Restriction endonucleases such as EcoRI, BamHI, BglII, HindIII, NdeI, XhoI, SacI, XbaI, and SpeI, T4 ligase, T4 phosphorylase (T4 PNK), and T7 RNA polymerase were purchased from Fermentas, and the corresponding buffers were included at the time of purchase. The Hieff Clone (registered trademark) One Step Clone Kit used in the examples was purchased from Shanghai Yisheng Biological Technology Co., Ltd. Unless otherwise stated, all inorganic salt chemical reagents were purchased from Shanghai Chemical Reagents Company of China Pharmaceutical Group. Kanamycin was purchased from Ameresco, ampicillin (Amp) was purchased from Ameresco, and 384-well and 96-well fluorescence detection black panels were purchased from Grenier. DFHBI-1T and DFHO were purchased from Lucerna. GTP and SAM were purchased from Sigma.

[0089] The DNA purification kit used in the examples was purchased from BBI (Canada), and the general plasmid mini extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The BL21 (DE3, Star) strain was purchased from Invitrogen. 293T / 17 cells and COS-7 cells were purchased from the Cell Library of the Committee for the Protection of Typical Cultures, Chinese Academy of Sciences. The BY4741 yeast strain was purchased from Shanghai Weidi Biological Technology Co., Ltd.

[0090] The equipment used in the examples includes Synergy Neo2 multifunction microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplification device (Biometra, Germany), bioimaging system (Kodak, USA), photometer (Wako, Japan), and nucleic acid electrophoresis device (Shanghai Shenneng Bocai Biological Technology Co., Ltd.).

[0091] The abbreviations are as follows: "h" stands for hour, "min" for minute, "s" for second, "d" for day, "μL" for microliter, "mL" for milliliter, "L" for liter, "bp" for base pair, "mM" for millimole, and "μM" for micromole.

[0092] Experimental methods and materials used in the examples

[0093] (1) Preparation of nucleic acid aptamer molecules: cDNA corresponding to the test RNA was amplified by a primer containing a T7 promoter, and the recovered double-stranded cDNA was transcribed as a template by T7 RNA polymerase (purchased from Fermentas) to obtain RNA. 10 μL 3M NaAc and 115 μL DEPC water were added to 20 μL of the transcription system, mixed uniformly, and then 150 μL of phenol-chloroform-isopropanol mixture (phenol:chloroform:isopropanol=25:24:1) was added, shaken to mix uniformly, centrifuged at 10000 rpm for 5 min, and the supernatant was removed. An equal volume of chloroform solution was added, shaken to mix uniformly, centrifuged at 10000 rpm for 5 min, and the supernatant was removed, and this procedure was repeated once. The supernatant was added with 2.5 volumes of absolute ethanol, left in a -20℃ refrigerator for 30 min, centrifuged at 12000 rpm at 4℃ for 5 min, the supernatant was discarded, and the mixture was washed and precipitated twice with 75% pre-cooled absolute ethanol. After the ethanol had completely evaporated, an appropriate amount of screening buffer was added, resuspended and precipitated, treated at 75℃ for 5 min, left at room temperature for 10 min or more, and used in subsequent experiments.

[0094] (II) Cell culture and transfection: All cells in this example were cultured in CO 2 The cells were cultured in a high glucose medium (DMEM) containing 10% fetal bovine serum (FBS) and streptomycin and penicillin in an incubator, and subcultured when they reached 80-90% confluence. Transfection was performed using (purchased from Promega) according to the manufacturer's instructions.

[0095] (III) Fluorescence imaging: In the examples, the main imaging experiments were performed using a Leica SP8 confocal laser microscope, an HCXPL APO 63.0×1.47 oil mirror, and a HyD detector. Imaging was performed using multiple lasers at 405 nm, 457 nm, 476 nm, 488 nm, 497 nm, 514 nm, 561 nm, and 633 nm. The complexes formed between each carrot and dye molecule were imaged using a laser with a wavelength close to the corresponding maximum excitation peak. GFP was imaged using a 488 nm laser, Hoechst and DAPI fluorescence was imaged using a 405 nm laser, and Rhodamine was imaged using a 561 nm laser.

[0096] (IV) Construction of recombinant plasmid by homologous recombination method 1. Preparation of linearized vector: Once a suitable cloning site is selected, the vector can be linearized to prepare the linearized vector using enzymatic digestion or inverse PCR amplification.

[0097] 2. Preparation of insert fragment by PCR amplification: By introducing 15-25 bp (not including the enzyme cleavage site) of homologous sequence to the linearized vector end at the 5' end of the forward and reverse PCR primers for the insert fragment, the 5' and 3' ends of the insert fragment PCR product each have a perfectly matched sequence to both ends of the linearized vector.

[0098] 3. Concentration measurement of linearized vector and insert fragment: The linearized vector and insert fragment amplification products are diluted in equal volume gradients, and 1 μL of each of the original and diluted products is taken and subjected to agarose gel electrophoresis. The band brightness is compared with a DNA molecular weight standard (DNA marker) to determine their approximate concentration.

[0099] 4. Recombination Reaction In the recombination reaction system, the optimal amount of vector used is 0.03 pmol, and the optimal molar ratio of vector to insert is 1:2 to 1:3, that is, the optimal amount of insert is 0.06 to 0.09 pmol.

[0100] [Table 1(3)]

[0101] X and Y are the linearized vector and the amount of insert calculated by the formula, respectively. After the system is prepared, the components are mixed uniformly and incubated at 50°C for 20 min. If the insert is >5 kb, the incubation temperature can be extended to 25 min. After the reaction is completed, it is recommended to place the reaction tube on ice for 5 min to cool. The reaction product can be directly converted or stored at -20°C and thawed and converted when needed.

[0102] (5) Functional detection of nucleic acid aptamers Carrot or Carrot variant nucleic acid aptamer molecules were prepared according to the conventional experimental method (1), and 5 μM of the nucleic acid aptamer molecule and 1 μM of the fluorophore molecule were dissolved in a detection buffer (40 mM HEPES, pH 7.4, 125 mM KCl, 5 mM MgCl 2 , 5% DMSO) and the maximum excitation peak and maximum emission peak of the nucleic acid aptamer-fluorophore molecule complex fluorescence were detected by Synergy Neo2 multifunctional microplate reader. Furthermore, the fluorescence intensity of the nucleic acid aptamer-fluorophore molecule complex at its maximum excitation and emission conditions was detected by Synergy Neo2 multifunctional microplate reader, and a control sample (1 μM fluorophore molecule without nucleic acid aptamer) was also measured under the same conditions, and the ratio of the fluorescence intensities was calculated. For example, the complex formed by 5 μM Carrot-1 nucleic acid aptamer and 1 μM IV-39 fluorophore molecule has a maximum fluorescence excitation peak of 524 nm and a maximum emission peak of 580 nm. The fluorescence intensity of this complex detected by the Synergy Neo2 multifunction microplate reader at 524±10 nm excitation and 580±10 nm emission was 5720, while the fluorescence intensity of the control (1 μM IV-39 fluorophore molecule) at the same detection conditions was 20, resulting in an activation fold of 286 for the Carrot-1 nucleic acid aptamer relative to the IV-39 fluorophore molecule.

[0103] Example 1. Secondary structure of Carrot nucleic acid aptamer molecule The secondary structure of the Carrot nucleic acid aptamer general formula is predicted and analyzed by the mFold online RNA structure analysis software (Figure 1), but it does not represent its true secondary structure. 1 , N 14 -N 15 -N 16 and N 23 The predicted secondary structures of Carrot-1 and Carrot-2 (SEQ ID NOs: 1 and 2) are shown in FIG.

[0104] Example 2. Characterization of Carrot-IV-39 Complex To detect the spectral characteristics of Carrot-IV-39 complex, Carrot-1 (SEQ ID NO:2) RNA was prepared according to conventional experimental method (1). 1 μM IV-39 was incubated with 5 μM Carrot-1. The detection result showed that the Carrot-IV-39 complex has an excitation maximum of 524 nm and an emission maximum of 580 nm (Figure 5a).

[0105] To detect the binding constant of Carrot-1 to IV-39, we incubated 20 nM Carrot-1 with different concentrations of IV-39 and detected their fluorescence values. The binding constant of Carrot-1 to IV-39 was found to be 58 nM (Figure 5b).

[0106] Mg in Carrot-IV-39 Complex 2+ To detect ion dependence, the Carrot-1-IV-39 complex was incubated with different Mg 2+ The fluorescence value was detected by placing it in a buffer containing ions. The detection results showed that the Mg content of the Carrot-1-IV-39 complex was higher than that of Pepper599 and Corn-DFHO. 2+ It has low ion dependence (Figure 5c).

[0107] Example 3. Fluorescence activation effect of different Carrot variants on IV-39 fluorophore molecules.

[0108] In order to detect the fluorescence activation effect of different Carrot mutants on IV-39 fluorophore molecules, point mutations were made on the Carrot-1 sequence as shown in Table 1, Carrot mutant RNAs containing different base mutations were prepared according to conventional experimental method (I), 1 μM IV-39 was incubated with 5 μM of different Carrot-1 mutant RNAs, and their fluorescence activation folds on IV-39 fluorophore molecules were detected according to conventional experimental method (V). The detection results showed that some Carrot-1 mutants containing single base mutations maintained a relatively strong fluorescence activation effect (>20 times) on IV-39 (Table 2). Some Carrot-1 mutants containing 2 to 5 base mutations still maintained a certain fluorescence activation effect (>10 times) on IV-39 (Table 3). For the above reasons, many single and multi-base mutants of Carrot-1 still retain the aptamer function of activating IV-39 fluorophore molecules.

[0109] [Table 2]

[0110] Note: Carrot-1 in Table 2 is a nucleic acid aptamer whose sequence is SEQ ID NO:1, and the other aptamers are point mutations made in the Carrot-1 sequence at the corresponding nucleotide positions of Carrot in Figure 1.

[0111] [Table 3]

[0112] Example 4. Activation effect of base modification on Carrot IV-39 To detect the activating effect of base modification on Carrot IV-39, Carrot-3 (SEQ ID NO: 5, this sequence,

[0113]

number

[0114] The bold and underlined bases in are deoxyribose nucleotide bases) and Carrot-4 (SEQ ID NO:6, this sequence,

[0115]

number

[0116] The bold and underlined bases in are bases modified with 2'-F) were synthesized (Figure 6a) (synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.). The fluorescence activation effect of these base modifications on Carrot's IV-39 fluorophore molecule was detected according to conventional experimental methods (5). The detection results showed that the base modifications Carrot-3 and Carrot-4 can still significantly activate the fluorescence of the IV-39 fluorophore molecule (Figure 6b).

[0117] Example 5. Carrot Concatemers To detect the fluorescence activation effect of Carrot concatemers on IV-39 fluorophore molecules, Carrot-2 was tandemly linked in different configurations, including the following three types:

[0118] (1) In the "series linkage 1" form (Figure 7a), the "head" and "tail" on the Carrot structure are linked in the form of "head-to-tail" to obtain nCarrot (where n is any copy of Carrot). In this embodiment, the coding cDNA of F30-2Carrot-2, F30-4Carrot-2, F30-6Carrot-2, F30-8Carrot-2 and F30-10Carrot-2 are respectively synthesized in whole genome (the sequences encoding the RNA aptamer are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, respectively), and after PCR amplification, the nucleic acid aptamer RNA is prepared according to conventional experimental method (1), and 0.2μM RNA aptamer is incubated with 5μM IV-39, and the fluorescence intensity is detected according to conventional experimental method (5). The detection results showed that the fluorescence of F30-nCarrot-IV-39 increased with increasing n (Figure 7d), indicating that the “series connection 1” configuration could improve the fluorescence intensity of the Carrot-IV-39 complex.

[0119] (2) In the "tandem linkage 2" form (Fig. 7b), Carrot is linked in series as one building block to obtain n x Carrot (wherein n can be any number of copies of Carrot). In this embodiment, the coding cDNA of 2 x Carrot-2, 4 x Carrot-2, 6 x Carrot-2, 8 x Carrot-2 and 10 x Carrot-2 are synthesized in whole genome (the sequences encoding RNA aptamers are SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, respectively), and the nucleic acid aptamer RNA is prepared according to conventional experimental method (1). 0.2 μM RNA aptamer is incubated with 5 μM IV-39, and then the fluorescence intensity is detected according to conventional experimental method (5). The detection results showed that the fluorescence of n × Carrot-IV-39 increased with increasing n (Figure 7e), indicating that the “series connection 2” configuration could improve the fluorescence intensity of the Carrot-IV-39 complex.

[0120] (3) In the "series connection 3" form (Figure 7c), the above "series connection 1" and "series connection 2" are combined, and the nCarrot obtained in "series connection 1" is used as one building block and serially connected in the form of "series connection 2" to obtain n2 x n1Carrot (wherein n1 and n2 may be any copy of Carrot). In this embodiment, the coding cDNA of 2 x 2Carrot-2, 2 x 4Carrot-2, 4 x 2Carrot-2, and 4 x 4Carrot-2 are each synthesized in whole genome (the sequences encoding the RNA aptamers are SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively), and the nucleic acid aptamer RNA is prepared according to conventional experimental method (1), and 0.2 μM RNA aptamer is incubated with 5 μM IV-39, and then the fluorescence intensity is detected according to conventional experimental method (5). According to the detection results, the fluorescence intensity of Carrot concatemer-IV-39 obtained by this "series linkage 3" form was clearly higher than that of Carrot-2-IV-39 (Figure 7f), indicating that the "series linkage 3" form can improve the fluorescence intensity of the Carrot-IV-39 complex.

[0121] Example 6. Characterization of IV-39 Analogs According to the conventional experimental method (I), the Carrot-1 RNA aptamer molecule was prepared, and the basic properties including the fluorescence spectrum, molar extinction coefficient, quantum yield and fluorescence activation factor were detected when it was combined with Carrot of IV-39 analog, and the detection results are shown in Table 4. The data in the table show that Carrot-1 activates the fluorescence intensity of IV-39 analog to different degrees.

[0122] [Table 4(1)] [Table 4(2)]

[0123] Example 7. Labeling of RNA in bacteria with Carrot-IV-4 complex To detect the effect of Carrot-IV-4 in bacteria, we first constructed a bacterial expression plasmid expressing F30-Carrot-2. The DNA sequence encoding F30-Carrot-2 was synthesized in its entirety and amplified by primers, and pET28a was amplified by primers to remove the promoter and multicloning site regions. The amplified F30-Carrot-2 DNA fragment was ligated with the pET28a linearized vector according to the experimental method (IV), and the resulting recombinant plasmid was named pET28a-T7-F30-Carrot-2.

[0124] The primers used for amplifying the F30-Carrot-2 fragment are as follows:

[0125] Upstream primer (P1): 5'-TAATACGACTCACTATAGGGTTGCCATGTGTATGTGGGA-3' Downstream primer (P2): 5'-CAAGGGGTTATGCTATTGCCATGAATGATCC-3' The primers used to amplify and linearize the pET28a vector are as follows:

[0126] Upstream primer (P3): 5'-TAGCATAACCCCTTGGGGCCTC-3' Downstream primer (P4): 5'-TAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCG-3' pET28a-T7-F30-Carrot-2 was introduced into the BL21 (DE3, Star) E. coli strain by transformation of the recombinant plasmid, and monoclonal clones were selected and cultured at 37°C to reach OD 600 When the pH reached about 0.2, 1 mM IPTG was added to induce expression of F30-F30-Carrot-2. After 4 hours, the bacteria were harvested and resuspended in PBS (10 mM Mg) containing 1 μM IV-4. 2+The cells were resuspended in a 100% PBS solution containing 100% ethanol. BL21(DE3,Star) E. coli transformed with pET28a empty vector served as a control. The results showed that the Carrot-IV-4 complex could be used for fluorescent labeling of RNA in bacteria, since only the bacteria expressing F30-F30-Carrot-2 and in the presence of IV-4 showed bright orange-red fluorescence (Figure 8).

[0127] Example 8 Labeling of RNA in yeast cells with Carrot-IV-4 complex In order to detect the effect of Carrot-IV-4 in yeast, firstly, construct yeast expression plasmid expressing F30-Carrot-2. Use primers to amplify the F30-Carrot-2 DNA fragment in Example 7, and insert the amplified F30-Carrot-2 fragment into pYES2.1 TOPO TA vector according to experimental method (IV), and obtain recombinant plasmid named pYES2.1-F30-Carrot-2.

[0128] The primers used for amplifying the F30-Carrot-2 fragment are as follows:

[0129] Upstream primer (P5): 5'-GGAATATTAAGCTCGCCCTTTTGCCATGTGTATGTGGG-3' Downstream primer (P6): 5'-TGACCTCGAAGCTCGCCCTTGTTGCCATGAATGATCC-3' pYES2.1-F30-Carrot-2 was introduced into the BY4741 strain by transformation of the recombinant plasmid, and monoclonal clones were selected and cultured at 30°C until OD 600 When the β-actin ratio reached approximately 0.1, 1 mM galactose was added to induce expression of F30-Carrot-2. After 10 h, the bacteria were harvested and resuspended in PBS (10 mM Mg) containing 1 μM IV-4. 2+The yeast cells were resuspended in a 100% PBS containing IV-4 (Figure 9). The untreated BY4741 strain served as a control. Imaging results showed that only yeast cells expressing F30-Carrot-2 and in the presence of IV-4 exhibited bright orange-red fluorescence (Figure 9), indicating that the Carrot-IV-4 complex can be used for fluorescent labeling of RNA in yeast cells.

[0130] Example 9. Labeling of RNA in mammalian cells with Carrot and IV-39 and its analogs To detect RNA labeling in mammalian cells by Carrot and IV-39, we constructed their mammalian cell expression plasmids using the reported Pepper and Corn nucleic acid aptamer molecules (conjugated with HBC599 and DFHO fluorophore molecules, respectively) as controls (Chen et al. Nature Biotechnology 2019.37:1287-1293; Song et al. Nature Chemical Biology 2017.13:1187-1194). DNA fragments encoding tRNA-Carrot-2 (encoding RNA sequence is SEQ ID No:4), tRNA-Pepper (encoding RNA sequence is SEQ ID No:22) and tRNA-Corn (encoding RNA sequence is SEQ ID No:21) were synthesized in whole genome, and these fragments were PCR amplified by primers P7 and P8, respectively, and pEGFP-N1 vector was amplified by primers P9 and P10 to remove its own CMV promoter and multiple cloning site region. According to the experimental method (IV), these fragments were inserted into the pEGFP-N1 vector from which the promoter and multicloning site region had been removed. Furthermore, these vectors were amplified and linearized by P11 and P12, and the U6 promoter (SEQ ID No: 23) was amplified using the pLKO.1puro vector as a template by P13 and P14. The U6 promoter was inserted into the linearized vector according to the experimental method (IV). The resulting plasmids were named pU6-tRNA-Carrot-2, pU6-tRNA-Pepper and pU6-tRNA-Corn, which express tRNA-Carrot-2, tRNA-Pepper and tRNA-Corn, respectively.pU6-tRNA-Carrot-2, pU6-tRNA-Pepper and pU6-tRNA-Corn were introduced into 293T / 17 cells by plasmid transfection, and 24 hours later, 0.5μM IV-39, 0.5μM HBC599 and 10μM DFHO were added to label tRNA-Carrot-2, tRNA-Pepper and tRNA-Corn, respectively. The corresponding RNA aptamer-unexpressing cells were used as controls to detect the labeling effect according to the experimental method (III). According to the results, the addition of 20mM Mg to the detection buffer. 2+ Regardless of the addition of , the tRNA-Carrot-IV-39 complex exhibited very bright orange-red fluorescence (Fig. 10a), and the fluorescence intensity was obviously higher than that of the tRNA-Pepper-HBC599 and tRNA-Corn-DFHO complexes (Fig. 10b), indicating that Carrot-IV-39 could work well in mammalian cells.

[0131] The primers used for amplifying tRNA-Carrot-2, tRNA-Pepper, and tRNA-Corn are as follows.

[0132] Upstream primer (P7): 5'-GCCGCCCCCTTCACCTCTAGAGCCCGGATAGCTCAGTCGG-3' Downstream primer (P8): 5'-GAGAATTCAAAAAAATGGCGCCCGAACAGGGAC-3' The primers used to amplify pEGFP-N1 and remove the promoter and multicloning site regions are as follows:

[0133] Upstream primer (P9): 5'-TTTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCA-3' Downstream primer (P10): 5'-GGTGAAGGGGGCGGCCGCTCGAGG-3' The primers used to amplify and linearize the vector and introduce the U6 promoter are as follows:

[0134] Upstream primer (P11): 5'-TCTAGAGCCCGGATAGCTCAGTCGGT-3' Downstream primer (P12): 5'-GGTGAAGGGGGCGGCCGCTCGAGG-3' The primers used to amplify the U6 promoter are as follows:

[0135] Upstream primer (P13): 5'-GCCGCCCCCTTCACCGAGGGCCTATTTCCCATG-3' Downstream primer (P14): 5'-TATCCGGGCTCTAGAGTTTCGTCCTTTCCACAAGATATAT-3' In order to detect the labeling of RNA in mammalian cells by Carrot and IV-39 analogues, a mammalian expression plasmid expressing F30-4Carrot-2 was constructed. In this example, the primers P15 and P16 were used to amplify the F30-4Carrot-2 fragment in Example 5, and these fragments were inserted into the pU6-tRNA-Carrot-2 vector that was enzymatically cut with XbaI and EcoRI according to experimental method (IV), and the resulting expression vector was named pU6-F30-4Carrot-2.

[0136] The primers used for amplifying the F30-4Carrot-2 fragment are as follows:

[0137] Upstream primer (P15): 5'-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGGA-3' Downstream primer (P16): 5'-TGTCTCGAGGTCGAGAATTCAAAAAAATTGCCATGAATGATCCCGAAG-3' pU6-F30-4Carrot-2 was introduced into 293T / 17 cells by plasmid transfection, and after 24 h, different IV-39 analogs were added for labeling, and the labeling effect was detected by experimental method (III). The results showed that all the different IV-39 analogs could specifically label the cells expressing F30-4Carrot-2, but did not label the control cells not expressing F30-4Carrot-2 (Figure 10c), indicating that Carrot and IV-39 and their analogs can be used to label RNA in mammalian cells.

[0138] Example 10. Probe construction with Carrot To construct a Carrot-based analyte probe, N in the general formula of Carrot-1 (SEQ ID No: 1) 14 -N 15 -N 16 The nucleotides were replaced with RNA aptamers that can specifically recognize and bind adenosine and S-adenosylmethionine (SAM), and these aptamers were linked to Carrot-1 by appropriate bases (Figure 11a). Probe RNAs were prepared according to conventional experimental methods (1), incubated with IV-39, and the fluorescence intensity in the presence or absence of adenosine or SAM was detected by a multi-function microplate reader. The detection results showed that the fluorescence of these probes in the presence of the target was significantly higher than that in the absence of the target (Figure 11b), indicating that they can be used as probes to detect adenosine and SAM, respectively. The corresponding probe RNA sequences are SEQ ID No: 24 and SEQ ID No: 25.

[0139] Example 11. Localization of RNA in traced cells with Carrot To confirm the results of using Carrot to localize RNA in trace cells, we first constructed an expression plasmid for chimeric RNA in which Carrot was fused with a different RNA. The 4Carrot-2 fragment was amplified with primers P17 and P18, and inserted into the pCDNA3.1 hygro(+) vector double-cut with HindIII and XhoI according to the experimental method (IV), to obtain the pCDNA3.1 hygro(+)-4Carrot-2 recombinant plasmid. GAPDH and ACTB gene fragments were synthesized in whole genome (the gene sequences encoding GAPDH and ACTB are Genebank: BC009081, BC016045, respectively), and the GAPDH and ACTB gene fragments were amplified by primers, respectively, and inserted into pCDNA3.1 hygro(+)-4Carrot-2 vector double enzyme-cut by NheI and HindIII to obtain pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 and pCDNA3.1 hygro(+)-ACTB-4Carrot-2 recombinant plasmids, which encode GAPDH-4Carrot-2 and ACTB-4Carrot-2 chimeric RNAs, respectively, and their sequences are SEQ ID No: 26 and 27.

[0140] The primers used for amplifying 4Carrot-2 are as follows:

[0141] Upstream primer (P17): 5'-TAGCGTTTAAACTTAAGCTTGGAAGATTGTAAACACGCC-3' Downstream primer (P18): 5'-ACGGGCCCTCTAGACTCGAGGGAAGTGTCCGCCGGAAGT-3' The primers used for amplifying GAPDH are as follows:

[0142] Upstream primer (P19): 5'-GGAGACCCAAGCTGGCTAGCATGGGGAAGGTGAAGGTCGG-3' Downstream primer (P20): 5'-CACGGACACATGGCAAGCTTAACCATGCTCTAGCGAGTGTTACTCCTTGGAGGCCATGT-3' The primers used for amplifying ACTB are as follows:

[0143] Upstream primer (P21): 5'-GGAGACCCAAGCTGGCTAGCATGGTGACGCTTGCTGAACT-3' Downstream primer (P22): 5'-CACGGACACATGGCAAGCTTAACCATGCTCTAGCGAGTGCTAGAAGCATTTGCGGTGGA-3' After constructing the above-mentioned plasmids, the inserted sequences were confirmed to be completely accurate by sequencing, and the plasmids were extracted using a transfection-grade plasmid extraction kit and used in subsequent transfection experiments.

[0144] The pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 and pCDNA3.1 hygro(+)-ACTB-4Carrot-2 recombinant plasmids constructed in this example were transfected into COS-7 cells, and 24 hours after transfection, 1μM IV-4 was added to label the cells, and the cells were imaged according to the fluorescent imaging method described in Experimental Method (III). The imaging results showed that the fluorescence of GAPDH-4Carrot-2 and ACTB-4Carrot-2 was mainly concentrated in the cytoplasm (Figure 12), which is consistent with previous reports and is also consistent with the results of fluorescent in situ hybridization (FISH) (Figure 12). These results indicate that Carrot can be used to trace the location of RNA.

[0145] Example 12. Detection of genomic DNA using Carrot To detect genomic DNA by Carrot, we first constructed recombinant plasmids expressing Carrot-2 and sgRNA chimeric RNA. cDNAs of sgRNA-Carrot-2-1, sgRNA-Carrot-2-2 and sgRNA-Carrot-2-3 containing centromere targeting sequences were synthesized in the whole genome, and their encoding RNA sequences are SEQ ID No: 28, 29 and 30, respectively. The cDNAs of the chimeric RNAs were amplified by primers P23 and P24, and the psgRNA plasmid (Shao et al. Nucleic acids research 2016.44:e86) was amplified by primers P25 and P26. The amplified cDNA was ligated with the linearized psgRNA vector according to the experimental method (IV), and the resulting plasmids were named psgRNA-Carrot-2-1, psgRNA-Carrot-2-2 and psgRNA-Carrot-2-3, respectively (Figure 13a). The dCas9-GFP gene fragment was amplified using pSLQ1645 (dCas9-GFP) (Shao et al. Nucleic Acids Research 2016.44:e86) as a template with primers P27 and P28, and inserted into the pCDNA3.1hygro(+) vector double-cut with HindIII and XhoI according to experimental method (IV). The resulting plasmid was named pCDNA3.1hygro(+)-dCas9-GFP.

[0146] The primers used to amplify cDNA corresponding to Carrot and sgRNA chimeric RNA are as follows:

[0147] Upstream primer (P23): 5'-AAAGGACGAAACACCGAATCTGCAAGTGGATATTGTTTGAG-3' Downstream primer (P24): 5'-TGATCTAGAAAAAAAGCACCGACTCGGTGCCAC-3' The primers used to amplify and linearize the psgRNA plasmid are as follows:

[0148] Upstream primer (P25): 5'-TTTTTTTCTAGATCATAATCAGCCATACC-3' Downstream primer (P26): 5'-GGTGTTTCGTCCTTTCCACAAG-3' The primers used to amplify SpdCas9-GFP are as follows:

[0149] Upstream primer (P27): 5'-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCACCATGGCCCC-3' Downstream primer (P28): 5'-ACGGGCCCTCTAGACTCGAGTTACTTGTACAGCTCGTCCATGC-3' pCDNA3.1 hygro(+)-dCas9-GFP was introduced into COS-7 cells by co-transfection of psgRNA-Carrot-2-1, psgRNA-Carrot-2-2, and psgRNA-Carrot-2-3 with the recombinant plasmid. 24 h after transfection, the cells were labeled with 1 μM IV-4 and Hoechst, and the fluorescence of Carrot-2-IV-4 complex, GFP, and Hoechst was observed under a fluorescent microscope. The imaging results showed that the fluorescence of Carrot-2-IV-4 was mainly concentrated in the cell nucleus and gathered in puncta (centromere), which was completely consistent with the fluorescence of dCas9-GFP (Figure 13b), indicating that Carrot can be used for imaging gene DNA.

[0150] Example 13. Detection of RNA-protein interactions using Carrot To detect the interaction between RNA and protein by Carrot, we took the recognition and binding of the MS2 RNA sequence of the phage capsid protein MCP as an example. The Carrot-1-MS2 fragment (the RNA sequence encoded by it is SEQ ID No:31) was synthesized in its entirety and amplified as a template by primers P29 and P30. The resulting fragment was inserted into pU6-tRNA-Carrot-2 that had been double-cut with XbaI and EcoRI according to experimental method (IV), and the resulting plasmid was named pU6-Carrot-1-MS2. The gene fragment encoding tdMCP protein (which is a dimeric form of MCP protein and its coding DNA sequence is SEQ ID No:32) and the gene fragment encoding NanoLuc protein (which is its coding DNA sequence is SEQ ID No:33) were synthesized in whole genome, the tdMCP gene fragment was amplified by primers P31 and P32, P33 and P34, respectively, and the NanoLuc gene fragment was amplified by primers P35 and P36. The amplified fragments were inserted into pCDNA3.1 hygro(+) vector double-cut with HindIII and XhoI according to the experimental method (IV). The resulting plasmid was named pCDNA3.1 hygro(+)-tdMCP-NanoLuc-tdMCP, and the gene sequence of the fusion protein encoded by it is SEQ ID No:34.

[0151] The primers used for amplifying the Carrot-1-MS2 fragment are as follows:

[0152] Upstream primer (P29): 5'-GGAAAGGACGAAACTCTAGAGGGAAGATTGTAAACAC-3' Downstream primer (P30): 5'-TGTCTCGAGGTCGAGAATTCAAAAAAAGGGAAGTGTCCGATGGG-3' The primers used for amplifying tdMCP are as follows:

[0153] Upstream primer (P31): 5'-TAGCGTTTAAACTTAAGCTTATGCTAGCCGTTAAAATGGC-3' Downstream primer (P32): 5'-ACTCCCTCCGCCACCTCCAGAATCCGCGTAGATGC-3' Another set of primers used to amplify tdMCP is as follows:

[0154] Upstream primer (P33): 5'-ACTCCCTCCCGCCAGAATGCGTTCGCAC-3' Downstream primer (P34): 5'-ACGGGCCCTCTAGACTCGAGTTATCCAGAATCCGCGTAG-3' The primers used for amplifying NanoLuc are as follows:

[0155] Upstream primer (P35): 5'-GGTGGCGGAGGGAGTATGGTCTTCACACTCGAAGA-3' Downstream primer (P36): 5'-ACTCCCTCCCGCCAGAATGCGTTCGCAC-3' pCDNA3.1 hygro(+)-tdMCP-NanoLuc-tdMCP was introduced into 293T / 17 cells by co-transfection of pU6-Carrot-1-MS2 and the recombinant plasmid. 24 h after transfection, 1 μM IV-4 fluorophore was added to label Carrot, and then imaging was performed immediately after adding 20 μM Furimazine to the medium. The imaging results showed that the tdMCP protein in the tdMCP-NanoLuc-tdMCP fusion protein recognized and bound to the MS2 RNA in the Carrot-1-MS2 chimeric RNA, and the distance between the Carrot-IV-4 complex and the NanoLuc protein was very close, and the light emitted by the catalytic substrate Furimazine of NanoLuc was transferred to the Carrot-IV-4 complex, so the efficiency of luminescence energy transfer (BRET) was relatively high, and the Carrot-IV-4 complex was excited to produce obvious orange-red fluorescence (Figure 14). As a control, Carrot-1 RNA alone without MS2 did not interact with the tdMCP-NanoLuc-tdMCP fusion protein, and the distance between the Carrot-IV-4 complex and the NanoLuc protein was relatively long, and the light emitted by Furimazine, the catalytic substrate of NanoLuc, was not transferred to the Carrot-IV-4 complex, so the efficiency of luminescence energy transfer (BRET) was relatively low and the Carrot-IV-4 complex could not be excited to produce obvious orange-red fluorescence (Figure 14).

[0156] Example 14. RNA Purification and Purification Tags with Carrot pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 in Example 11 was introduced into COS-7 cells by recombinant plasmid transfection, and the cells were harvested after 24 h and incubated in 40 mM HEPES, pH 7.4, 125 mM KCl, 5 mM MgCl 2The cells were resuspended in 100 μL of PBS (handled on ice). Activated Thiol Sepharose 4B (GE Healthcare) was taken and washed twice with 500 μL of PBS, then incubated with PBS containing 10 mM TCEP (Sigma) at room temperature for 1 h. After washing twice with 500 μL of PBS, maleamide-containing IV-39 fluorophore molecule (Mal-IV-39) was added and reacted at room temperature for 30 min, and washed three times with 500 μL of PBS. The resuspended cells were disrupted and incubated with treated beads at 4°C, and after 30 min, centrifuged at 4000 rpm for 2 min. The supernatant was discarded and the cells were incubated in pre-cooled 40 mM HEPES, pH 7.4, 125 mM KCl, 5 mM MgCl 2 The agarose beads were washed six times with this buffer, centrifuged each time and the supernatant was discarded. The beads were resuspended in DEPC water, treated at 70°C for 10 min, centrifuged at 4000 rpm for 2 min, and the supernatant was collected. 1 / 10 volume of NaAc and 2.5 volumes of absolute ethanol were added to the collected supernatant, left in a -80°C refrigerator for 20 min, centrifuged at 14000 rpm for 10 min at 4°C, the precipitate was left, the supernatant was discarded, the precipitate was washed with pre-cooled 70% ethanol solution, centrifuged at 14000 rpm for 10 min at 4°C, the precipitate was left, the supernatant was discarded, and this procedure was repeated once. The precipitate was left at room temperature for 5 min, and after the ethanol had finished evaporating, a small volume of DEPC water was added to resuspend the precipitate.

[0157] The supernatant after cell lysis and the eluate after the final high-temperature elution were incubated with fluorophore IV-39 and the fluorescence was detected, respectively, and the supernatant after blank cell lysis was used as a control. The detection results showed that the fluorescence after incubation of the eluate with IV-39 was obviously higher than that of the supernatant after lysis before sample loading (Figure 15), indicating that GAPDH-4Carrot-2 RNA was successfully enriched and that Carrot could be used as a tag for RNA isolation and purification.

[0158] Example 15. Synthesis of IV-39 and its analogs Compound IV-1:

[0159] [ka] Compound 1 (0.504 g, 2 mmol) and p-cyanobenzaldehyde (0.626 g, 5 mmol) were placed in a 250 ml round flask, and 100 ml of tetrahydrofuran was added to dissolve the compound. Under Ar protection, anhydrous zinc chloride (0.545 g, 4 mmol) was added, and the system was heated to reflux in an oil bath at 80°C. The reaction was measured by TLC. After the reaction was completed, the solvent was removed in vacuum, and the residue was separated by column chromatography to obtain the desired product (0.292 g, 40%). 1 H NMR (400MHz, DMSO-d 6 ) δ 11.07(s,1H),8.10(d,J=8.5Hz,2H),8.06(dd,J=7.8,2.1Hz,2H),8.03(s,1H),7.9 4(d,J=8.3Hz,2H),7.44(d,J=15.9Hz,1H),7.03(s,1H),3.29(s,3H).MS(ESI):m / z Calcd.For C 20 H 13 F 2 N 3 O 2 365.0976;found 364.0902,[MH] - . Compound IV-2:

[0160] [ka] The procedure for the synthesis of compound IV-1 was followed (0.475 g, 65%). 1 H NMR (400MHz, DMSO-d 6 ) δ 11.04(s,1H),8.49(d,J=1.9Hz,1H),8.21(dt,J=8.0,1.4Hz,1H),8.10-8.02(m,2H),8.00(s,1H),7.88(dt ,J=7.7,1.4Hz,1H),7.67(t,J=7.8Hz,1H),7.43(d,J=16.0Hz,1H),7.01(s,1H),3.29(s,3H).MS(ESI):m / z Calcd.For C20 H 13 F 2 N 3 O 2 365.0976; found 364.0903, [M-H] - . Compound IV-3:

[0161]

Chem.

[0162]

Chem.

[0163]

Chem.

[0164]

Chem.

[0165] [ka] Compound IV-5 (0.716 g, 2.0 mmol), t-butyldimethylchlorosilane (0.450 g, 3.0 mmol), and imidazole (0.204 g, 3.0 mmol) were placed in a 100 ml round flask, and 50 ml of dry dimethylformamide was added to dissolve the compound. The mixture was stirred at room temperature under Ar protection conditions and measured by TLC. After the reaction was completed, the system was poured into 150 ml of water and extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was separated by column chromatography to obtain compound 2 (0.927 g, 98%). 1 H NMR (400MHz, DMSO-d 6 ) δ 8.16(d,J=8.5Hz,2H),7.99-7.86(m,3H),7.31(t,J=8.9Hz,2H),7.18(d,J=15.9Hz,1H),6.95(s,1H),6.83-6.72(m ,2H),3.59(t,J=5.9Hz,2H),3.51(t,J=5.9Hz,2H),3.27(s,3H),3.05(s,3H),1.50(s,9H),0.2(s,6H).MS(ESI):m / z Calcd.For C 25 H 28 F 3 N 2 O 2 Si 473.2; found 473.2, [M+H] + .

[0166] [ka] Compound 2 (0.473g, 1mmol) and Lawesson's reagent (0.808g, 2mmol) were placed in a 250ml three-neck flask, dissolved in 100ml of toluene, added with 2 drops of aniline, heated under reflux in an oil bath under Ar protection, and measured by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was dissolved in 50ml of dichloromethane, added with tetrabutylammonium fluoride (0.313g, 1.2mmol), stirred at room temperature under Ar protection, and measured by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain compound IV-7 (0.209g, 56%). 1 H NMR (400MHz, DMSO-d 6 ) δ 8.17(d,J=8.5Hz,2H),7.98-7.86(m,3H),7.31(t,J=8.9Hz,2H),7.18(d,J=15.9Hz,1H),6.95(s,1H),6 .83-6.72(m,2H),3.59(t,J=5.9Hz,2H),3.51(t,J=5.9Hz,2H),3.27(s,3H),3.05(s,3H).MS(ESI):m / z Calcd.For C 19 H 13 F 3 N 2 NaOS 397.0598;found 397.0597,[M+Na] + . Compound IV-8: Compound 3:

[0167] [ka] The procedure for the synthesis of compound 2 was followed (0.932 g, 99%). 1 H NMR (400MHz, DMSO-d 6 ) δ 8.10(d,J=8.5Hz,2H),8.06(dd,J=7.8,2.1Hz,2H),8.03(s,1H),7.94(d,J=8.3Hz,2H),7 .44(d,J=15.9Hz,1H),7.03(s,1H),3.29(s,3H),1.51(s,9H),0.29(s,9H).MS(ESI):m / z Calcd.For C 26 H 28 F2 N 3 O 2 Si 480.2; found 480.2, [M+H] + .

[0168] [ka] The procedure for the synthesis of compound 2 was followed (0.332 g, 49%). 1 H NMR (400MHz, DMSO-d 6 ) δ 11.00(s,1H),8.11(d,J=8.5Hz,2H),8.07(dd,J=7.8,2.1Hz,2H),8.04(s,1H),7.9 4(d,J=8.3Hz,2H),7.44(d,J=15.9Hz,1H),7.03(s,1H),3.29(s,3H).HMS(ESI):m / z Calcd.For C 20 H 13 F 2 N 3 NaOS 404.0645;found 404.0646,[M+Na] + . Compound IV-9: Compound 5:

[0169] [ka] 3-Fluoro-4-hydroxy-benzaldehyde (0.560g, 4.0mmol) was placed in a 100ml round flask, dissolved in 40ml of anhydrous ethanol, added with 10g of anhydrous sodium sulfate and 5ml of 33% aqueous methylamine solution, stirred at room temperature for 24h under Ar protection, filtered, removed the organic solvent under pressure, dissolved the residue in 10ml of anhydrous ethanol, added compound 4 (0.790g, 5.0mmol), stirred at room temperature overnight under Ar protection, filtered the next day, and rinsed with cold ethanol three times to obtain compound 5 (0.796g, 85%). 1 H NMR (400MHz, DMSO-d 6) δ 10.52(s,1H),8.19(m,1H),7.76(m,1H),6.99(t,J=8.8Hz,1H),6.89(s,1H),3.09(s,3H),2.34(s,3H).MS(ESI):m / z Calcd.For C 12 H 10 FN 2 O 2 234.2;found 234.2,[M-H] - .

[0170]

Chem.

[0171]

Chem.

[0172]

Chem.

[0173]

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[0174]

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[0175]

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[0176]

Chem.

[0177]

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[0178]

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[0179]

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[0180]

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[0181]

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[0182]

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[0183]

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[0184]

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[0185]

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[0186]

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[0188]

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[0189]

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[0190]

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[0191]

Chem.

[0192]

Chem.

[0193] [ka] The procedure for the synthesis of compound IV-7 was followed (0.152 g, 72%). 1 H-NMR (400MHz, DMSO-d 6): δ 8.43 (d, J = 1.8 Hz, 1H), 8.16 (t, J = 8.5 Hz, 3H), 7.93 (d, J = 15.9 Hz, 1H), 7.85 (dt, J = 7.8, 1.4 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.40 (d, J = 15.9 Hz, 1H), 6.98 (s, 1H), 6.86 - 6.71 (m, 2H), 3.81 (s, 2H), 3.60 (t, J = 5.9 Hz, 2H), 3.52 (t, J = 5.9 Hz, 2H), 3.41 (s, 3H), 3.06 (s, 3H). MS(ESI): m / z Calcd. For C 26 H 27 N 4 OS 443.1906; found 443.1905, [M + H] + . Compound IV - 24: Compound 15:

[0194]

Chem.

[0195]

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[0196]

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[0197]

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[0198]

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[0199]

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[0201]

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[0202]

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[0203]

Chem.

[0204] [ka] The procedure for the synthesis of compound IV-1 was followed (0.333 g, 26%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.75(d,J=1.5Hz,1H),8.32(dt,J=2.1,8.2Hz,1H),7.95(d,J=16.0Hz,1H ),7.60(s,2H),7.16(s,1H),7.05(dd,J=2.5,8.4Hz,1H),6.95(d,J=16.0 Hz,1H),6.44(d,J=8.26Hz,1H),3.60(t,J=8.46Hz,2H),3.06(dd,J=18.05,9.48Hz,2H),4.01(t,J=8.4Hz,2H),2.93(t,J=8.4Hz,2H).MS(ESI):m / z Calcd.For C 22 H 22 FN 4 O 2 393.1727;found 393.1726,[M+H] + . Compound IV-29: Compound 21:

[0205] [ka] Compound IV-21 (0.792 g, 2.0 mmol), p-toluenesulfonyl chloride (0.476 g, 2.5 mmol), and triethylamine (0.303 g, 3.0 mmol) were placed in a 250 ml round flask, dissolved in 100 ml dry dichloromethane, stirred at room temperature under Ar protection conditions, and measured by TLC. After the reaction was completed, the system was poured into 200 ml water and extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was separated by column chromatography to obtain compound 27 (0.822 g, 76%). 1 H-NMR (400MHz, DMSO-d 6):δ 8.18(d,J=8.5Hz,2H),8.08-8.02(m,2H),7.98-7.90(m,3H),7.40(d,J=15.9Hz,1H),7.18(d,J=8.2Hz,2H),7.00(s,1H),6.86-6.7 2(m,2H),6.47(d,J=8.2Hz,2H),4.06(t,J=6.1Hz,2H),3.49(t,J=6.1Hz,2H),3.28(s,3H),2.77(s,3H),2.31(s,3H).MS(ESI):m / z Calcd.For C 30 H 29 N 4 O 4 S 541.2; found 541.2, [M+H] + .

[0206] [ka] Compound 21 (0.541 g, 1.0 mmol) and sodium sulfite (0.630 g, 5.0 mmol) were placed in a 100 ml round flask, 20 ml of anhydrous DMF was added, and the mixture was heated in an oil bath at 50°C for 24 hours under Ar protection conditions, and the reaction was measured by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by reverse phase chromatography to obtain the product (0.301 g, 60%). 1 H-NMR (400MHz, DMSO-d 6 ):δ 8.18(d,J=8.5Hz,2H),8.08-8.02(m,2H),7.98-7.90(m,3H),7.40(d,J=15.9Hz,1H),7.00(s,1H),6.86-6.72(m,2) H),3.85(m,4H),3.60(t,J=5.9Hz,2H),3.52(t,J=5.6Hz,2H),3.28(s,3H),3.16(m,4H),2.77(s,3H).MS(ESI):m / z Calcd.For C 23 H 21 N 4 O 4 S 499.1289;found 499.1288,-[MH] - . Compound IV-30: Compound 22:

[0207] [Chemical formula] Synthesized according to the procedure for Compound 5 (0.572 g, 80%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.60 (s, 2H), 7.04 (s, 1H), 6.44 (d, J = 8.26 Hz, 1H), 3.60 (t, J = 8.46 Hz, 2H), 3.06 (dd, J = 18.05, 9.48 Hz, 2H), 4.57 (q, J = 9.25, 2H), 2.93 (s, 3H), 2.42 (s, 3H). MS (ESI): m / z Calcd. For C 16 H 17 F 3 N 3 O 324.1; found 324.1, [M+H] + .

[0208] [Chemical formula] Synthesized according to the procedure for Compound IV-1 (0.433 g, 21%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.75 (d, J = 1.5 Hz, 1H), 8.32 (dt, J = 2.1, 8.2 Hz, 1H), 7.95 (d, J = 16.0 Hz, 1H), 7.60 (s, 2H), 7.11 (dd, J = 2.5, 8.4 Hz, 1H), 7.04 (s, 1H), 6.95 (d, J = 16.0 Hz, 1H), 6.44 (d, J = 8.26 Hz, 1H), 3.60 (t, J = 8.46 Hz, 2H), 3.06 (dd, J = 18.05, 9.48 Hz, 2H), 4.57 (q, J = 9.25, 2H), 2.93 (s, 3H). MS (ESI): m / z Calcd. For C 22 H 19 BrF 3 N 4 O 491.0694; found 491.0693, [M+H] + . Compound IV-31:

[0209] [Chemical formula] Compound IV-21 (0.386 g, 1.0 mmol), compound 31 (0.265 g, 1.2 mmol), EDCI (0.382 g, 2.0 mmol), and DMAP (0.183 g, 1.5 mmol) were placed in a 100 ml round flask, dissolved in 30 ml of anhydrous DMF, stirred at room temperature under Ar protection conditions, and measured by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain the product (0.490 g, 83%). 1 H-NMR (400MHz, DMSO-d 6 ):δ 8.18(d,J=8.5Hz,2H),8.08-8.02(m,2H),7.98-7.90(m,3H),7.40(d,J=15.9Hz,1H),7.00(s,1H),6.86-6.72(m,2H),4.17(s ,2H),3.75(s,3H),3.6-3.7(m,10H),3.57(m,2H),3.52(t,J=5.6Hz,2H),3.38(s,3H),3.28(s,3H),3.06(s,3H).MS(ESI):m / z Calcd.For C 32 H 39 N 4 O 7 S 591.2819;found 591.2820,[M+H] + . Compound IV-32: Compound 31:

[0210] [ka] The procedure for the synthesis of compound 5 was followed (0.612 g, 87%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.15(d,J=9.0Hz,2H),8.14(d,J=9.0Hz,2H),7.21(s,1H),4.23(s,2H),4.11(s,3H), 3.38(t,J=6.4Hz,2H),3.01(s,3H),2.92(t,J=6.4Hz,2H),2.41(s,3H).MS(ESI):m / z Calcd.For C 18 H 25 N4 O 3 345.2; found 345.2, [M+H] + .

[0211]

Chem.

[0212]

Chem.

[0213] [ka] Compound 25 (0.534 g, 1.0 mmol)) was placed in a 100-mL round flask, dissolved in 35 mL of DMF, and carefully added NaN 3 (0.195g, 3.0mmol) was added and heated in an oil bath at 50°C overnight under Ar protection conditions. The next day, the system was cooled to room temperature, poured into 100ml of water, extracted with DCM three times, the organic phases were combined, washed with saturated brine twice, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue did not need to be purified and was used directly in the next reaction.

[0214] The residue was dissolved in 30 ml THF and 3 P (0.524 g, 2.0 mmol) and 2 ml of water were added, and the mixture was stirred overnight at room temperature under Ar protection. The next day, the solvent was removed under pressure, and the residue was separated by column chromatography to obtain the desired product (0.301 g, 79%). 1 H-NMR (400MHz, DMSO-d 6):δ 8.17(d,J=8.6Hz,2H),7.91(d,J=15.8Hz,1H),7.79(dt,J=10.6,2.1Hz,1H),7.66(d,J=7.8Hz,1H),7.50(td,J=8.0,6.2Hz,1H),7.3 4-7.20(m,2H),6.97(s,1H),6.84-6.73(m,2H),3.38(t,J=6.4Hz,2H),2.92(t,J=6.4Hz,2H),3.27(s,3H),3.06(s,3H).MS(ESI):m / z Calcd.For C 22 H 24 FN 4 O 379.1934;found 379.1935,[M+H] + . Compound IV-34:

[0215] [ka] Compound 25 (0.534 g, 1.0 mmol) was placed in a 100 ml round flask, dissolved in 50 ml of ethanol, and then 33% dimethylamine solution was added. The mixture was heated to reflux in an oil bath at 90° C. under Ar protection conditions and measured by TLC. After the reaction was completed, the system was returned to room temperature, the solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain the desired product (0.276 g, 68%). 1 H-NMR (400MHz, DMSO-d 6 ):δ 8.17(d,J=8.6Hz,2H),7.91(d,J=15.8Hz,1H),7.79(dt,J=10.6,2.1Hz,1H),7.66(d,J=7.8Hz,1H),7.50(td,J=8.0,6.2Hz,1H),7.3 4-7.20(m,2H),6.97(s,1H),6.84-6.73(m,2H),3.47(t,J=7.6Hz,2H),2.96(s,3H),2.49(t,J=7.6Hz,2H),2.31(s,6H).MS(ESI):m / z Calcd.For C 24 H 28 FN 4 O 407.2247;found 407.2246,[M+H] + . Compound IV-35: Compound 26:

[0216] [ka] The procedure for the synthesis of compound IV-1 was followed (0.732 g, 39%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.19(d,J=8.5Hz,2H),8.07(m,4H),8.04(s,1H),7.94(d,J=8.3Hz,2H),7.44(d,J=15.9Hz,1H),7.03(s,1H),3.29(s,3H)..MS(ESI):m / z Calcd.For C 20 H 15 N 4 O 3 359.1;found 359.1,[M+H] + .

[0217] [ka] Compound 26 (0.718g, 2.0mmol) and stannous chloride (0.758g, 4.0mmol) were placed in a 250ml round flask, dissolved in 100ml of ethyl acetate, stirred at room temperature under Ar protection, and measured by TLC. After the reaction was completed, the system was poured into 150ml of water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was separated and purified by column chromatography to obtain the desired product (0.586g, 89%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.19(d,J=8.5Hz,2H),7.97(m,4H),8.04(s,1H),7.94(d,J=8.3Hz,2H),7.44(d,J=15.9Hz,1H),7.03(s,1H),3.29(s,3H).MS(ESI):m / z Calcd.For C 20 H 17 N 4 O 329.1402;found 329.1403,[M+H] + . Compound IV-36:

[0218] [Chem.] Synthesized according to the procedure for Compound 18 (0.322 g, 79%). 1 H-NMR (400 MHz, DMSO-d 6 ): δ 8.16 (d, J = 8.5 Hz, 2H), 7.93 (t, J = 4.4 Hz, 2H), 7.90 (d, J = 6.1 Hz, 1H), 7.31 (t, J = 8.9 Hz, 2H), 7.18 (d, J = 15.9 Hz, 1H), 6.95 (s, 1H), 6.83 - 6.72 (m, 2H), 4.21 (s, 2H), 3.59 (t, J = 5.9 Hz, 2H), 3.51 (t, J = 5.9 Hz, 2H), 3.27 (s, 3H), 3.05 (s, 3H). MS (ESI): m / z Calcd. For C 25 H 25 FN 3 O 2 418.1931; found 418.1932, [M + H] + . Compound IV-37:

[0219] [Chem.] Synthesized according to the procedure for Compound IV-1 (0.275 g, 75%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.00 (s, 1H), 8.08 - 8.04 (m, 2H), 8.02 (d, J = 15.9 Hz, 1H), 7.92 - 7.87 (m, 2H), 7.49 - 7.45 (m, 2H), 7.26 (d, J = 15.9 Hz, 1H), 6.98 (s, 1H), 3.29 (s, 3H). MS (ESI): m / z Calcd. For C 19 H 13 F 2 N 3 O 2 339.0951; found 339.0950, -[M - H] - . Compound IV-38:

[0220] [Chemistry] Synthesis was carried out according to the procedure for Compound IV-1 (0.327 g, 55%). 1 1H NMR (400 MHz, DMSO-d 6 ) δ 9.70 (s, 1H), 8.05 (d, J = 8.9 Hz, 2H), 7.98 - 7.89 (m, 1H), 7.30 (t, J = 6.2 Hz, 1H), 7.26 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 15.8 Hz, 1H), 6.97 (s, 1H), 6.87 (d, J = 7.5 Hz, 1H), 3.27 (s, 3H). MS (ESI): m / z Calcd. For C 18 1 12 9 2 1 3 2 2 340.0903; found 340.090, -[M - H] - . Compound IV-39:

[0221] [Chemistry] Synthesis was carried out according to the procedure for Compound IV-1 (0.342 g, 46%). 1 1H-NMR (400 MHz, DMSO-d 6 ): δ = 8.21 (d, 2H, J = 8.8 Hz), 8.00 (d, 1H, J = 16 Hz), 7.85 (d, 2H, J = 8.0 Hz), 7.50 - 7.43 (m, 2H), 7.42 (d, J = 2.6 Hz, 1H), 7.24 (s, 1H), 7.01 (s, 1H), 6.92 (d, 2H, J = 8.8 Hz), 3.85 (t, 2H, J = 5.6 Hz), 3.60 (t, 2H, J = 5.6 Hz), 3.10 (s, 3H). MS (ESI): m / z Calcd. For C 22 2 24 0 3 2 2 362.1869; found 362.1868, [M + H] + . Compound IV-40:

[0222] [Chemistry] The procedure for the synthesis of compound IV-1 was followed (0.412 g, 46%). 1 H-NMR (400MHz, DMSO-d 6 ):δ =8.72(s,2H),8.00(d,1H,J=16Hz),7.50-7.43(m,2H),7.42(d,J=2.6Hz,1H),7.24(s,1H),7.01(s, 1H),6.92(d,2H,J=8.8Hz),3.75(t,J=6.8Hz,2H),3.60(t,2H,J=6.8Hz),3.05(s,3H).MS(ESI):m / z Calcd.For C 21 H 21 lN 6 O 373.1777;found 373.1778,[M+H] + .

[0223] Unless otherwise specified, the amounts used, reaction conditions, etc. in each example of this specification are approximate values, and it should be understood that slight changes can be made according to the actual situation to obtain similar results. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as understood by those skilled in the art. All documents mentioned in this specification are incorporated by reference into this application. The present specification describes preferred embodiments for illustrative purposes, and those skilled in the art can practice the present invention using methods and materials similar to those described in this specification to obtain the same or similar results, and various changes or modifications made to the present invention are still included within the scope defined in the claims attached to this application.

Claims

1. A nucleic acid aptamer molecule comprising a nucleotide sequence (a), a nucleotide sequence (b), or a nucleotide sequence (c), The nucleotide sequence (a) is １ AGAUUGUAAACAN １４ -N １５ -N １６ GACACUN ２３ Where N １ , N １４ , N １５ , N １６ and N ２３ represents a fragment equal to or greater than one nucleotide in length, and N １ and N ２３ At least one pair of bases in the nucleotide sequence forms a complementary base pair, １４ and N １６ At least one pair of bases in the nucleotide sequence forms a complementary base pair; the nucleotide sequence (b) has at least 70%, 72%, 77%, 83%, 88%, 94% or 100% identity to the nucleotide sequence (a); The nucleotide sequence (c) is a nucleotide sequence (a) containing N １ , N １４ , N １５ , N １６ and N ２３ and having aptamer function, and derived from the nucleotide sequence (a), Nucleic acid aptamer molecule.

2. The nucleotide sequence (c) is a nucleotide sequence (a) containing N １ , N １４ , N １５ , N １６ and N ２３ The nucleic acid aptamer molecule of claim 1, which is a nucleic acid aptamer molecule obtained by substituting, deleting and / or inserting 5, 4, 3, 2 or 1 nucleotides at a position that does not contain:

3. N in nucleotide sequence (a) １ and N ２３ If N forms a complementary base pair, １ The orientation of the nucleotide sequence is 5'-3', ２３ The orientation of the nucleotide sequence is 3'-5', １４ and N １６ If N forms a complementary base pair, １４ The orientation of the nucleotide sequence is 5'-3', １６ The nucleic acid aptamer molecule of claim 1, wherein the nucleotide sequence is 3'-5' oriented.

4. N １ and N ２３ If the length of at least one fragment in １ and N ２３ At least two pairs of bases in the nucleotide sequence form complementary base pairs, １４ and N １６ If the length of at least one fragment in １４ and N １６ The nucleic acid aptamer molecule of claim 3 , wherein at least two pairs of bases in the nucleotide sequence form complementary base pairs.

5. Nucleotide substitutions into nucleotide sequence (a) are A4U, A4G, A4C, U5A, U5G, U5C, G7C, G7A, G7U, U8C, A10U, A10G, A10C, A11U, A11G, A11C, C12G, C12A, C12U, A13U, A13G, A13C, G17A, C19A, C19U, A20 C, A4C / U5A, A4C / U5C, A4C / A11G, A4C / C12A, A4C / A13C, U5A / A11G, U5A / C12A, U5A / A13C , U5G / A13C, U5C / G7U, U5C / A11G, U5C / C12G, U5C / C12A, U5C / C12U, U5C / A13C, G7U / A13C , A11G / A13C, C12G / A13C, C12A / A13C, C12U / A13C, A4C / U5C / A13C, U5C / G7U / A13C, U5C / C12G / A13C, U5C / C12U / A13C, U5C / A11G / A13C, U5C / C12A / A13C, A4C / U5C / A11G / A13C, A The nucleic acid aptamer molecule of claim 1, which is one selected from the group consisting of 4C / U5C / C12A / A13C, A4C / U5C / G7U / A11G / A13C, A4C / U5C / G7U / C12A / A13C, A4C / U5C / A11G / C12A / A13C, and A4C / U5C / A11G / C12A / A13C.

6. N in nucleotide sequence (a) １ and N ２３ The nucleic acid aptamer molecule of claim 1 , wherein the nucleotide sequence of the position is F30 or a tRNA scaffold RNA sequence.

7. The nucleic acid aptamer molecule of claim 1 , wherein the aptamer molecule is an RNA molecule or a base-modified RNA molecule.

8. The nucleic acid aptamer molecule according to claim 1, which is a DNA-RNA hybrid molecule or a base-modified DNA-RNA molecule.

9. N １４ -N １５ -N １６ The nucleic acid aptamer molecule of claim 1, which comprises one nucleotide sequence capable of recognizing a target molecule.

10. The nucleic acid aptamer molecule of claim 9 , wherein the target molecule comprises a protein, a nucleic acid, a lipid molecule, a carbohydrate, a hormone, a cytokine, a chemokine, a metabolite, or a metal ion.

11. The N １４ -N １５ -N １６ The nucleic acid aptamer molecule of claim 9, wherein: is a nucleotide sequence capable of discriminating between S-adenosylmethionine and adenosine molecules.

12. The nucleic acid aptamer molecule of claim 1, wherein the aptamer function is that the nucleic acid aptamer can enhance the fluorescence intensity of the fluorophore molecule with excitation light of an appropriate wavelength by at least 2-fold, at least 5-10-fold, at least 20-50-fold, at least 100-200-fold, or at least 200-fold or more.

13. The nucleic acid aptamer molecule of claim 1, comprising a concatemer capable of binding to a plurality of fluorophore molecules, the concatemers being linked by a spacer sequence of suitable length, the number of which can be 2, 3, 4, 5, 6, 7, 8 or more, and the nucleotides of the concatemers are selected from the sequences SEQ ID No: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

14. The nucleic acid aptamer molecule of claim 1, wherein the nucleic acid aptamer molecule has the sequence SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30 or 31.

15. A complex of a nucleic acid aptamer molecule and a fluorophore molecule, the nucleic acid aptamer molecule being the nucleic acid aptamer molecule of claim 1, the fluorophore molecule having a structure represented by the following formula (I): 【Chemistry 1】 In the formula: Ar １ , Ar ２ are independently a 6-membered aryl group, a 6-membered heteroaryl group, and D- is HO- or N(X １ ) (X ２ )-, and X １ , X ２ are each independently selected from hydrogen, an alkyl group, and a modified alkyl group; １ , X ２ may be bonded to each other to form an aliphatic heterocycle together with the N atom, and D- is N(X １ ) (X ２ )-, Ar １ is a phenyl group, X １ , X ２ independently form a saturated or unsaturated ester heterocycle with a benzene ring, D- is HO-, and Ar １ is a phenyl group, at least one hydrogen atom adjacent to HO- is replaced with a halogen, Y is O, S, and R １ is hydrogen, an alkyl group, or a modified alkyl group; R ２ is a hydrogen atom, a halogen atom, -OH, or -CN, and each of the alkyl groups is independently １ -C １０ is a linear or branched alkyl group, The modified alkyl groups each independently have any carbon atom of the alkyl group substituted with a halogen atom, -OH, -CO-, -O-, -CN, or -SO ３ a modified alkyl group having 1 to 10 carbon atoms, with the proviso that the carbon-carbon single bonds may be independently replaced with a carbon-carbon double bond or a carbon-carbon triple bond, and the carbon atom being replaced means that the carbon atom, or the carbon atom together with the hydrogen atom thereon, is replaced with the corresponding group.

16. The fluorophore molecule has the formula 【Chemistry 2(1)】 【Chemistry 2(2)】 The complex of claim 15, wherein the complex is selected from

17. 16. The complex of claim 15, wherein the aptamer molecule in the complex comprises the nucleotide sequence SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30 or 31.

18. A kit comprising at least one of the nucleic acid aptamer molecule of claim 1, the complex of claim 15, an expression vector, or a host cell, The expression vector comprises a DNA molecule that transcribes the nucleic acid aptamer molecule; The host cell comprises the expression vector.

19. The complex of claim 15 for detecting or labeling a target nucleic acid molecule in vitro or in vivo, detecting or labeling a target molecule extracellularly or intracellularly, imaging of genomic DNA, detecting interactions between RNA and proteins, detecting genomic DNA, and purifying and purifying RNA.

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