WO2021190654A1 - 一种新型rna检测与定量的方法 - Google Patents

一种新型rna检测与定量的方法 Download PDF

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WO2021190654A1
WO2021190654A1 PCT/CN2021/083579 CN2021083579W WO2021190654A1 WO 2021190654 A1 WO2021190654 A1 WO 2021190654A1 CN 2021083579 W CN2021083579 W CN 2021083579W WO 2021190654 A1 WO2021190654 A1 WO 2021190654A1
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carrot
molecule
nucleic acid
rna
nucleotide sequence
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PCT/CN2021/083579
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French (fr)
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杨弋
陈显军
谢鑫
苏倪
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华东理工大学
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Priority to EP21776483.6A priority Critical patent/EP4130267A4/en
Priority to JP2022557982A priority patent/JP2023519282A/ja
Priority to US17/907,055 priority patent/US20230133910A1/en
Publication of WO2021190654A1 publication Critical patent/WO2021190654A1/zh

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Definitions

  • the present invention relates to an aptamer nucleic acid molecule, a method for detecting RNA, DNA or other target molecules in or outside the cell, and a kit containing the aptamer.
  • the aptamer of the present invention can specifically bind a small fluorophore molecule, and significantly increase its fluorescence intensity under excitation by light of a suitable wavelength.
  • RNA is one of the most important biological molecules. It has a wide variety of types including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), and long non-coding RNA (lncRNA).
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • siRNA small interfering RNA
  • lncRNA long non-coding RNA
  • RNAs present in cells such as short-chain interfering RNA (siRNA), small microRNA (microRNA), and long-chain non-coding RNA (lncRNA), play an important role in the regulation of gene expression at the post-transcriptional level.
  • siRNA short-chain interfering RNA
  • microRNA small microRNA
  • lncRNA long-chain non-coding RNA
  • RNA binding protein-fluorescent protein technology RNA binding protein-fluorescent protein technology
  • RNA aptamer-fluorescent dye technology Fluorescence in situ hybridization is a technology based on fluorophore-modified oligonucleotides to directly label RNA. Its basic principle is that fluorophore-modified oligonucleotides have a sequence complementary to the target RNA, which is achieved through base pairing. Marking of target RNA (Raj et al. Nature Methods 2018.5:877-879). The advantage is that the target RNA itself does not need to be modified or genetically recombined, and it is directly labeled.
  • oligonucleotides are not easy to enter cells, and need auxiliary steps to introduce cells, which are easy to cause damage to cells; background fluorescence is high, and complex elution steps are required, which can only be used for fixed cell research, not for real-time Monitor the dynamic changes of RNA in living cells.
  • Molecular beacon technology is an oligonucleotide probe based on a hairpin structure. When it binds to the target RNA, the quenching group labeled at one end eliminates the quenching effect of the fluorescent group labeled at the other end. The group produces fluorescence, or the FRET of the fluorophores at both ends disappears (Chen et al. Nucleic Acids Res 2010.38: e148).
  • molecular beacons have a higher signal-to-noise ratio than other oligonucleotide probes because they have no background fluorescence. Its disadvantages are not only difficult to enter the cell, but also tend to accumulate in the nucleus to cause non-specific fluorescence, susceptible to the influence of RNA secondary structure, and need to customize oligonucleotide probes for each RNA (You et al. Ann. Rev. Biophys 2015.44:187–206), these shortcomings limit the application of this technology.
  • RNA binding protein-fluorescent protein technology is currently a commonly used method for RNA labeling in living cells.
  • Commonly used RNA binding protein systems include MCP/MS2 phage system (Bertrand et al. Molecular Cell 1998.2:437-445), ⁇ N /boxB phage system ( Daigle et al. Nature Methods 2007.4:633-636) and dCas9-sgRNA system (Nelles et sl. Cell 2016.165:488-496).
  • the basic principle is to fuse a fluorescent protein with an RNA-binding protein that can bind to a specific RNA motif to obtain an RNA-binding protein-fluorescent protein fusion protein.
  • the specific RNA sequence is fused with the target RNA through RNA binding
  • the combination of protein and specific RNA sequence achieves the purpose of labeling target RNA with fluorescent protein.
  • this method is widely used for RNA labeling in living cells, the unbound RNA binding protein-fluorescent protein fusion protein will cause higher imaging background fluorescence.
  • scientists have used multiple RNA-binding protein-recognized RNA sequences in series to bind multiple fluorescent proteins to a target RNA to improve the imaging signal-to-noise ratio, the load of larger complexes may affect the normal physiological functions of RNA.
  • Fluorescent RNA technology based on RNA aptamer-fluorescent dyes is an ideal RNA labeling technology.
  • the basic principle is that the fluorescence of the fluorescent dye is low before it is combined with the RNA aptamer, and the fluorescence intensity is increased after it is combined with the RNA aptamer.
  • the advantage of this technology is that the system is simple and only needs to be fused with the RNA aptamer and the target RNA. It also has genetic coding characteristics and modularity, and can be universally applied to different RNAs.
  • RNA labeling in living cells especially mammalian cells.
  • S. Jaffrey's research group obtained an RNA aptamer called Spinach based on the fluorescent protein chromophore analogue (DFHBI) (Paige et al. Science 2011.333:642-646).
  • DFHBI Compared with the previous fluorescent RNA, DFHBI has the advantages of low background fluorescence, easy cell entry and no cytotoxicity. Subsequently, the same research group developed Spinach2, Broccoli and Corn fluorescent RNA (Song et al. Nature Chemical Biology2017.13:1187-1194; 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 a variety of high-abundance RNAs in mammalian cells, they still have fluorescence instability and easy quenching, low brightness, dependence on Mg 2+ and aggregate properties, etc. These shortcomings limit them Is widely used.
  • the invention provides a nucleic acid aptamer molecule, a DNA molecule encoding the nucleic acid aptamer molecule, a complex of a nucleic acid aptamer molecule and a fluorophore molecule, and uses of the complex.
  • the present invention relates to a nucleic acid aptamer molecule comprising the following nucleotide sequence (a), nuclear
  • the nucleotide sequence (a) is N 1 AGAUUGUAAACAN 14 -N 15 -N 16 GACACUN 23 (referred to as the general Carrot structure), where N 1 , N 14 , N 15 , N 16 and N 23 represent length greater than or equal to 1. At least one pair of bases in the nucleotide sequence of N 1 and N 23 forms a complementary pair, and at least one pair of bases in the nucleotide sequence of N 14 and N 16 forms a complementary pair;
  • the nucleotide sequence (a) does not include the positions of N 1 , N 14 , N 15 , N 16 and N 23 , after one or several nucleotide substitutions, deletions and/or additions, and has an aptamer Functional nucleic acid sequence (c) derived from nucleotide sequence (a).
  • nucleic acid aptamer molecule and the nucleotide sequence (a) of the Carrot structure nucleotide sequence have at least 72%, 77%, 83%, 88%, 94% % Or 100% sequence identity.
  • the nucleotide sequence (c) does not include N 1 , N 14 , N 15 , N 16 in the Carrot structure nucleotide sequence defined by the nucleotide sequence (a) And N 23 position, a nucleic acid aptamer molecule obtained by substitution, deletion and/or addition of 5, 4, 3, 2 or 1 nucleotides.
  • nucleotide sequence (c) does not include the positions of N 1 , N 14 , N 15 , N 16 and N 23 in the nucleotide sequence (a)
  • a nucleic acid aptamer molecule obtained by the substitution of one, three, two or one nucleotides.
  • N 1 and N 23 in the nucleotide sequence (a) are complementary paired, the direction of the N 1 nucleotide sequence is 5'-3', and N 23 nucleotides The direction of the sequence is 3'-5'; when N 14 and N 16 are complementary paired, the direction of the N 14 nucleotide sequence is 5'-3', and the direction of the N 16 nucleotide sequence is 3'-5'.
  • N 1 and N 23 when the length of at least one of N 1 and N 23 is greater than or equal to 5 nucleotide bases, then there is at least N 1 and N 23 nucleotide sequence Two pairs of bases form a complementary pair; when the length of at least one of N 14 and N 16 is greater than or equal to 5 nucleotide bases, then there are at least two pairs of bases in the N 14 and N 16 nucleotide sequence Form complementary pairs.
  • nucleic acid aptamer molecule the nucleotide substitution of the general formula Carrot structure is selected from one of the following groups: 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, A4C U5A/A11G, U5A/C12A, U5A/A13C, U5G/A13C, U5C/G7U, U5C/A11G, U5C/C12G, U5C/C12A, U5C/C12A, U5
  • the nucleotide substitution of the general formula Carrot structure is selected from one of the following group: 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, U5U5C /C12A, U5C/C12U, U5C/A13C, G7U/A13C, A11G/A13C, C12G/A13C, C12A/A13C, C12U/A13C, A4C/U5C/A13C, U5C/G7
  • nucleotide substitution of the general formula Carrot structure is selected from one of the following group: 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, U5A13A/C12A .
  • nucleotide sequence at N 1 and N 23 in the nucleotide sequence (a) is F30 or tRNA scaffold RNA sequence.
  • the aptamer molecule therein is an RNA molecule or a base-modified RNA molecule.
  • the aptamer molecule therein is a DNA-RNA hybrid molecule or a base-modified DNA-RNA molecule.
  • N 14 -N 15 -N 16 contains a nucleotide sequence capable of recognizing the target molecule.
  • the target molecules therein include, but are not limited to: proteins, nucleic acids, lipid molecules, carbohydrates, hormones, cytokines, chemokines, metabolites, and metal ions.
  • N 14 -N 15 -N 16 are nucleotide sequences capable of recognizing S-adenosylmethionine and adenosine molecules.
  • the aptamer function means that the nucleic acid aptamer can increase the fluorescence intensity of the fluorophore molecule under excitation light of a suitable wavelength by at least 2 times, at least 5-10 times, and at least 20-fold. 50 times, at least 100-200 times, or at least 200 times.
  • the nucleic acid aptamer molecule may also include a concatemer capable of binding multiple fluorophore molecules.
  • the concatemers are connected together by spacers of appropriate length, and the number can be 2, 3, 4, 5, 6, 7, 8, or more.
  • the nucleotides of the concatemer can be selected from but not limited to the sequence SEQ ID No: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • nucleic acid aptamer molecule 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.
  • 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 the following formula (I) structure:
  • Ar 1, Ar 2 are independently a six-membered aryl group or a six-membered heteroaryl group; D- is HO- or N(X 1 )(X 2 )-, X 1 , X 2 are each independently selected from hydrogen, Alkyl and modified alkyl; X 1 and X 2 are optionally connected to each other to form an aliphatic heterocyclic ring together with the N atom; when D- is N(X 1 )(X 2 )- and Ar 1 is phenyl, X 1 , X 2 independently forms a saturated or unsaturated ester heterocycle with a benzene ring; when D- is HO- and Ar 1 is a phenyl group, at least one hydrogen atom adjacent to HO- is substituted by halogen; Y is O , S; R 1 is hydrogen, alkyl and modified alkyl; R 2 is hydrogen, halogen, -OH, -CN;
  • the "alkyl” is each independently a C 1 -C 10 linear or branched alkyl; alternatively, a C 1 -C 7 linear or branched alkyl; alternatively, a C 1 -C 5 straight or branched chain alkyl; optionally selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl , 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methyl Pentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl Butyl, 1,3-didi
  • any carbon atom of the "modified alkyl group" each independently being an alkyl group is selected from halogen atoms, -OH, -CO-, -O-, -CN,- SO 3 H, a primary amino group, a secondary amino group, a tertiary amino group replaced by one or more groups, the modified alkyl group has 1-10 carbon atoms, wherein the carbon-carbon single bond is optionally independently Carbon-carbon double bond or carbon-carbon triple bond replacement;
  • the carbon atom is replaced means that the carbon atom or the carbon atom and the hydrogen atom on it are replaced by the corresponding group;
  • Ar 1 is a structure selected from the following formulas (II-1) to (II-15):
  • Ar 2 is selected from the following structures (III-1) ⁇ (III-25):
  • the aptamer molecule in the complex includes 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.
  • the complex is used for the detection or labeling of target nucleic acid molecules in vitro or in vivo.
  • the complex is used for the detection or labeling of extracellular or intracellular target molecules.
  • a said complex for detecting the interaction of RNA and protein 25.
  • a host cell comprising the expression vector.
  • a kit comprising the nucleic acid aptamer molecule and/or the expression vector and/or the host cell and/or the complex.
  • a method for detecting target molecules comprising the steps:
  • a method of detecting genomic DNA comprising imaging the genomic DNA using the complex.
  • a method for extracting and purifying RNA comprising extracting and purifying RNA by using the complex.
  • the inventor designed a new nucleic acid aptamer molecule and synthesized a new fluorophore molecule to form a new fluorophore-nucleic acid aptamer complex. After the aptamer molecule is combined with the fluorophore molecule, the fluorophore molecule can be significantly improved. The fluorescence intensity under suitable wavelength excitation light, they overcome 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 strong affinity for fluorophore molecules and exhibits different fluorescence spectra.
  • nucleic acid aptamer-fluorophore molecular complexes can be used for real-time labeling and imaging of RNA/DNA in prokaryotic and eukaryotic cells, detecting RNA-protein interactions, or labeling for RNA extraction and purification.
  • Figure 1 The secondary structure prediction of the general structure of the Carrot nucleic acid aptamer molecule.
  • Figure 2 The secondary structure prediction of Carrot-1 and Carrot-2 aptamer molecules.
  • Figure 4 The secondary structure prediction of tRNA-Carrot-2.
  • FIG. 6 The activation effect of Carrot with different bases on IV-39.
  • Carrot schematics modified with different bases For Carrot-3, the dark bases in the structural schematic are deoxyribonucleotide bases; for Carrot-4, the dark bases in the structural schematic are 2'- F modified bases;
  • the "control” is to use buffer to replace Carrot-3 or Carrot-4 aptamer RNA.
  • FIG. 7 The activation effect of different Carrot concatemers on IV-39.
  • Figure 8 The labeling effect of F30-Carrot-2-IV-4 complex for RNA in bacteria.
  • Figure 9 The labeling effect of F30-Carrot-2-IV-4 complex for RNA in yeast cells.
  • Figure 10 The labeling effect of Carrot, IV-39 and their analogs on RNA in mammalian cells.
  • the tRNA-Carrot-2-IV-39 complex is used for the labeling effect of RNA in mammalian cells;
  • C The effect of F30-4Carrot-2 and IV-39 analogues in labeling RNA in mammalian cells.
  • Probe construction based on Carrot-1 (a) Schematic diagram of probe construction; (b) the activation effect of the probe on the fluorescence of IV-39 fluorophore molecules in the presence or absence of a target.
  • Carrot is used to track RNA localization in cells.
  • 4Carrot-2 is used to detect the location of GAPDH mRNA in cells;
  • 4Carrot-2 is used to detect the location of ACTB mRNA in cells.
  • Figure 13 The imaging results of Carrot used to detect genomic DNA.
  • nucleotide and “nucleotide base” are used interchangeably to indicate the same meaning.
  • the "aptamer molecule" of the present invention is also called “aptamer molecule”.
  • the nucleic acid aptamer molecule comprises (a) the nucleotide sequence is N 1 AGAUUGUAAACAN 14 -N 15 -N 16 GACACUN 23 (corresponding to the general formula Carrot structure of Figure 1); or (b) and (a) described
  • the nucleotide sequence is a sequence with at least 70% identity; wherein at least one pair of bases in the nucleotide sequence of N 1 and N 23 form a reverse complementary pair, that is, the direction of the nucleotide sequence of N 1 is 5'-3' , The direction of the N 23 nucleotide sequence is 3'-5'.
  • At least one pair of bases is required to form a complementary pair; when the length of at least one nucleotide base of N 1 and N 23 is greater than or equal to 5, At least two pairs of bases are required to form a complementary pair.
  • at least one pair of bases in the N 14 and N 16 nucleotide sequences form a reverse complementary pair, that is , the direction of the N 14 nucleotide sequence is 5'-3', and the direction of the N 16 nucleotide sequence is 3'- 5'.
  • N 15 is a nucleotide base of any length and any composition; or (c) any position of the nucleotide sequence (a) is substituted, deleted and/or increased by 1-5 nucleotides .
  • the nucleic acid aptamer molecule contains the substitution of the nucleotide of the general formula Carrot structure, and the substitution is selected from one of the following group: 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/C12A13, A4C , U5A/A11G, U5A/C12A, U5A/A13C, U5G/A13C, U5C/G7U, U5C/A11G, U5C/C12G, U5C/C12A, U5C/C12
  • A4U indicates that the 4th adenine nucleotide A of Carrot is replaced with uracil nucleotide U , That is, Carrot (A4U) in Table 1;
  • U5C/A13C means that the 5th U of Carrot is replaced by C, and the 13th A is replaced by C, which is Carrot (U5C/A13C) in Table 1.
  • Table 1 The sequence of the aptamer with the general structure of Carrot after 1 nucleotide substitution
  • Aptamer molecules are single-stranded nucleic acid molecules that have a secondary structure composed of one or more base pairing regions (stems) and one or more unpaired regions (loops) ( Figure 1).
  • the nucleic acid aptamer molecule of the present invention contains a secondary structure as predicted in FIG. 1.
  • the 5'end or 3'end of the structure can be fused with any target RNA molecule for detecting the target RNA molecule outside or inside the cell.
  • the 5'end of the nucleic acid aptamer molecule is fused with ACTB RNA sequence (Genebank: BC016045); in another preferred embodiment of the present invention, the 5'end of the nucleic acid aptamer molecule is fused GAPDH RNA sequence (Genebank: BC009081).
  • the stem-loop structure (N 14 -N 15 -N 16 ) in Figure 1 serves to stabilize the molecular structure of the entire nucleic acid aptamer, and can be replaced with other nucleosides of any length and composition that can form a stem-loop structure Acid sequence.
  • the aptamer molecule of the present invention may also include other nucleotide sequences inserted at positions N 14 -N 15 -N 16 , and the inserted nucleotide sequence replaces the stem-loop structure (N 14- N 15 -N 16 ).
  • the nucleotide sequence can specifically recognize/bind the target molecule.
  • the ability of the aptamer molecule to bind to the fluorophore molecule is weak, resulting in weak fluorescence of the fluorophore molecule; when the target molecule is present, the binding of the target molecule and the aptamer will promote the The combination of the aptamer and the fluorophore molecule significantly increases the fluorescence intensity of the fluorophore molecule under the appropriate wavelength of excitation light.
  • the target molecule may be a small molecule, a signal molecule on the surface of a cell, or the like. These nucleic acid aptamers are non-covalently bound to specific target molecules.
  • This non-covalent binding is mainly dependent on the ionic force, dipole force, hydrogen bond, van der Waals force, electron-positron interaction, stacking effect or more between molecules. A combination of several forces.
  • the stem-loop structure (N 14 -N 15 -N 16 ) can be replaced with an RNA sequence that recognizes the target molecule for detecting the target molecule outside or inside the cell.
  • 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 their mutation sequences that can combine with fluorophore molecules to significantly increase their fluorescence under suitable wavelength excitation light .
  • the nucleic acid aptamer molecule of the present invention may also include a nucleotide sequence that increases its stability.
  • F30 scaffold RNA sequence 3
  • tRNA scaffold RNA sequence 4
  • the nucleotide sequence identity calculation of the two aptamers of the present invention does not include N 1 , N 14 , N 15 , N 16 , and N 23 in the sequence (a).
  • the degree of identity between two nucleotide sequences uses Needle such as the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277)
  • the program is preferably determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in version 3.0.0 or higher.
  • the optional parameters used are gap penalty of 10, gap extension penalty of 0.5 and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). Use the output of Needle marked as "longest identity” (obtained with the -nobrief option) as the percentage identity, and calculate it as follows:
  • the sequences of Carrot-1 and Carrot-1 (U5C) in Table 1 of the present invention are N 1 AGAUUGUAAACAN 14 -N 15 -N 16 GACACUN 23 and N 1 AGACUGUAAACAN 14 -N 15 -N 16 GACACUN 23 , compare their identities
  • the nucleotide bases N 1 , N 14 -N 15 -N 16 and N 23 should not be included, so their sequence identity comparison result is 94.4% (with a difference of 1 Nucleotides).
  • fluorophore molecule in the present invention is also called “fluorophore” or “fluorescent molecule”.
  • fluorophore molecules in the present invention are a type of fluorophore molecules that can be conditionally activated. They show lower quantum yield in the absence of nucleic acid aptamers.
  • the quantum yield of the fluorophore is less than 0.1, more preferably less than 0.01, and most preferably less than 0.001; when the fluorophore is bound by a specific aptamer After that, the quantum yield of the fluorophore is increased by more than 2 times, more preferably by more than 10 times, and most preferably by more than 100 times.
  • the fluorophore molecule is preferably water-soluble, non-toxic to cells and easily penetrates the membrane.
  • the fluorophore of the present invention is preferably able to enter the cytoplasm or periplasm through active transport or passive diffusion through the cell membrane or cell wall.
  • the fluorophore can penetrate the outer and inner membranes of Gram-negative bacteria, the cell walls and cell membranes of plant cells, fungi and cell walls and cell membranes, the cell membranes of animal cells, and the GI and endothelium of living animals.
  • Cell membrane the outer and inner membranes of Gram-negative bacteria, the cell walls and cell membranes of plant cells, fungi and cell walls and cell membranes, the cell membranes of animal cells, and the GI and endothelium of living animals.
  • the nucleic acid aptamer molecule of the present invention can specifically bind to a fluorophore and significantly increase its fluorescence value under excitation at a specific wavelength.
  • the fluorophore molecule is selected from structure (I):
  • Ar 1 and Ar 2 are independently a six-membered aryl group or a six-membered heteroaryl group; D- is HO- or N(X 1 )(X 2 )-, X 1 , X 2 are each independently selected From hydrogen, alkyl and modified alkyl; X 1 and X 2 are optionally connected to each other to form an aliphatic heterocyclic ring together with the N atom; when D- is N(X 1 )(X 2 )-, Ar 1 is phenyl When, X 1 and X 2 independently form a saturated or unsaturated ester heterocycle with the benzene ring; when D- is HO- and Ar 1 is phenyl, at least one hydrogen atom adjacent to HO- is substituted by halogen; Y is O, S; R 1 is hydrogen, alkyl and modified alkyl; R 2 is hydrogen, halogen, -OH, -CN;
  • the "alkyl” is each independently a C 1 -C 10 linear or branched alkyl; alternatively, a C 1 -C 7 linear or branched alkyl; alternatively, a C 1 -C 5 straight or branched chain alkyl; optionally selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl , 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methyl Pentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl Butyl, 1,3-didi
  • the "modified alkyl group” is independently any carbon atom of an alkyl group selected from halogen atoms, -OH, -CO-, -O-, -CN, -SO 3 H, primary amino, secondary amino , A group obtained by replacing one or more groups of a tertiary amino group, the modified alkyl group has 1-10 carbon atoms, wherein the carbon-carbon single bond is optionally independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond Replacement
  • the carbon atom is replaced means that the carbon atom or the carbon atom and the hydrogen atom on it are replaced by the corresponding group;
  • Ar 1 is a structure selected from the following formulas (II-1) to (II-15):
  • Ar 2 is selected from the following structures (III-1) ⁇ (III-25):
  • 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.
  • “Increase fluorescence signal”, “increase fluorescence”, “increase fluorescence intensity”, and “increase fluorescence intensity” in the present invention refer to the increase in the quantum yield of the fluorophore under the irradiation of excitation light of the appropriate wavelength, or the shift of the maximum emission peak of the fluorescence signal (relative to Ethanol or the emission peak of the fluorophore itself in an aqueous solution), or an increase in the molar extinction coefficient, or two or more of the above.
  • the quantum yield is increased by at least 2 times; in another preferred embodiment of the present invention, the quantum yield is increased by at least 5-10 times; in another more preferred embodiment of the present invention In another more preferred embodiment of the present invention, the increase in quantum yield is at least 20-50 times; in another more preferred embodiment of the present invention, the increase in quantum yield is at least 100-200 times; in another more preferred embodiment of the present invention, the quantum yield The rate increase is at least 200 times.
  • the light source used to excite fluorophores to generate fluorescent signals can be any suitable lighting equipment, such as LED lamps, incandescent lamps, fluorescent lamps, and lasers; the excitation light can be directly emitted from these devices or indirectly obtained through other fluorophores , Such as the donor fluorophore for fluorescence resonance energy transfer (FERT), or the donor luminophore for luminescence resonance energy transfer (BRET).
  • FET fluorescence resonance energy transfer
  • BRET donor luminophore for luminescence resonance energy transfer
  • the target molecule of the present invention can be any biological material or small molecule, including but not limited to: protein, nucleic acid (RNA or DNA), lipid molecule, carbohydrate, hormone, cytokine, chemokine, metabolite, Metal ions, etc.
  • the target molecule may be a molecule related to a disease or pathogen infection.
  • the inserted nucleotide sequence replaces the stem-loop structure of N 14 -N 15 -N 16 in Figure 1, and the nucleoside
  • the acid sequence can specifically recognize/bind the target molecule.
  • the aptamer molecule When the target molecule does not exist, the aptamer molecule does not bind to the fluorophore molecule or the binding ability is weak, and cannot significantly improve the fluorescence of the fluorophore molecule under the excitation light of the appropriate wavelength; when the target molecule exists, the target molecule and the nucleoside
  • the combination of acid sequences will promote the combination of aptamer molecules and fluorophore molecules, and significantly increase the fluorescence of fluorophore molecules under excitation light at a suitable wavelength, so as to realize the detection, imaging and quantitative analysis of target molecules.
  • the target molecule can also be a whole cell or a molecule expressed on the surface of the whole cell. Typical cells include but are not limited to cancer cells, bacterial cells, fungal cells and normal animal cells.
  • the target molecule can also be a virus particle.
  • many aptamers of the above-mentioned target molecules have been identified, and they can be integrated into various nucleic acid aptamers of the present invention.
  • the RNA aptamers that have been reported to bind to target molecules include but are not limited to: T4 RNA polymerase aptamers, HIV reverse transcriptase aptamers, and phage R17 capsid protein aptamers.
  • Target nucleic acid molecule
  • Target nucleic acid molecule also known as “target nucleic acid molecule” refers to the nucleic acid molecule to be detected, which can be intracellular or extracellular; including target RNA molecules and target DNA molecules.
  • the target nucleic acid molecule is connected to the nucleic acid aptamer molecule, and the fluorophore molecule is combined with the nucleic acid aptamer molecule to significantly increase the fluorescence value of the fluorophore molecule under excitation light of a suitable wavelength, thereby realizing the detection of the target nucleic acid molecule.
  • the content and the purpose of distribution is not limited to distribution.
  • Target RNA molecule in the present invention includes any RNA molecule, including but not limited to pre-mRNA, mRNA encoding the cell itself 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 can be fused to the 5'end or 3'end or the position of N 14 -N 15 -N 16 of the RNA aptamer molecule of the present invention.
  • sgRNA refers to a single guide RNA (sgRNA) formed by transforming tracrRNA and crRNA in the CRISPR/Cas9 system.
  • the sequence of about 20 nt at the 5'end is used to target DNA through base complementary pairing.
  • the site prompts the Cas9 protein to induce a DNA double-strand break at this site.
  • the nucleic acid aptamer molecule of the present invention may further include a concatemer that can bind multiple fluorophore molecules.
  • the series bodies are connected together by a spacer sequence of appropriate length, and the number of the series Carrot structures can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. There can be multiple forms of the concatenation.
  • the concatenation form is "series 1", as shown in Figure 7a, and the preferred nucleotide sequence is SEQ ID NO: 7, 8, 9 , 10 or 11; where 2Carrot-2 represents a series body 1 with two Carrot-2 structures; in another preferred embodiment of the present invention, the series form is "series 2", as shown in Figure 7b, preferably The nucleotide sequence of is SEQ ID NO: 12, 13, 14, 15 or 16, where 2 ⁇ Carrot-2 represents the concatemer 2 with two Carrot-2 structures; in another preferred embodiment of the present invention , The tandem form is "tandem 3", as shown in Figure 7c, the preferred nucleotide sequence is SEQ ID NO: 17, 18, 19 or 20; where 2 ⁇ 2Carrot-2 means that there are 4 Carrot-2 structures The series 3; no matter what the form, the interval sequence between the series can be replaced.
  • the monomeric aptamer in the present invention refers to an aptamer containing only one Carrot structure, that is, only one aptamer as shown in FIG. 1.
  • An aptamer in the form of a multimer refers to an aptamer containing more than one Carrot structure, including but not limited to the aptamer constructed in the tandem form shown in FIG. 7.
  • the aptamer-fluorophore complex of the present invention includes one nucleic acid aptamer molecule and one or more fluorophore molecules.
  • the molecular complex comprising a nucleic acid molecule and a 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, Carrot-1-IV-25, Carrot-1-
  • the nucleic acid molecule of the concatemer and multiple fluorophore molecules form a complex, for example, F30-4Carrot-2 and F30-4Carrot-2 containing 4 aptamer units formed in a "tandem 1" manner
  • the complex formed by 4 fluorophore molecules 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-37), 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) and F30-4Carrot-2-4 ⁇ (IV-22).
  • the molecular complex can exist in the form of two separate
  • the aptamer function of the present invention means that it can significantly increase the fluorescence intensity of fluorophore molecules under excitation light of a suitable wavelength, and the common experimental methods in the specific examples (5) Functional detection of nucleic acid aptamers can be used to perform detection on the aptamers. Detection.
  • the increase in fluorescence intensity is at least 2 times (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 In another more preferred embodiment of the present invention, the increase in fluorescence intensity is at least 20-50 times; in another more preferred embodiment of the present invention, the increase in fluorescence intensity is at least 100-200 times; in another embodiment of the present invention In a more preferred embodiment, the increase in fluorescence intensity is at least 200 times.
  • the stem structure in the secondary structure refers to the partial double-stranded structure formed by hydrogen bond complementary pairing in certain regions within the single strand of the nucleic acid aptamer molecule.
  • the formation of a double-stranded structure does not require that all the nucleotides in the region are complementary paired; in general, at least 50% of the sequence of N 1 and N 23 , and N 14 and N 16 Complementary pairing of the nucleotides with another fragment can form a stem structure. If N 1 and N 23 are single nucleotides, N 1 and N 23 need to be completely complementary to form a stem structure (as shown in Figure 1).
  • the DNA molecule contains a DNA sequence that can encode the nucleic acid aptamer molecule of the present invention.
  • the DNA molecule comprises the nucleotide sequence R 1 AGATTGTAAACAR 14 -R 15 -R 16 GACACTR 23 and a nucleotide sequence having at least 70% identity.
  • R 1 encodes N 1 in the general Carrot structure
  • R 14 encodes N 14 in the general Carrot structure
  • R 15 encodes N 15 in the general Carrot structure
  • R 16 encodes N 16 and R in the general Carrot structure
  • 23 encodes N 23 in the Carrot structure.
  • the DNA molecule may also include a promoter that controls DNA transcription, and the promoter is operably linked to the DNA sequence encoding the nucleic acid aptamer.
  • the DNA molecule includes a U6 promoter; in another specific embodiment of the present invention, the DNA molecule includes a CMV promoter.
  • the DNA molecule may further include a DNA sequence encoding any target nucleic acid molecule.
  • the DNA molecule encoding the target RNA contains a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ⁇ -actin (sequence of chimeric RNA) Respectively SEQ ID No: 26, 27).
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • ⁇ -actin sequence of chimeric RNA
  • the "promoter” in the present invention includes eukaryotic cell and prokaryotic cell promoters.
  • the promoter sequence of eukaryotic cells is completely different from the promoter sequence of prokaryotic cells.
  • eukaryotic promoters cannot be recognized by RNA polymerase in prokaryotic cells to mediate RNA transcription.
  • prokaryotic promoters cannot be recognized by RNA polymerase in eukaryotic cells and mediate RNA transcription.
  • the strength of different promoters varies greatly (strength refers to the ability to mediate transcription). Depending on the actual application, strong promoters can be used to achieve high-level transcription.
  • a high level of expression is better, and if the transcription behavior is evaluated, a lower level of transcription can allow cells to regulate the transcription process in time.
  • one or more suitable promoters can be selected.
  • T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, PR and PL promoters in lambda phage, and other promoters but Not limited to: lacUV5 promoter, ompF promoter, bla promoter, lpp promoter, etc.
  • a hybrid trp-lacUV5 promoter tac promoter
  • coli promoters obtained by recombinant or synthetic DNA technology can be used to transcribe the RNA aptamer of the present invention.
  • Some operator sequences in bacteria can be combined with promoter sequences to form inducible promoters.
  • specific inducers need to be added to induce transcription of DNA molecules.
  • the lac operator needs to add lactose or lactose analogue (IPTG) to induce its expression, and other operators include trp, pro, etc.
  • the regulatory sequence at the 5'end of the coding sequence of the DNA molecule is a promoter. Whether it is to obtain RNA aptamers by in vitro transcription or express aptamers in cultured cells or tissues, it is necessary to select a suitable promoter according to the strength of the promoter. Since the expression of aptamers in vivo can be genetically manipulated, another type of promoter is an inducible promoter that induces DNA transcription in response to a specific environment, such as expression in a specific tissue, a specific time, and a specific developmental stage. These different promoters can be recognized by RNA polymerase I, II or III.
  • RNA polymerase transcribes genes differently, and its transcription terminator is also very different. However, to screen for a suitable 3'transcription terminator region, the basic experimental skills of those skilled in the art can be achieved.
  • the "expression system” of the present invention also referred to as "expression vector”, includes a DNA molecule integrated with an expression nucleic acid aptamer.
  • the expression system of the present invention can be a plasmid or a virus particle.
  • the "expression vector" recombinant virus can be obtained by transfecting a plasmid into a virus-infected cell.
  • Suitable vectors include but are not limited to viral vectors such as lambda vector system 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 system), pET28 series, pACYCDuet1, pCDFDuet1, pRSET series, pBAD series, pQE, pIH821, pGEX, pIIIEx426RPR, etc.
  • the host vector system includes, but is not limited to: transformed phage DNA, or plasmid DNA, or coliform plasmid DNA; yeast containing yeast vectors; virus-infected mammals Animal cells (such as adenovirus, adeno-associated virus, retrovirus); mammalian cells transfected with plasmids; insect cells (such as baculovirus) infected with viruses; plant cells infected with bacteria or transformed by particle bombardment.
  • the strength and characteristics of the expression elements in the vectors vary greatly. Any one or more suitable transcription elements are selected according to the host-vector system used.
  • methods include, but are not limited to, transformation, transduction, conjugation, fixation, electrotransduction, etc.
  • expression plasmids pET28a-T7-F30-Carrot-2 and pYES2.1-F30-Carrot-2 containing DNA molecules encoding F30-Carrot-2 RNA are provided.
  • an expression plasmid pU6-tRNA-Carrot-2 containing a DNA molecule encoding tRNA-Carrot-2 RNA is provided.
  • an expression plasmid pU6-F30-4Carrot-2 containing a DNA molecule encoding F30-4Carrot-2 RNA is provided.
  • an expression plasmid pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 and pCDNA3.1 hygro() containing DNA molecules encoding GAPDH-4Carrot-2 and ACTB-4Carrot-2 is provided +)-ACTB-4Carrot-2.
  • an expression plasmid psgRNA-Carrot-2-1 containing DNA molecules encoding sgRNA-Carrot-2-1, sgRNA-Carrot-2-2 and sgRNA-Carrot-2-3 is provided , PsgRNA-Carrot-2-2 and psgRNA-Carrot-2-3.
  • an expression plasmid pU6-Carrot-1-MS2 containing a DNA molecule encoding Carrot-1-MS2 is provided.
  • the present invention also provides an expression vector that integrates DNA molecules encoding nucleic acid aptamers, but the coding DNA sequence of the target RNA molecule is vacant.
  • the coding DNA sequence vacancy of the target RNA molecule allows the user to select the DNA sequence of the target RNA molecule to be detected.
  • the coding DNA sequence corresponding to GAPDH mRNA the DNA sequence is inserted into the expression vector of the present invention using standard recombinant DNA technology, and the obtained expression vector is introduced into the host cell (transfection, transformation, infection, etc.) to detect the target The content and distribution of RNA.
  • “Host cells” in the present invention include but are not limited to bacteria, yeast, mammalian cells, insect cells, plant cells, zebrafish cells, Drosophila cells, and nematode cells.
  • the host cells are more preferably cultured in vitro cells or whole in vivo living tissues.
  • the host cell of the present invention includes mammalian cells including but not limited to 297T, COS-7, BHK, CHO, HEK293, HeLa, H1299, fertilized egg stem cells, induced pluripotent stem cells, and the original directly isolated from mammalian tissues. Generation cells, etc.; it contains E. coli cells including but not limited to BL21 (DE3), BL21 (DE3, Star), TOP10, Mach1, DH5 ⁇ ; it contains yeast cells including but not limited to BY4741, BY4742, AH109.
  • the detection array of the present invention comprises one or more nucleic acid aptamer molecules of the present invention, wherein the nucleic acid aptamer molecules are anchored at discrete positions on the array surface, and the array surface is composed of solid supports, including but not limited to Glass, metal, ceramics, etc.
  • the nucleic acid aptamer molecule of the present invention can be anchored to the array surface by, but not limited to, the following methods: (1) Use biotin to label the 5'or 3'end of the nucleic acid aptamer molecule to bind streptavidin to The aptamer molecules are anchored by the specific binding of biotin and streptavidin; (2) the phage capsid protein MCP recognizes the binding sequence MS2, the phage capsid protein PCP recognition binding sequence PP7 or lambda phage transcription terminator protein N recognition binding sequence boxB RNA sequence is fused to the 5', 3'or stem-loop structure of the nucleic acid aptamer molecule to recognize the bound protein MCP, PP7 or lambda N The protein is coated on the surface of the array, and the nucleic acid aptamer molecule is anchored by the specific action of MS2 and MCP protein, PP7 and PCP protein or boxB RNA and ⁇ N protein; (3) A piece of RNA or DNA sequence
  • the detection array can be used to detect the presence or absence and concentration of target molecules. Therefore, only in the presence of the target molecules, the nucleic acid aptamer molecules can bind to the fluorophore molecules, which significantly improves their ability to excite light at a suitable wavelength. Within a certain range, the higher the concentration of target molecules, the higher the fluorescence intensity.
  • the kit of the present invention includes the nucleic acid aptamer molecule and/or fluorophore molecule of the present invention, and corresponding instructions; or an expression system and/or fluorophore molecule for expressing the nucleic acid aptamer molecule, and Corresponding instructions; or a host cell and/or fluorophore molecule containing an expression system for expressing nucleic acid aptamer molecules, and corresponding instructions.
  • the nucleic acid aptamer molecule and the fluorophore molecule in the kit are in separate solutions, or the nucleic acid aptamer molecule and the fluorophore molecule are in the same solution.
  • 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 for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Jereh Bioengineering Technology Co., Ltd. The expression plasmids constructed in the examples have all undergone sequence determination, which was completed by Jie Li Sequencing Company.
  • the Taq DNA polymerase used in each example was purchased from Shanghai Yisheng Biotechnology Co., Ltd., and the PrimeSTAR DNA polymerase was purchased from TaKaRa Company.
  • the three polymerases were purchased with corresponding polymerase buffer and dNTP.
  • Restriction enzymes such as EcoRI, BamHI, BglII, HindIII, NdeI, XhoI, SacI, XbaI, SpeI, T4 ligase, T4 phosphorylase (T4 PNK), and T7 RNA polymerase were purchased from Fermentas, with the purchase attached The corresponding buffer, etc.
  • the Hieff CloneTM One Step cloning kit used in the examples was purchased from Shanghai Yisheng Biotechnology Co., Ltd.
  • inorganic salt chemical reagents were purchased from Sinopharm Shanghai Chemical Reagent Company.
  • Kanamycin was purchased from Ameresco Company;
  • Ampicillin was purchased from Ameresco Company;
  • 384-well and 96-well fluorescence detection blackboards were purchased from Grenier Company.
  • DFHBI-1T and DFHO were purchased from Lucerna Company.
  • GTP and SAM were purchased from Sigma Company.
  • the DNA purification kit used in the examples was purchased from BBI Company (Canada), and the common plasmid mini-purge 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 bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.
  • the BY4741 yeast strain was purchased from Shanghai Weidi Biotechnology Co., Ltd.
  • the main instruments used in the examples Synergy Neo2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), PCR Thermal cycler (Biometra, Germany), in vivo imaging system (Kodak, U.S.), photometer (Wako, Japan), and nucleic acid electrophoresis (Shenergy).
  • the cDNA corresponding to the RNA to be detected is amplified using primers containing T7 promoter, and RNA is transcribed using T7 RNA polymerase (purchased from Fermentas) using the recovered double-stranded cDNA as a template.
  • T7 RNA polymerase purchased from Fermentas
  • Add 10 ⁇ L 3M NaAc, 115 ⁇ L DEPC water to 20 ⁇ L transcription system, mix well, add 150 ⁇ L phenol chloroform-isopropanol mixture (phenol:chloroform:isopropanol 25:24:1), shake and mix, centrifuge at 10000rpm Take the supernatant after 5 min. Add an equal volume of chloroform solution, shake and mix, centrifuge at 10,000 rpm for 5 minutes, take the supernatant, and repeat once.
  • the cells in this example were all cultured in a CO 2 incubator with 10% fetal bovine serum (FBS) and streptomycin and penicillin high glucose medium (DMEM), and the cells were subcultured when the growth reached 80-90% confluence nourish.
  • FBS fetal bovine serum
  • DMEM penicillin high glucose medium
  • the main imaging experiment in the examples is to use Leica SP8 confocal laser microscope to shoot, use HCXPL APO 63.0x1.47 oil lens and HyD detector, and can use 405nm, 457nm, 476nm, 488nm, 497nm, 514nm, 561nm, 633nm and more
  • This kind of laser is used for imaging, and the complex formed by each Carrot and dye molecule is imaged with a laser with a wavelength close to its maximum excitation peak.
  • GFP uses a 488nm laser
  • Hoechst and DAPI use a 405nm laser
  • Rhodamine uses a 561nm laser.
  • Preparation of linearized vector select a suitable cloning site and linearize the vector.
  • the linearized vector can be prepared by restriction enzyme digestion or reverse PCR amplification.
  • the 'and 3'ends respectively have identical sequences corresponding to the two ends of the linearized vector.
  • the optimal amount of vector used in the recombination reaction system is 0.03pmol; the optimal molar ratio of vector to insert is 1:2-1:3, that is, the optimal amount of insert used is 0.06-0.09pmol.
  • X and Y are calculated according to the formula to obtain the linearized vector and insert fragment. After the preparation of the system is complete, mix the components and place at 50°C to react for 20 minutes. When inserts> 5kb, the incubation temperature can be extended to 25min. After the reaction is complete, it is recommended to cool the reaction tube on ice for 5 minutes. The reaction product can be converted directly, or stored at -20°C, and thawed for conversion when needed.
  • the fluorescence maximum excitation peak of the complex formed by 5 ⁇ M Carrot-1 nucleic acid aptamer and 1 ⁇ M IV-39 fluorophore molecule is 524 nm, and the maximum emission peak is 580.
  • Neo2 multifunctional microplate reader to detect the fluorescence intensity of the complex under 524 ⁇ 10nm excitation and 580nm ⁇ 10nm emission conditions is 5720, while the fluorescence intensity of the control (1 ⁇ M IV-39 fluorophore molecule) under the same detection conditions is 20, then the activation factor of Carrot-1 aptamer to IV-39 fluorophore molecule is 286 times.
  • Example 1 The secondary structure of the Carrot nucleic acid aptamer molecule
  • Carrot-1 SEQ ID NO: 2 RNA was prepared according to the common experimental method (1). Incubate 1 ⁇ M IV-39 with 5 ⁇ M Carrot-1. The test results show that the maximum excitation light of the Carrot-IV-39 complex is 524nm and the maximum emission light is 580nm ( Figure 5a).
  • Carrot-1 in Table 2 is a nucleic acid aptamer with the sequence SEQ ID NO:1; other aptamers are point mutations made in the Carrot-1 sequence at the corresponding nucleotide positions of Carrot in Figure 1.
  • Carrot-2 was connected in series according to different forms, including the following three types:
  • RNA After PCR amplification, prepare nucleic acid aptamers according to common experimental methods (1) For RNA, after incubating 0.2 ⁇ M RNA aptamer with 5 ⁇ M IV-39, detect the fluorescence intensity according to the common experimental method (5). The test results show that with the increase of n, the fluorescence of F30-nCarrot-IV-39 also increases ( Figure 7d), indicating that the fluorescence intensity of the Carrot-IV-39 complex can be increased through the “tandem 1” method.
  • the common experimental method (1) prepare the nucleic acid aptamer RNA, and add 0.2 ⁇ M After the RNA aptamer was incubated with 5 ⁇ M IV-39, the fluorescence intensity was measured according to the common experimental method (5). The test results showed that with the increase of n, the fluorescence of n ⁇ Carrot-IV-39 also increased ( Figure 7e), indicating that the fluorescence intensity of the Carrot-IV-39 complex can be increased through the "tandem 2" method.
  • 2 ⁇ 2Carrot-2, 2 ⁇ 4Carrot-2, 4 ⁇ 2Carrot-2, 4 ⁇ 4Carrot-2 coding cDNA were synthesized respectively (the sequence of coding RNA aptamer is SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20), prepare nucleic acid aptamer RNA according to common experimental method (1), after incubating 0.2 ⁇ M RNA aptamer with 5 ⁇ M IV-39, Detect the fluorescence intensity according to the commonly used experimental method (5).
  • Carrot-1 RNA aptamer molecules were prepared according to the commonly used experimental methods (1), and the basic properties of IV-39 analogues binding to Carrot were detected by using it, including fluorescence spectrum, molar extinction coefficient, quantum yield and fluorescence activation multiple, test results As shown in Table 4, it can be seen from the data in the table that Carrot-1 can activate the fluorescence intensity of IV-39 analogs to varying degrees.
  • Example 7 Carrot-IV-4 complex is used for the labeling of RNA in bacteria
  • a bacterial expression plasmid expressing F30-Carrot-2 was first constructed. Full-gene synthesis of F30-Carrot-2 encoding DNA sequence, using primers to amplify it, using primers to amplify pET28a to remove the promoter and multiple cloning site region, the amplified F30-Carrot-2 DNA fragment Connect with the pET28a linearized vector according to the experimental method (4), and the obtained recombinant plasmid is named pET28a-T7-F30-Carrot-2.
  • the primers used to amplify the F30-Carrot-2 fragment are:
  • Upstream primer (P1) 5’-TAATACGACTCACTATAGGGTTGCCATGTGTATGTGGGA-3’
  • the primers used to amplify the pET28a vector to linearize it are:
  • Example 8 Carrot-IV-4 complex is used for the labeling of RNA in yeast cells
  • a yeast expression plasmid expressing F30-Carrot-2 was first constructed.
  • the F30-Carrot-2 DNA fragment in Example 7 was amplified using primers, and the amplified F30-Carrot-2 fragment was inserted into the pYES2.1 TOPO TA vector according to the experimental method (4) to obtain the recombinant plasmid Named pYES2.1-F30-Carrot-2.
  • the primers used to amplify the F30-Carrot-2 fragment are:
  • Upstream primer (P5): 5’-GGAATATTAAGCTCGCCCTTTTGCCATGTGTATGTGGG-3’
  • the pYES2.1-F30-Carrot-2 recombinant plasmid was transformed into BY4741 strain, and a single clone was picked and cultured at 30°C.
  • OD 600 0.1
  • 1 mM galactose was added to induce the expression of F30-Carrot-2, and the bacteria were harvested after 10 hours.
  • the untreated BY4741 strain was used as a control.
  • the imaging results show that only when F30-Carrot-2 is expressed and in the presence of IV-4, yeast cells can show bright orange-red fluorescence (Figure 9), indicating that the Carrot-IV-4 complex can be used for RNA in yeast cells Fluorescent label.
  • Example 9 Carrot, IV-39 and their analogs are used for the labeling of RNA in mammalian cells
  • RNA-Carrot-2 encoding RNA sequence is SEQ ID No: 4
  • tRNA-Pepper encoding RNA sequence is SEQ ID No: 22
  • tRNA-Corn encoding RNA sequence is SEQ ID No: 21
  • primers P7 and P8 were used for PCR amplification of these fragments
  • primers P9 and P10 were used to amplify the pEGFP-N1 vector to remove its own CMV promoter and multiple cloning site region.
  • experimental method four
  • these fragments were inserted into the pEGFP-N1 vector with the promoter and multiple cloning site regions removed.
  • the resulting plasmids are named pU6-tRNA-Carrot-2, pU6-tRNA-Pepper and pU6-tRNA-Corn, and these plasmids express tRNA-Carrot-2, tRNA-Pepper and tRNA-Corn, respectively.
  • the primers used to amplify tRNA-Carrot-2, tRNA-Pepper and tRNA-Corn are:
  • Upstream primer 5’-GCCGCCCCCTTCACCTCTAGAGCCCGGATAGCTCAGTCGG-3’
  • the primers used to amplify pEGFP-N1 to remove the promoter and multiple cloning site regions are:
  • Upstream primer (P9): 5’-TTTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCA-3’
  • the primers used to amplify the vector to linearize and introduce the U6 promoter are:
  • the primers used to amplify the U6 promoter are:
  • Upstream primer (P13): 5’-GCCGCCCCCTTCACCGAGGGCCTATTTCCCATG-3’
  • a mammalian expression plasmid expressing F30-4Carrot-2 was constructed.
  • the primers P15 and P16 in this example were used to amplify the F30-4Carrot-2 fragment in Example 5, and these fragments were inserted into the pU6-tRNA-Carrot- cleaved by XbaI and EcoRI using experimental method (4).
  • the resulting expression vector was named pU6-F30-4Carrot-2.
  • the primers used to amplify the F30-4Carrot-2 fragment are:
  • Upstream primer 5’-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGGA-3’
  • the pU6-F30-4Carrot-2 plasmid was transfected into 293T/17 cells, and 24 hours later, different IV-39 analogues were added for labeling, and the labeling effect was tested by experimental method (3).
  • the results showed that different IV-39 analogs can specifically label cells expressing F30-4Carrot-2, but not control cells that do not express F30-4Carrot-2 ( Figure 10c), indicating that Carrot and IV-39 and their Analogs can be used to label RNA in mammalian cells.
  • the N 14 -N 15 -N 16 nucleotides in the general formula of Carrot-1 are replaced with those that can specifically recognize and bind adenosine (adenosine) and S-adenosylmethionine (SAM) RNA aptamers, these aptamers and Carrot-1 are connected with appropriate bases ( Figure 11a), and probe RNA is prepared according to the common experimental method (1), and the Incubate with IV-39, and use a multifunctional microplate reader to detect their fluorescence intensity in the presence or absence of adenosine or SAM.
  • SEQ ID No: 1 SEQ ID No: 1
  • SAM S-adenosylmethionine
  • test results show that the fluorescence of these probes when the target is present is significantly higher than when there is no target ( Figure 11b), indicating that they can be used as probes for detecting adenosine and SAM, respectively.
  • the corresponding probe RNA sequence is SEQ ID No. : 24 and SEQ ID No: 25.
  • Example 11 Carrot is used to track RNA localization in cells
  • the expression plasmid of chimeric RNA in which Carrot is fused with different RNAs is first constructed.
  • GAPDH and ACTB coding gene sequences are Genebank: BC009081, BC016045, respectively
  • use primers to amplify GAPDH and ACTB gene fragments and insert them into pCDNA3 digested with NheI and HindIII .1
  • pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 and pCDNA3.1 hygro(+)-ACTB-4Carrot-2 recombinant plasmids are obtained, which respectively code for GAPDH-4Carrot -2 and ACTB-4Carrot-2 chimeric RNA, their sequence is SEQ ID No: 26 and 27
  • the primers used to amplify 4Carrot-2 are:
  • Upstream primer (P17): 5’-TAGCGTTTAAACTTAAGCTTGGAAGATTGTAAACACGCC-3’
  • the primers used to amplify GAPDH are:
  • Upstream primer (P19): 5’-GGAGACCCAAGCTGGCTAGCATGGGGAAGGTGAAGGTCGG-3’
  • the primers used to amplify ACTB are:
  • Downstream primer (P22): 5’-CACGGACACATGGCAAGCTTAACCATGCTCTAGCGAGTGCTAGAAGCATTTGCGGTGGA-3’
  • the inserted sequence was identified by sequencing to be completely correct, and the plasmid was extracted with a transfection-grade plasmid extraction kit for subsequent transfection experiments.
  • the pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 and pCDNA3.1 hygro(+)-ACTB-4Carrot-2 recombinant plasmids constructed in this example were respectively transfected into COS-7 cells, and 1 ⁇ M IV was added 24h after transfection -4 Near labeling, imaging the cells according to the fluorescence imaging method described in the specific experimental method (3).
  • Example 12 Carrot is used to detect genomic DNA
  • a recombinant plasmid expressing the chimeric RNA of Carrot-2 and sgRNA was first constructed.
  • the cDNAs of sgRNA-Carrot-2-1, sgRNA-Carrot-2-2 and sgRNA-Carrot-2-3 containing centromeric targeting sequences were synthesized by the whole gene, and the encoded RNA sequences are SEQ ID No: 28, 29 and 30.
  • Use primers P23 and P24 to amplify the cDNA of the chimeric RNA use primers P25 and P26 to amplify the psgRNA plasmid (Shao et al.
  • the primers used to amplify the cDNA corresponding to the chimeric RNA of Carrot and sgRNA are:
  • Upstream primer 5’-AAAGGACGAAACACCGAATCTGCAAGTGGATATTGTTTGAG-3’
  • the primers used to amplify the psgRNA plasmid to linearize it are:
  • Upstream primer (P25): 5’-TTTTTTTCTAGATCATAATCAGCCATACC-3’
  • the primers used to amplify SpdCas9-GFP are:
  • Upstream primer 5’-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCACCATGGCCCC-3’
  • Example 13 Carrot is used to detect RNA-protein interactions
  • the phage capsid protein MCP In order to use Carrot to detect the RNA-protein interaction, take the phage capsid protein MCP to recognize the binding sequence MS2 RNA as an example.
  • the whole gene synthesis of the Carrot-1-MS2 fragment (the encoded RNA sequence is SEQ ID No: 31), the primers P29 and P30 are used as templates to amplify, and the obtained fragments are inserted into the XbaI and In the pU6-tRNA-Carrot-2 digested by EcoRI, the plasmid obtained is named pU6-Carrot-1-MS2.
  • tdMCP protein coding gene fragment (it is the dimer form of MCP protein, and its coding DNA sequence is SEQ ID No: 32) and NanoLuc protein coding gene fragment (its coding DNA sequence SEQ ID No: 33), using primers P31 and P32, P33 and P34 to amplify tdMCP gene fragments, and P35 and P36 to amplify NanoLuc gene fragments .
  • pCDNA3.1 hygro(+) vector digested with HindIII and XhoI insert the amplified fragment into the pCDNA3.1 hygro(+) vector digested with HindIII and XhoI, and the resulting plasmid is named pCDNA3.1 hygro(+)-tdMCP-NanoLuc- tdMCP, the gene sequence of the fusion protein encoded by it is SEQ ID No: 34.
  • the primers used to amplify the Carrot-1-MS2 fragment are:
  • Upstream primer (P29): 5’-GGAAAGGACGAAACTCTAGAGGGAAGATTGTAAACAC-3’
  • the primers used to amplify tdMCP are:
  • Upstream primer (P31): 5’-TAGCGTTTAAACTTAAGCTT ATGCTAGCCGTTAAAATGGC-3’
  • Another set of primers used to amplify tdMCP is:
  • Upstream primer 5’-ACTCCCTCCCGCCAGAATGCGTTCGCAC-3’
  • the primers used to amplify NanoLuc are:
  • the pCDNA3.1 hygro(+)-tdMCP-NanoLuc-tdMCP and pU6-Carrot-1-MS2 recombinant plasmid were co-transfected into 293T/17 cells, 24h after transfection, 1 ⁇ M IV-4 fluorophore was added to label Carrot. Then add 20 ⁇ M Furimazine to the medium and image immediately.
  • the imaging results show that because the tdMCP protein in the tdMCP-NanoLuc-tdMCP fusion protein will recognize and bind the MS2 RNA in the Carrot-1-MS2 chimeric RNA, the distance between the Carrot-IV-4 complex and the NanoLuc protein is very close.
  • Example 14 Carrot tags used for RNA extraction and purification
  • the pCDNA3.1 hygro(+)-GAPDH-4Carrot-2 recombinant plasmid in Example 11 was transfected into COS-7 cells, and the cells were collected 24 hours later, using a buffer of 40mM HEPES, pH 7.4, 125mM KCl, and 5mM MgCl 2 Resuspend (operation on ice). After Activated Thiol Sepharose 4B (GE Healthcare) was washed twice with 500 ⁇ L PBS, it was added with PBS containing 10 mM TCEP (Sigma) and incubated for 1 h at room temperature.
  • the maleamide-containing IV-39 fluorophore molecule (Mal-IV-39) was added and reacted at room temperature for 30 minutes, and washed three times with 500 ⁇ L PBS.
  • the resuspended cells were disrupted and incubated with the above-treated beads at 4°C. After 30 minutes, centrifuged at 4000 rpm for 2 minutes, discarded the supernatant, and washed the agarose with a pre-cooled buffer of 40 mM HEPES, pH 7.4, 125 mM KCl, and 5 mM MgCl 2 Beads were centrifuged 6 times, and the supernatant was removed by centrifugation each time.
  • the beads were re-selected with DEPC water, treated at 70°C for 10 minutes, centrifuged at 4000 rpm for 2 minutes, and collected the supernatant.
  • 3-Fluoro-4-hydroxy-benzaldehyde (0.560g, 4.0mmol) was dissolved in a 100ml round bottom flask by adding 40ml of absolute ethanol, adding 10g of anhydrous sodium sulfate, 5ml of 33% methylamine aqueous solution, and stirring at room temperature under Ar protection After 24h, filter and pressurize to remove the organic dissolution. The residue was dissolved in 10ml of absolute ethanol, compound 4 (0.790g, 5.0mmol) was added, and the mixture was stirred overnight under Ar protection at room temperature. The next day was filtered and washed with cold ethanol three times to obtain compound 5. (0.796 g, 85%).

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Abstract

本发明提供一种适配体核酸分子，一种包含该适配体和荧光团小分子的复合物，该适配体核酸分子检测细胞内或者细胞外的RNA、DNA或者其他靶分子的方法，以及包含该适配体的试剂盒。本发明的适配体可以特异性结合一种荧光团小分子，并显著提高其在合适波长光激发下的荧光强度。

Description

一种新型RNA检测与定量的方法 技术领域
本发明涉及一种适配体核酸分子，该适配体核酸分子用于检测细胞内或者细胞外的RNA、DNA或者其他靶分子的方法，以及包含该适配体的试剂盒。本发明的适配体可以特异性结合一种荧光团小分子，并显著提高其在合适波长光激发下的荧光强度。
背景技术
RNA是最重要的生物分子之一，它种类繁多包括信使RNA(mRNA)、转运RNA(tRNA)、核糖体RNA(rRNA)、小干扰RNA(siRNA)、长链非编码RNA(lncRNA)等。近些年来，RNA在多种生命活动中发挥至关重要的功能被科学家们逐渐发掘出来，包括很多RNA-蛋白质复合体，如端粒酶、剪接酶、核酶和核糖开关等。此外，细胞中存在的大量非编码RNA，如短链干扰RNA(siRNA)、小微RNA(microRNA)和长链非编码RNA(lncRNA)等，在转录后水平上对于基因表达的调控发挥着不可替代的作用。因此，实时监测细胞中RNA的运输和代谢过程对于研究RNA的定位与基因表达以及细胞调控过程的关系是至关重要的。
目前用于标记RNA的方法包括原位杂交技术、RNA结合蛋白-荧光蛋白技术和RNA适配体-荧光染料技术。荧光原位杂交技术是基于荧光团修饰的寡核苷酸对RNA进行直接标记的技术，它的基本原理是荧光团修饰的寡核苷酸有着一段于靶标RNA互补的序列，通过碱基配对实现对靶标RNA的标记(Raj et al.Nature Methods 2018.5:877–879)。其优点是不需要对靶标RNA本身进行修饰或基因重组，直接进行标记。缺点是寡核苷酸不易进入细胞，需要导入细胞的辅助操作步骤，易对细胞造成损伤；背景荧光较高，需要复杂的洗脱操作步骤，只能用于固定细胞的研究，不能用于实时监测活细胞中RNA的动态变化过程。分子信标技术是基于发夹结构的寡核苷酸探针，当其与靶标RNA结合后，标记在一端的淬灭基团对标记在另一端的荧光基团的淬灭作用消除，荧光基团产生荧光，或者两端荧光基团的FRET消失(Chen et al.Nucleic Acids Res 2010.38:e148)。其优点是由于本身没有背景荧光，分子信标比其他的寡核苷酸探针有更高的信噪比。其缺点除了难以进入细胞外，还趋向于聚集在细胞核造成非特异性荧光，易受 RNA二级结构的影响且需要对每条RNA专门定制寡核苷酸探针等缺点(You et al.Ann.Rev.Biophys 2015.44:187–206)，这些缺点限制了该技术的应用。
RNA结合蛋白-荧光蛋白技术是目前常用的活细胞RNA标记方法，常用的RNA结合蛋白系统有MCP/MS2噬菌体系统(Bertrand et al.Molecular Cell 1998.2:437-445)、λ N/boxB噬菌体系统(Daigle et al.Nature Methods 2007.4:633-636)和dCas9-sgRNA系统(Nelles et sl.Cell 2016.165:488-496)。其基本原理是将荧光蛋白与一个能够结合特定RNA基序的RNA结合蛋白进行融合，得到RNA结合蛋白-荧光蛋白的融合蛋白，同时将这段特定的RNA序列与靶标RNA进行融合，通过RNA结合蛋白与特定RNA序列的结合，达到用荧光蛋白标记靶标RNA的目的。该方法虽然被广泛用于活细胞RNA标记，但未结合的RNA结合蛋白-荧光蛋白的融合蛋白会造成较高的成像本底荧光。虽然科学家们通过串联多个RNA结合蛋白识别的RNA序列，让一条靶标RNA上结合多个荧光蛋白来提高成像信噪比，但负载较大的复合物可能影响RNA正常的生理功能。
基于RNA适配体-荧光染料的荧光RNA技术是比较理想的RNA标记技术。其基本原理是，荧光染料在与RNA适配体结合之前，荧光较低，在与RNA适配体结合后，荧光强度增强。这一技术的优点是，系统简单，只需要将RNA适配体与靶标RNA进行融合，还具有遗传编码特性和模块性，可通用于不同的RNA。早期的荧光RNA是基于噻唑橙及其类似物或荧光团-淬灭基团开发的(Dolgosheina et al.ACS chemical biology 2014.9：2412-2420；Sunbul et al.Angewandte Chemie 2013.52:13401-13404)，这些荧光染料本身存在背景荧光高、细胞毒性大或进细胞困难等缺点，使得它们不适合活细胞RNA标记，特别是哺乳动物细胞。2011年S.Jaffrey课题组基于荧光蛋白生色团类似物(DFHBI)筛选得到称为Spinach的RNA适配体(Paige et al.Science 2011.333:642-646)。相对此前的荧光RNA，DFHBI具有背景荧光低、进细胞容易且无细胞毒性等优点。随后同一课题组又开发了Spinach2、Broccoli和Corn荧光RNA(Song et al.Nature Chemical Biology2017.13:1187-1194；Filonov et al.Journal of the American Chemical Society 2014.136:16299-16308；Strack et al.Nature methods 2013.10:1219-1224)。尽管这些荧光RNA可用于哺乳动物细胞中对多种高丰度RNA进行标记成像，但仍存在荧光不稳定易淬灭，亮度低、依赖于Mg 2+和聚体性质等，这些缺点限制了它们的广泛使用。2019年，Chen et al.开发了称为Pepper的荧光RNA，它具有高亮度、高稳定性等优点，可用于活细胞中多种RNA的标记与成像(Chen et al.Nature Biotechnology 2019.37:1287-1293)。然而， 在活细胞中，很多细胞功能的执行需要多种RNA的同时参与，因此需要开发具有优良性质且可以生物正交的多种荧光RNA来实现对细胞中多种RNA进行标记与成像，探究它们的生物功能。
发明的简述
本发明提供了一种核酸适配体分子，编码该核酸适配体分子的DNA分子，一种核酸适配体分子与荧光团分子的复合物，以及该复合物的用途。
本发明提供的技术方案如下：
1.本发明涉及一种核酸适配体分子，所述适配体分子包含下述核苷酸序列(a)、核
苷酸序列(b)或核苷酸序列(c)：
核苷酸序列(a)为N 1AGAUUGUAAACAN 14-N 15-N 16GACACUN 23(称为通式Carrot结构)，其中N 1、N 14、N 15、N 16和N 23代表长度大于或等于1个的核苷酸片段，并且N 1与N 23核苷酸序列中至少有一对碱基形成互补配对，N 14与N 16核苷酸序列中至少有一对碱基形成互补配对；
与核苷酸序列(a)具有至少70％同一性的核苷酸序列(b)；
在核苷酸序列(a)中不包括N 1、N 14、N 15、N 16和N 23的位置，经过一个或几个核苷酸的取代、缺失和/或添加，且具有适配体功能的由核苷酸序列(a)衍生的核酸序列(c)。
2.在本申请的一些实施例中，所述核酸适配体分子与核苷酸序列(a)所述的Carrot结构核苷酸序列具有至少72％，77％，83％，88％，94％或100％同一性的序列。
3.在本申请的一些实施例中，其中核苷酸序列(c)是在核苷酸序列(a)限定的Carrot结构核苷酸序列中不包括N 1、N 14、N 15、N 16和N 23的位置，经过5个、4个、3个、2个或1个核苷酸的取代、缺失和/或添加而得到的核酸适配体分子。
4.在本申请的一些实施例中，其中核苷酸序列(c)是在核苷酸序列(a)中不包括N 1、N 14、N 15、N 16和N 23的位置，经过4个、3个、2个或1个核苷酸的取代而得到的核酸适配体分子。
5.在本申请的一些实施例中，其中核苷酸序列(a)中的N 1与N 23互补配对时，N 1核苷酸序列的方向为5’-3’，N 23核苷酸序列的方向为3’-5’；N 14与N 16互补配对时，N 14核苷酸序列的方向为5’-3’，N 16核苷酸序列的方向为3’-5’。
6.在本申请的一些实施例中，其中当N 1与N 23中的至少一条片段的长度大于或等于5个核苷酸碱基时，则N 1与N 23核苷酸序列中至少有两对碱基形成互补配对；当 N 14与N 16中的至少一条片段的长度大于或等于5个核苷酸碱基时，则N 14与N 16核苷酸序列中至少有两对碱基形成互补配对。
7.上述核酸适配体分子，对通式Carrot结构的核苷酸取代选自下组中的一种：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/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、A4C/U5C/C12A/A13C、A4C/U5C/G7U/A11G/A13C、A4C/U5C/G7U/C12A/A13C、A4C/U5A/A11G/C12A/A13C、A4C/U5C/A11G/C12A/A13C。
8.在本申请的一些实施例中，其中对通式Carrot结构的核苷酸取代选自下组中的一种：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、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、A4C/U5C/C12A/A13C。
9.在本申请的一些实施例中，其中对通式Carrot结构的核苷酸取代选自下组中的一种：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。
10.上述核酸适配体分子，其中核苷酸序列(a)中的N 1与N 23处的核苷酸序列为F30或tRNA脚手架RNA序列。
11.在本申请的一些实施例中，其中的适配体分子是RNA分子或经碱基修饰的RNA分子。
12.在本申请的一些实施例中，其中的适配体分子是DNA-RNA杂交分子或经碱基修饰的DNA-RNA分子。
13.上述核酸适配体分子，其中的N 14-N 15-N 16包含一个能够识别靶标分子的核苷酸序列。
14.在本申请的一些实施例中，其中的靶标分子包括但不限于：蛋白质，核酸，脂质分子，碳水化合物，激素，细胞因子，趋化因子，代谢物，金属离子。
15.在本申请的一些实施例中，其中的N 14-N 15-N 16为能够识别S-腺苷蛋氨酸和腺苷分子的核苷酸序列。
16.在本申请的一些实施例中，所述的适配体功能是指核酸适配体能提高荧光团分子在合适波长激发光下的荧光强度至少2倍，至少5-10倍，至少20-50倍，至少100-200倍或者至少200倍以上。
17.在本申请的一些实施例中，所述核酸适配体分子还可包含能够结合多个荧光团分子的串联体，所述串联体通过适当长度的间隔序列连在一起，个数可以是2、3、4、5、6、7、8或者更多。所述串联体的核苷酸可选自但不限于序列SEQ ID No：7、8、9、10，11、12、13、14、15、16、17、18、19和20。
18.上述核酸适配体分子，其中的核酸适配体分子具有序列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或31。
19.一种核酸适配体分子与荧光团分子的复合物，其中所述的核酸适配体分子为上述核酸适配体分子，所述的荧光团分子具有下述式(I)所述的结构：
Figure PCTCN2021083579-appb-000001
其中：Ar 1、Ar 2独立地为六元芳基、六元芳杂基；D-为HO-或N(X 1)(X 2)-，X 1、X 2各自独立地选自氢、烷基和改性烷基；X 1，X 2任选相互连接，与N原子一起形成脂杂环；当D-为N(X 1)(X 2)-，Ar 1为苯基时，X 1，X 2独立地与苯环形成饱和或不饱和的酯杂环；当D-为HO-，Ar 1为苯基时，与HO-相邻的至少一个氢原子被卤素取代；Y为O、S；R 1为氢、烷基和改性烷基；R 2为氢原子、卤原子、-OH、-CN；
其中：所述“烷基”各自独立地为C 1-C 10直链或支链烷基；可选地，为C 1-C 7直链或支链烷基；可选地，为C 1-C 5直链或支链烷基；可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基 丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
20.在本申请的一些实施例中，所述“改性烷基”各自独立地为烷基的任意碳原子被选自卤原子、-OH、-CO-、-O-、-CN、-SO 3H、伯氨基、仲氨基、叔氨基的一种或多种基团置换所得的基团，所述改性烷基具有1-10个碳原子，其中碳碳单键任选独立地被碳碳双键或碳碳三键置换；
所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
所述“改性亚烷基”为C 1-C 10(优选为C 1-C 6)亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中的基团置换所得的基团；
可选地，所述“改性烷基”为含有选自-OH、-O-、乙二醇单元(-(CH 2CH 2O) n-)、单糖单元、-O-CO-、-NH-CO-、-SO 2-O-、-SO-、Me 2N-、Et 2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、氰基中的一种或多种基团；
可选地，Ar 1为选自下式(Ⅱ-1)～(Ⅱ-15)中的结构：
Figure PCTCN2021083579-appb-000002
Ar 2选自下式(Ⅲ-1)～(Ⅲ-25)中的结构：
Figure PCTCN2021083579-appb-000003
可选地，式(I)所示的化合物选自下式化合物：
Figure PCTCN2021083579-appb-000004
Figure PCTCN2021083579-appb-000005
21.在本申请的一些实施例中，其中复合物中的适配体分子包含核苷酸序列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或31。
22.一种所述的复合物用于体外或体内目标核酸分子的检测或标记。
23.一种所述的复合物用于细胞外或细胞内靶标分子的检测或标记。
24.一种所述的复合物用于对基因组DNA进行成像。
25.一种所述的复合物用于检测RNA与蛋白质的相互作用。
26.一种DNA分子，其转录上述核酸适配体分子。
27.一种表达载体，其包含所述的DNA分子。
28.一种宿主细胞，其包含所述的表达载体。
29.一种试剂盒，包含所述核酸适配体分子和/或所述表达载体和/或所述的宿主细胞和/或所述的复合物。
30.一种检测靶标分子的方法，包括步骤：
a)在包含靶标分子的溶液中加入所述的复合物；
b)用合适波长的光激发复合物；
c)检测复合物的荧光。
31.一种检测基因组DNA的方法，包含利用所述复合物对基因组DNA进行成像。
32.一种提取与纯化RNA的方法，包含利用所述复合物提取与纯化RNA。
本发明人设计了全新的核酸适配体分子，并合成全新的荧光团分子，组成全新的荧光团-核酸适配体复合物，适配体分子结合荧光团分子之后可以显著提高荧光团分子在合适波长激发光下的荧光强度，它们克服了此前荧光团-核酸适配体复合物的缺点，能有效用于活细胞中的RNA/DNA实时标记。本发明的核酸适配体对荧光团分子具有较强的亲和力，并展现出了不同的荧光光谱。这些核酸适配体-荧光团分子复合物可以对原核和真核细胞中RNA/DNA进行实时标记与成像，检测RNA-蛋白质的相互作用，或用于RNA的提取与纯化的标签等作用。
附图说明
图1.Carrot核酸适配体分子的通用结构的二级结构预测。
图2.Carrot-1和Carrot-2核酸适配体分子的二级结构预测。
图3.F30-Carrot-2的二级结构预测。
图4.tRNA-Carrot-2的二级结构预测。
图5.Carrot-Ⅳ-39复合物性质鉴定。(a)Carrot-1-Ⅳ-39复合物的荧光激发光谱和发射光谱；(b)Carrot-1与IV-39结合的解离常数测定；(c)Carrot-1-Ⅳ-39复合物对Mg 2+的依赖性测定。
图6.不同碱基修饰的Carrot对IV-39的激活效果。(a)经不同碱基修饰的Carrot示意图，对于Carrot-3，结构示意图中深色碱基为脱氧核糖核苷酸碱基；对于Carrot-4，结构示意图中深色碱基为经2’-F修饰的碱基；(b)Carrot-3和Carrot-4对IV-39荧光团分子荧光的激活效果，“对照”是利用缓冲液替换Carrot-3或Carrot-4适配体RNA。
图7.不同Carrot串联体对Ⅳ-39的激活效果。(a)串联方式1示意图；(b)串联方式2示意图；(c)串联方式3示意图；(d)串联方式1的Carrot串联体对IV-39荧 光团分子荧光的激活效果；(e)串联方式2的Carrot串联体对IV-39荧光团分子荧光的激活效果；(f)串联方式3的Carrot串联体对IV-39荧光团分子荧光的激活效果。
图8.F30-Carrot-2-IV-4复合物用于细菌中RNA的标记效果。
图9.F30-Carrot-2-IV-4复合物用于酵母细胞中RNA的标记效果。
图10.Carrot与IV-39及其类似物用于哺乳动物细胞中RNA的标记效果。(a)tRNA-Carrot-2-IV-39复合物用于哺乳动物细胞中RNA的标记效果；(b)tRNA-Carrot-2-IV-39复合物用于哺乳动物细胞中RNA标记的统计结果；(c)F30-4Carrot-2与IV-39类似物在哺乳动物细胞中标记RNA的效果。
图11.基于Carrot-1的探针构建。(a)探针的构建示意图；(b)探针在有无靶标存在下对IV-39荧光团分子荧光的激活效果。
图12.Carrot用于示踪细胞中RNA定位。(a)4Carrot-2用于检测细胞中GAPDH mRNA的定位；(b)4Carrot-2用于检测细胞中ACTB mRNA的定位。
图13.Carrot用于检测基因组DNA的成像结果。(a)不同嵌合sgRNA示意图；(b)不同嵌合sgRNA对活细胞中基因位点的成像结果。
图14.Carrot用于检测RNA-蛋白质相互作用的结果。
图15.Carrot用于RNA的提取与纯化的结果。
发明详述
本发明在此通过对使用下述定义和实施例的引用进行详细描述。所有在本文中提及的专利和公开文献的内容，包括在这些专利和公开中披露的所有序列，明确地通过提述并入本文。下文中，“核苷酸”与“核苷酸碱基”互换使用，表示相同意思。
核酸适配体分子
本发明所述的“核酸适配体分子”也称为“适配体分子”。该核酸适配体分子包含(a)核苷酸序列为N 1AGAUUGUAAACAN 14-N 15-N 16GACACUN 23(对应于图1的通式Carrot结构)；或(b)与(a)所述的核苷酸序列具有至少70％同一性的序列；其中N 1与N 23核苷酸序列中至少有一对碱基形成反向互补配对，即N 1核苷酸序列的方向为5’-3’，N 23核苷酸序列的方向为3’-5’。当N 1与N 23至少一条核苷酸碱基长度小于或等于4时，需要至少一对碱基形成互补配对；当N 1与N 23至少一条核苷酸碱基长度大于或等于5时，需要 至少两对碱基形成互补配对。其中N 14与N 16核苷酸序列中至少有一对碱基形成反向互补配对，即N 14核苷酸序列的方向为5’-3’，N 16核苷酸序列的方向为3’-5’。当N 14与N 16至少一条核苷酸碱基长度小于或等于4时，需要至少一对碱基形成互补配对；当N 14与N 16至少一条核苷酸碱基长度大于或等于5时，需要至少两对碱基形成互补配对。其中N 15为任意长度任意组成的核苷酸碱基；或(c)在所述的核苷酸序列(a)的任一位置经过1-5个核苷酸的取代、缺失和/或增加。
核酸适配体分子包含对通式Carrot结构的核苷酸的取代，该取代选自下组中的一种：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/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、A4C/U5C/C12A/A13C、A4C/U5C/G7U/A11G/A13C、A4C/U5C/G7U/C12A/A13C、A4C/U5A/A11G/C12A/A13C、A4C/U5C/A11G/C12A/A13C(即表1中的适配体分子结构)。这些突变体能特异性结合荧光团分子，并在结合之后可以显著提高荧光团分子在合适波长激发光下的荧光强度。其中核苷酸的位置序列对应于图1中的位置。
上述的突变表示在通式Carrot结构的适配体核苷酸序列的相应位点发生核苷酸替换，如A4U表示Carrot的第4位腺嘌呤核苷酸A被取代为尿嘧啶核苷酸U，也即表1中的Carrot(A4U)；U5C/A13C表示Carrot的第5位U被取代为C，同时第13位A被取代为C，也即表1中的Carrot(U5C/A13C)。
表1：Carrot通式结构经过1个核苷酸取代的适配体序列
Carrot通式结构的取代 取代后的适配体结构通式(加粗为取代后碱基)
Carrot(A4U) N 1AGUUUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(A4G) N 1AGGUUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(A4C) N 1AGCUUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(U5A) N 1AGAAUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(U5G) N 1AGAGUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(U5C) N 1AGACUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(G7C) N 1AGAUUCUAAACAN 14-N 15-N 16GACACUN 23
Carrot(G7A) N 1AGAUUAUAAACAN 14-N 15-N 16GACACUN 23
Carrot(G7U) N 1AGAUUUUAAACAN 14-N 15-N 16GACACUN 23
Carrot(U8C) N 1AGAUUGCAAACAN 14-N 15-N 16GACACUN 23
Carrot(A10U) N 1AGAUUGUAUACAN 14-N 15-N 16GACACUN 23
Carrot(A10G) N 1AGAUUGUAGACAN 14-N 15-N 16GACACUN 23
Carrot(A10C) N 1AGAUUGUACACAN 14-N 15-N 16GACACUN 23
Carrot(A11U) N 1AGAUUGUAAUCAN 14-N 15-N 16GACACUN 23
Carrot(A11G) N 1AGAUUGUAAGCAN 14-N 15-N 16GACACUN 23
Carrot(A11C) N 1AGAUUGUAACCAN 14-N 15-N 16GACACUN 23
Carrot(C12G) N 1AGAUUGUAAAGAN 14-N 15-N 16GACACUN 23
Carrot(C12A) N 1AGAUUGUAAAAAN 14-N 15-N 16GACACUN 23
Carrot(C12U) N 1AGAUUGUAAAUAN 14-N 15-N 16GACACUN 23
Carrot(A13U) N 1AGAUUGUAAACUN 14-N 15-N 16GACACUN 23
Carrot(A13G) N 1AGAUUGUAAACGN 14-N 15-N 16GACACUN 23
Carrot(A13C) N 1AGAUUGUAAACCN 14-N 15-N 16GACACUN 23
Carrot(G17A) N 1AGAUUGUAAACAN 14-N 15-N 16AACACUN 23
Carrot(C19A) N 1AGAUUGUAAACAN 14-N 15-N 16GAAACUN 23
Carrot(C19U) N 1AGAUUGUAAACAN 14-N 15-N 16GAUACUN 23
Carrot(A20C) N 1AGAUUGUAAACAN 14-N 15-N 16GACCCUN 23
Carrot(A4C/U5A) N 1AGCAUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5C) N 1AGCCUGUAAACAN 14-N 15-N 16GACACUN 23
Carrot(A4C/A11G) N 1AGCUUGUAAGCAN 14-N 15-N 16GACACUN 23
Carrot(A4C/C12A) N 1AGCUUGUAAAAAN 14-N 15-N 16GACACUN 23
Carrot(A4C/A13C) N 1AGCUUGUAAACCN 14-N 15-N 16GACACUN 23
Carrot(U5A/A11G) N 1AGAAUGUAAGCAN 14-N 15-N 16GACACUN 23
Carrot(U5A/C12A) N 1AGAAUGUAAAAAN 14-N 15-N 16GACACUN 23
Carrot(U5A/A13C) N 1AGAAUGUAAACCN 14-N 15-N 16GACACUN 23
Carrot(U5G/A13C) N 1AGAGUGUAAACCN 14-N 15-N 16GACACUN 23
Carrot(U5C/G7U) N 1AGACUUUAAACAN 14-N 15-N 16GACACUN 23
Carrot(U5C/A11G) N 1AGACUGUAAGCAN 14-N 15-N 16GACACUN 23
Carrot(U5C/C12G) N 1AGACUGUAAAGAN 14-N 15-N 16GACACUN 23
Carrot(U5C/C12A) N 1AGACUGUAAAAAN 14-N 15-N 16GACACUN 23
Carrot(U5C/C12U) N 1AGACUGUAAAUAN 14-N 15-N 16GACACUN 23
Carrot(U5C/A13C) N 1AGACUGUAAACCN 14-N 15-N 16GACACUN 23
Carrot(G7U/A13C) N 1AGAUUUUAAACCN 14-N 15-N 16GACACUN 23
Carrot(A11G/A13C) N 1AGAUUGUAAGCCN 14-N 15-N 16GACACUN 23
Carrot(C12G/A13C) N 1AGAUUGUAAAGCN 14-N 15-N 16GACACUN 23
Carrot(C12A/A13C) N 1AGAUUGUAAAACN 14-N 15-N 16GACACUN 23
Carrot(C12U/A13C) N 1AGAUUGUAAAUCN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5C/A13C) N 1AGCCUGUAAACCN 14-N 15-N 16GACACUN 23
Carrot(U5C/G7U/A13C) N 1AGACUUUAAACCN 14-N 15-N 16GACACUN 23
Carrot(U5C/C12G/A13C) N 1AGACUGUAAAGCN 14-N 15-N 16GACACUN 23
Carrot(U5C/C12U/A13C) N 1AGACUGUAAAUCN 14-N 15-N 16GACACUN 23
Carrot(U5C/A11G/A13C) N 1AGACUGUAAGCCN 14-N 15-N 16GACACUN 23
Carrot(U5C/C12A/A13C) N 1AGACUGUAAAACN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5C/A11G/A13C) N 1AGCCUGUAAGCCN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5C/C12A/A13C) N 1AGCCUGUAAAACN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5C/G7U/A11G/A13C) N 1AGCCUUUAAGCCN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5C/G7U/C12A/A13C) N 1AGCCUUUAAAACN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5A/A11G/C12A/A13C) N 1AGCAUGUAAGACN 14-N 15-N 16GACACUN 23
Carrot(A4C/U5C/A11G/C12A/A13C) N 1AGCCUGUAAGACN 14-N 15-N 16GACACUN 23
适配体分子是单链核酸分子，它们有着一个或多个碱基配对区域(茎)以及一个或多个非配对的区域(环)构成的二级结构(图1)。本发明所述的核酸适配体分子包含一个如图1所预测的二级结构。所述结构的5’端或3’端可以与任意目标RNA分子融合，用于细胞外或细胞内检测目标RNA分子。在本发明一优选的实施方案中，核酸适配体分子的5’端融合ACTB RNA序列(Genebank:BC016045)；在本发明另一优选的实施方案中，核酸适配体分子的5’端融合GAPDH RNA序列(Genebank:BC009081)。
图1中的茎-环结构(N 14-N 15-N 16)起到稳定整个核酸适配体分子结构的作用，可以被替换成其他可以形成茎-环结构的任意长度任意组成的核苷酸序列。本发明所述的适配体分子还可包含插入到N 14-N 15-N 16位置的其他核苷酸序列，该插入的核苷酸序列替换图1中的茎-环结构(N 14-N 15-N 16)。所述核苷酸序列可以特异性识别/结合靶标分子。当靶标分子不存在时，所述适配体分子与荧光团分子的结合能力弱，导致荧光团分子显示弱荧光；当靶标分子存在时，靶标分子与所述适配体的结合会促进所述适配体与荧光团分子的结合，显著提高荧光团分子在合适波长激发光下的荧光强度。所述靶标分子可以是一种小分子、一种细胞表面的信号分子等。这些核酸适配体与特定的靶标分子通过非共价结合，这种非共价结合主要是依赖分子间的离子力、偶极力、氢键、范德华力、正负电子相互作用、堆积作用或者以上几种作用力的结合。所述茎-环结构(N 14-N 15-N 16)可被替换成识别靶标分子的RNA序列，用于细胞外或细胞内检测靶标分子。
本发明优选的实施方案中，所述核酸适配体分子优选为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或31，或者可以结合荧光团分子显著提高其在合适波长激发光下荧光的它们的突变序列。
本发明所述的核酸适配体分子还可包含一段增加其稳定性的核苷酸序列。在本发明的一优选的实施方案中，采用F30脚手架RNA(序列3)，其与所述核酸适配体分子的连接方式如图3所示；在在本发明的另一优选的实施方案中，采用tRNA脚手架RNA(序列4)，其与所述核酸适配体分子的连接方式如图4所示。
同一性
“同一性”在本发明中描述两个核苷酸序列之间的相关性。本发明的两个适配体核苷酸序列的同一性计算中不包括(a)序列中的N 1、N 14、N 15、N 16、N 23。就本发明而言，两个核苷酸序列之间的同一性程度使用如EMBOSS软件包(EMBOSS：The European Molecular Biology Open Software Suite，Rice等，2000，Trends in Genetics 16：276-277)的Needle程序，优选3.0.0版或更高版本中执行的Needleman-Wunsch算法(Needleman和Wunsch，1970，J.Mol.Biol.48：443-453)来确定。使用的任选参数为缺口罚分(gap penalty)10，缺口延伸罚分(gap extension penalty)0.5和EBLOSUM62取代矩阵(BLOSUM62的EMBOSS版)。使用Needle标记为“最高同一性(longest identity)”(使用-nobrief选项获得)的输出结果作为百分比同一性，并计算如下：
(相同的残基×100)/(比对长度-比对中缺口的总数)。
如本发明表1中Carrot-1和Carrot-1(U5C)的序列为N 1AGAUUGUAAACAN 14-N 15-N 16GACACUN 23和N 1AGACUGUAAACAN 14-N 15-N 16GACACUN 23，对它们同一性比对的时候，按照本发明的定义，应不包含 N 1、N 14-N 15-N 16和N 23 的核苷酸碱基，因此它们的序列同一性比对结果为94.4％(相差1个核苷酸)。
荧光团分子
本发明所述的“荧光团分子”也称为“荧光团”或“荧光分子”。“荧光团分子”在本发明中是一类可被条件性激活的荧光团分子。它们在没有核酸适配体的情况下显示出较低的量子产率。在具体的实施方式中，当没有与特定适配体结合时，荧光团的量子产率低于0.1，更优的低于0.01，最优的低于0.001；当荧光团被特定适配体结合后，荧光团的量子产率提高2倍以上，更优的提高10倍以上，最优的提高100倍以上。荧光团分子优选水溶性的，对细胞无毒且易穿透膜的。本发明的荧光团优选能够通过主动运输或者被动扩散通过细胞膜或细胞壁进入细胞浆或细胞周质。在本发明的实施方式中，荧光团可以透过革兰氏阴性菌的外膜和内膜，植物细胞的细胞壁和细胞膜，真菌和细胞壁和细胞膜，动物细胞的细胞膜，以及活体动物的GI和内皮细胞膜。
本发明所述的核酸适配体分子可以特异性结合一种荧光团，显著增加其在特定波长激发下的荧光值。所述荧光团分子选自结构(Ⅰ)：
Figure PCTCN2021083579-appb-000006
(Ι)其中：Ar 1、Ar 2独立地为六元芳基、六元芳杂基；D-为HO-或N(X 1)(X 2)-，X 1、X 2各自独立地选自氢、烷基和改性烷基；X 1，X 2任选相互连接，与N原子一起形成脂杂环；当D-为N(X 1)(X 2)-，Ar 1为苯基时，X 1，X 2独立地与苯环形成饱和或不饱和的酯杂环；当D-为HO-，Ar 1为苯基时，与HO-相邻的至少一个氢原子被卤素取代；Y为O、S；R 1为氢、烷基和改性烷基；R 2为氢原子、卤原子、-OH、-CN；
其中：所述“烷基”各自独立地为C 1-C 10直链或支链烷基；可选地，为C 1-C 7直链或支链烷基；可选地，为C 1-C 5直链或支链烷基；可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
其中：所述“改性烷基”各自独立地为烷基的任意碳原子被选自卤原子、-OH、-CO-、-O-、-CN、-SO 3H、伯氨基、仲氨基、叔氨基的一种或多种基团置换所得的基团，所述改性烷基具有1-10个碳原子，其中碳碳单键任选独立地被碳碳双键或碳碳三键置换；
其中：所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；所述“改性亚烷基”为C 1-C 10(优选为C 1-C 6)亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中的基团置换所得的基团；
可选地，所述“改性烷基”为含有选自-OH、-O-、乙二醇单元(-(CH 2CH 2O) n-)、单糖单元、-O-CO-、-NH-CO-、-SO 2-O-、-SO-、Me 2N-、Et 2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、氰基中的一种或多种基团；
可选地，Ar 1为选自下式(Ⅱ-1)～(Ⅱ-15)中的结构：
Figure PCTCN2021083579-appb-000007
Figure PCTCN2021083579-appb-000008
Ar 2选自下式(Ⅲ-1)～(Ⅲ-25)中的结构：
Figure PCTCN2021083579-appb-000009
可选地，式(I)所示的化合物选自下式化合物：
Figure PCTCN2021083579-appb-000010
Figure PCTCN2021083579-appb-000011
本发明优选的实施方式中，所述荧光团分子包含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。“提高荧光信号”、“荧光增加”、“提高荧光强度”、“增加荧光强度”在本发明中指合适波长激发光照射下荧光团量子产率的提高，或者荧光信号最大发射峰的迁移(相对乙醇或者水溶液中荧光团本身的发射峰)，或者摩尔消光系数的增加，或者以上的两种或更多。在本发明一优选实施方式中，量子产率的增加至少是2倍；在本发明另一优选实施方式中，量子产率的增加至少是5-10倍；在本发明另一更优选实施方式中，量子产率的增加至少是20-50倍；在本发明另一更优选实施方式中，量子产率的增加至少是100-200倍；在本发明另一更优选实施方式中，量子产率的增加至少是200倍以上。用于激发荧光团产生荧光信号的光源可以是任意合适的光照设备，如包括LED灯、白炽灯、荧光灯、激光；激发光既可以是直接从这些设备中发出，也可以间接通过其他荧光团获取，如荧光共振能量转移(FERT)的供体荧光团，或发光能量共振转移(BRET)的供体发光团。
靶标分子
本发明所述的靶标分子可以是任意的生物材料或者小分子，包括但不限于：蛋白质，核酸(RNA或者DNA)，脂质分子，碳水化合物，激素，细胞因子，趋化因子，代谢物、金属离子等。靶标分子可以是与疾病或者病原菌感染相关的分子。
通过在本发明所述的适配体分子，如图1所示的结构中，插入的核苷酸序列替换了图1中的N 14-N 15-N 16的茎-环结构，该核苷酸序列可以特异性识别/结合靶标分子。当靶标分子不存在时，适配体分子与荧光团分子不结合或结合能力弱，不能显著提高荧光团分子在合适波长激发光下的荧光；当靶标分子存在时，靶标分子与所述核苷酸序列的结合会促进适配体分子与荧光团分子的结合，显著提高荧光团分子在合适波长激发光下的荧光，实现对靶标分子的检测、成像和定量分析。
靶标分子也可以是整个细胞或表达在整个细胞表面的分子。典型的细胞包括但不限于癌症细胞、细菌细胞，真菌细胞以及正常动物细胞。靶标分子也可以是病毒颗粒。目前很多的上述靶标分子的适配体被鉴定出来，它们可以被整合到本发明中的多种核酸适配体中。目前已报道的可以结合靶标分子的RNA适配体包括但不限于：T4RNA聚合酶适配体，HIV逆转录酶适配体，噬菌体R17衣壳蛋白适配体。
目标核酸分子
“目标核酸分子”又称“靶标核酸分子”是指待检测的核酸分子，可以是细胞内的，也可以是细胞外的；包括目标RNA分子和目标DNA分子。本发明通过将目标核酸分子与所述核酸适配体分子连接，通过荧光团分子与核酸适配体分子结合，显著提高荧光团分子在合适波长激发光下的荧光值，进而实现检测目标核酸分子的含量与分布的目的。
“目标RNA分子”在本发明中包括任意的RNA分子，包括但不限于pre-mRNA，编码细胞本身或外源表达产物的mRNA，pre-rRNA，rRNA，tRNA，hnRNA，snRNA，miRNA，siRNA，shRNA，sgRNA，crRNA，lncRNA，噬菌体衣壳蛋白MCP识别结合序列MS2RNA、噬菌体衣壳蛋白PCP识别结合序列PP7RNA，λ噬菌体转录终止蛋白N识别结合序列boxB RNA等。靶标RNA可以融合在本发明RNA适配体分子的5’端或3’端或 N 14-N 15-N 16的位置 。
“sgRNA”在本发明中指CRISPR/Cas9系统中将tracrRNA和crRNA经改造后形成的单一的引导RNA(single guide RNA,sgRNA)，其5’端20nt左右的序列通过碱基互补配对来靶向DNA位点，促使Cas9蛋白在该位点诱发DNA双链断裂。
核酸适配体的串联体
本发明所述的核酸适配体分子进一步还可包含可以结合多个荧光团分子的串联体。所述串联体通过适当长度的间隔序列连在一起，串联的Carrot结构的个数可以是2，3，4，5，6，7，8，9，10或者更多。串联体的形式可以有多种，在本发明一优选的实施方案中，串联的形式为“串联1”，如图7a所示，优选的核苷酸序列为SEQ ID NO：7、8、9、10或11；其中的2Carrot-2表示具有2个Carrot-2结构的串联体1；在本发明另一优选的实施方案中，串联的形式为“串联2”，如图7b所示，优选的核苷酸序列为SEQ ID NO：12、13、14、15或16；其中的2×Carrot-2表示具有2个Carrot-2结构的串联体2；在本发明另一优选的实施方案中，串联的形式为“串联3”，如图7c所示，优选的核苷酸序列为SEQ ID NO：17、18、19或20；其中的2×2Carrot-2表示具有4个Carrot-2结构的串联体3；无论何种形式，串联体之间的间隔序列可以进行更换。
本发明所述的单体形式的适配体是指仅含有1个Carrot结构的适配体，也就是仅含有1个图1所示的适配体。
多聚体形式的适配体是指含有1个以上Carrot结构的适配体，包含但不限于图7所示的串联形式构成的适配体。
适配体-荧光团复合物
本发明的适配体-荧光团复合物包含1个核酸适配体分子以及1个或多个荧光团分子。在本发明的一具体实施方式中，包含1个核酸分子以及1个荧光团分子的分子复合物为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、Carrot-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。
在本发明的另一具体实施方式中，串联体的核酸分子与多个荧光团分子的形成复合物，例如以“串联1”方式形成的包含4个适配体单元的F30-4Carrot-2与4个荧光团分子形成的复合物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-37)、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)和F30-4Carrot-2-4×(IV-22)。所述分子复合物可以在体外以单独的两种溶液形式存在，或者在同一种溶液中存在，也可以存在于细胞内。
核酸适配体功能
本发明的适配体功能指可以显著提高荧光团分子在合适波长激发光下的荧光强度，可以采用具体实施例中的常用实验方法(五)核酸适配体的功能检测来对适配体进行检测。在本发明的一个优选实施方式中，荧光强度的增加至少是2倍(荧光强度按照实验方法(五)进行检测)；在本发明另一优选实施方式中，荧光强度的增加至少是5-10倍；在本发明另一更优选实施方式中，荧光强度的增加至少是20-50倍；在本发明另一更优选实施方式中，荧光强度的增加至少是100-200倍；在本发明另一更优选实施方式中，荧光强度的增加至少是200倍以上。
核酸适配体二级结构
本专利中的核酸适配体的二级结构是利用mFold在线分析软件模拟预测得到的(http://unafold.rna.albany.edu/？q＝mfold)。二级结构中的茎结构是指核酸适配体分子单链内某些区域靠氢键互补配对形成局部双链结构。一般情况下，双链结构的形成并不需要该段区域内的所有核苷酸均发生互补配对；一般情况下，N 1与N 23，以及N 14与N 16中其中一段序列的至少50％的核苷酸与另一片段发生互补配对即可形成茎结构。如果N 1与N 23是单个核苷酸，则需要N 1与N 23完全互补才能形成茎结构(如图1所示)。
表达核酸适配体的DNA分子
所述DNA分子包含可以编码本发明的核酸适配体分子的DNA序列。所述DNA分子包含核苷酸序列R 1AGATTGTAAACAR 14-R 15-R 16GACACTR 23，以及其具有至少70％同一性的核苷酸序列。其中R 1编码通式Carrot结构中的N 1，R 14编码通式Carrot结构中的N 14，R 15编码通式Carrot结构中的N 15，R 16编码通式Carrot结构中的N 16，R 23编码通式Carrot结构中的N 23。所述DNA分子还可包含一个控制DNA转录的启动子，启动子与编码核酸适配体的DNA序列之间可操作性连接。在本发明的一具体实施方式中，DNA分子包含U6启动子；在本发明的另一具体实施方式中，DNA分子包含CMV启动子。DNA分子除了包含所述DNA分子外，进一步还可包含编码任意目标核酸分子的DNA序列。在本发明的一具体实施方式中，编码目标RNA的DNA分子包含编码甘油醛-3-磷酸脱氢酶(GAPDH)或β-肌动蛋白(β-actin)的DNA序列(嵌合RNA的序列分别为SEQ ID No：26、27)。
启动子
“启动子”在本发明中包括真核细胞与原核细胞启动子。真核细胞的启动子序列与原核细胞的启动子序列完全不同。一般地，真核启动子不能被原核细胞中的RNA聚合酶所识别介导RNA的转录。同理，原核启动子也不能被真核细胞中的RNA聚合酶所识别介导RNA的转录。不同启动子的强度差别很大(强度指介导转录的能力)。根据实际应用的不同，可以使用强启动子达到高水平转录。比如用于标记时，高水平表达就比较好，而如果评估转录行为，较低水平的转录可以允许细胞及时的调节转录过程。根据宿主细胞的不同，可以选用一种或者多种合适的启动子。例如，当在大肠杆菌细胞中使用时，T7噬菌体启动子，lac启动子，trp启动子，recA启动子，核糖体RNA启动子，λ噬菌 体中的PR和PL启动子，以及其他启动子，但不限于:lacUV5启动子，ompF启动子，bla启动子，lpp启动子等。此外，一个杂交的trp-lacUV5启动子(tac启动子)或者其他通过重组或合成DNA技术得到的大肠杆菌启动子，均可用于转录本发明所述的RNA适配体。细菌中本身的一些操作子序列可以与启动子序列结合在一起构成诱导型启动子，此时需要加入特定的诱导物才能诱导DNA分子的转录。比如lac操作子需要加入乳糖或者乳糖类似物(IPTG)诱导其表达，其他的操作子还有trp，pro等。
如上所述，DNA分子编码序列的5’端的调控序列是启动子。无论是体外转录获得RNA适配体，还是在培养的细胞或组织中表达适配体，都需要依据启动子的强度选择合适的启动子。由于体内表达适配体可以被遗传操作，另一类型的启动子就是响应特定环境诱导DNA转录的诱导型启动子，如表达在特定的组织，特定的时间，特定的发育阶段等。这些不同的启动子可以被RNA聚合酶I，II或III识别。
真核细胞中转录的启动也需要合适的启动子，包括但不限于β-球蛋白启动子，CAG启动子，GAPDH启动子，β-肌动蛋白启动子，肌动蛋白启动子，Cstf2t启动子，SV40启动子，PGK启动子，MMTV启动子，腺病毒Ela启动子，CMV启动子等。真核细胞中转录的终止依赖于RNA序列中特定的切割位点。同样的，RNA聚合酶转录基因的不同，其转录终止子也差别很大。然而，筛选合适的3’转录终止子区域，本领域人员基础的实验技能就能实现。
表达系统
本发明的“表达系统”，也称为“表达载体”，包含整合有表达核酸适配体的DNA分子。本发明的表达系统，可以是一个质粒，也可以是病毒颗粒。
“表达载体”重组病毒可以通过将质粒转染进入感染病毒的细胞中获得。合适的载体包括但不限于病毒载体如λ载体系统gt11，gt WES.tB，Charon 4；质粒载体包括pBR322，pBR325，pACYC177，pACYC184，pUC8，pUC9，pUC18，pUC19，pLG399，pR290，pKC37，pKC101，pBluescript II SK+/-或KS+/-(见Stratagene克隆系统)，pET28系列，pACYCDuet1，pCDFDuet1，pRSET系列，pBAD系列，pQE，pIH821,pGEX，pIIIEx426RPR等。
大量的宿主表达系统可以用于表达本发明所述的DNA分子。主要地，载体系统必须要与所用的宿主细胞相容，宿主载体系统包括但不限于：转化的噬菌体DNA，或质粒DNA，或考斯质粒DNA的细菌；包含酵母载体的酵母；感染病毒的哺乳动物细胞(如 腺病毒，腺相关病毒，逆转录病毒)；转染了质粒的哺乳动物细胞；感染病毒的昆虫细胞(如杆状病毒)；感染细菌或者通过粒子轰击转化的植物细胞。所述的载体中的表达元件的强度和特性差异很大。根据使用的宿主-载体系统选用任意一种或多种合适的转录元件。
一旦构建的DNA分子被克隆到载体系统中，很容易将它们转入宿主细胞中。根据不同的载体或宿主细胞系统，方法包括但不限于转化，转导，接合，固定，电转等。
在本发明的一具体实施方式中，提供含编码F30-Carrot-2 RNA的DNA分子的表达质粒pET28a-T7-F30-Carrot-2和pYES2.1-F30-Carrot-2。在本发明的另一具体实施方式中，提供含编码tRNA-Carrot-2 RNA的DNA分子的表达质粒pU6-tRNA-Carrot-2。在本发明的另一具体实施方式中，提供含编码F30-4Carrot-2 RNA的DNA分子的表达质粒pU6-F30-4Carrot-2。在本发明的另一具体实施方式中，提供含编码GAPDH-4Carrot-2和ACTB-4Carrot-2的DNA分子的表达质粒pCDNA3.1 hygro(+)-GAPDH-4Carrot-2和pCDNA3.1 hygro(+)-ACTB-4Carrot-2。在本发明的另一具体实施方式中，提供含编码sgRNA-Carrot-2-1、sgRNA-Carrot-2-2和sgRNA-Carrot-2-3的DNA分子的表达质粒psgRNA-Carrot-2-1、psgRNA-Carrot-2-2和psgRNA-Carrot-2-3。在本发明的另一具体实施方式中，提供含编码Carrot-1-MS2的DNA分子的表达质粒pU6-Carrot-1-MS2。
本发明也提供整合了编码核酸适配体DNA分子，但目标RNA分子编码DNA序列空缺的表达载体，目标RNA分子的编码DNA序列空缺是让用户可以自行选择所需检测的目标RNA分子的DNA序列，例如GAPDH mRNA对应的编码DNA序列，用标准的重组DNA技术将DNA序列插入本发明的这种表达载体中，将获得的表达载体导入到(转染、转化、感染等)宿主细胞，检测目标RNA的含量及分布。
宿主细胞
“宿主细胞”在本发明中包括但不限于细菌，酵母，哺乳动物细胞，昆虫细胞，植物细胞，斑马鱼细胞，果蝇细胞，线虫细胞。宿主细胞更优选培养的体外细胞或整个体内活体组织。本发明中的宿主细胞，其包含的哺乳动物细胞包括但不限于297T，COS-7，BHK，CHO，HEK293，HeLa，H1299，受精卵干细胞，诱导全能干细胞，从哺乳动物组织中直接分离的原代细胞等；其包含的大肠杆菌细胞包括但不限于BL21(DE3)、BL21(DE3,Star)、TOP10、Mach1、DH5α；其包含的酵母细胞包含但不限于BY4741,BY4742,AH109。
检测阵列
本发明所述的检测阵列包含一个或多个本发明的核酸适配体分子，其中核酸适配体分子被锚定在阵列表面的离散位置，阵列表面是由固体支撑物构成，包括但不限于玻璃、金属、陶瓷等。将本发明所述核酸适配体分子锚定到阵列表面可通过但不限于以下方法：(1)利用生物素标记所述核酸适配体分子的5’或3’端，将链霉亲和素包被在阵列表面，通过生物素与链霉亲和素的特异性结合将所述核酸适配体分子进行锚定；(2)将噬菌体衣壳蛋白MCP识别结合序列MS2、噬菌体衣壳蛋白PCP识别结合序列PP7或λ噬菌体转录终止蛋白N识别结合序列boxB RNA序列融合在所述核酸适配体分子的5’、3’或茎环结构，将它们识别结合的蛋白MCP、PP7或λ N蛋白包被在阵列表面，通过MS2与MCP蛋白、PP7与PCP蛋白或boxB RNA与λ N蛋白的特异性作用将所述核酸适配体分子进行锚定；(3)将一段RNA或DNA序列融合在所述核酸适配体分子的5’或3’端，将与该段RNA序列互补配对的RNA序列或与该段DNA序列互补配对的DNA序列锚定在阵列表面，通过分子杂交的原理将所述核酸适配体分子锚定在阵列表面。所述检测阵列可用于检测靶标分子的存在与否以及浓度高低，因此，只有在靶标分子存在下，所述核酸适配体分子才会与荧光团分子结合，显著提高其在合适激发光波长下的荧光强度，且在一定范围内，靶标分子的浓度越高，荧光强度越高。
试剂盒
本发明的试剂盒，包含本发明所述的核酸适配体分子和/或荧光团分子，及相应的说明书；或者包含表达所述核酸适配体分子的表达系统和/或荧光团分子，及相应的说明书；或者包含表达核酸适配体分子表达系统的宿主细胞和/或荧光团分子，及相应的说明书。试剂盒中的核酸适配体分子与荧光团分子分别存在于单独的溶液中，或者核酸适配体分子与荧光团分子在同一溶液中。
具体实施方式
以下用实施例对本发明作进一步阐述。这些实施例仅仅用于举例说明，而不对本发明的范围构成任何限制。实施例中主要采用常规的基因工程分子生物学克隆方法，这些方法是本领域普通技术人员所熟知的，例如：简·罗斯凯姆斯等的《分子生物学实 验参考手册》和J.萨姆布鲁克，D.W.拉塞尔著,黄培堂等译：《分子克隆实验指南》(第三版，2002年8月，科学出版社出版，北京)中的有关章节。本领域普通技术人员按照以下实施例，不难根据具体情况略作修改和变换而成功实施本发明。
实施例中所用的pCDNA3.1 hygro(+)质粒载体购自Invitrogen公司，pLKO.1-puro质粒载体购自Sigma公司，pET28a质粒载体购自Novagen公司，pYES2.1 TOPO TA质粒载体购自Invitrogen。所有用于PCR的引物均由上海杰瑞生物工程技术有限公司合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定，序列测定由杰李测序公司完成。各实施例所用的Taq DNA聚合酶购自上海翊圣生物科技有限公司，PrimeSTAR DNA聚合酶购自TaKaRa公司，三种聚合酶购买时都附带赠送对应聚合酶缓冲液和dNTP。EcoRI、BamHI、BglII、HindIII、NdeI、XhoI、SacI、XbaI、SpeI等限制性内切酶、T4连接酶、T4磷酸化酶(T4 PNK)、T7 RNA聚合酶购自Fermentas公司，购买时附带有相对应的缓冲液等。实施例中所用的Hieff Clone TM One Step克隆试剂盒购自上海翊圣生物科技有限公司。除非特别声明，无机盐类化学试剂均购自国药集团上海化学试剂公司。卡那霉素(Kanamycin)购自Ameresco公司；氨苄青霉素(Amp)购自Ameresco公司；384孔和96孔荧光检测黑板购自Grenier公司。DFHBI-1T和DFHO购自Lucerna公司。GTP和SAM购自Sigma公司。
实施例中所用的DNA纯化试剂盒购自BBI公司(加拿大)，普通质粒小抽试剂盒购自天根生化科技(北京)有限公司。BL21(DE3,Star)菌株购自Invitrogen公司。293T/17细胞和COS-7细胞购自中国科学院典型培养物保藏委员会细胞库。BY4741酵母菌株购自上海唯地生物技术有限公司。
实施例中用到的主要仪器：Synergy Neo2多功能酶标仪(美国Bio-Tek公司)，X-15R高速冷冻离心机(美国Beckman公司)，Microfuge22R台式高速冷冻离心机(美国Beckman公司)，PCR扩增仪(德国Biometra公司)，活体成像系统(美国Kodak公司)，光度计(日本和光公司)，核酸电泳仪(申能博彩公司)。
缩写词意义如下：“h”指小时，“min”指分钟，“s”指秒，“d”指天,“μL”指微升，“mL”指毫升，“L“指升，“bp”指碱基对，“mM”指毫摩尔，“μM”指微摩尔。
实施例中常用实验方法及材料
(一)核酸适配体分子制备：
利用含T7启动子的引物对待检测RNA对应的cDNA进行扩增，利用T7 RNA聚 合酶(购自Fermentas公司)以回收得到的双链cDNA为模板转录获得RNA。在20μL的转录体系中加入10μL 3M NaAc，115μL DEPC水，混匀后加入150μL酚氯仿-异丙醇混合液(苯酚：氯仿：异丙醇＝25：24：1)，振荡混匀，10000rpm离心5min后取上清。加入等体积氯仿溶液，振荡混匀，10000rpm离心5min后取上清，反复一次。在上清中加入2.5倍体积的无水乙醇，-20℃冰箱放置30min，4℃12000rpm离心5min，弃上清，利用75％预冷的无水乙醇清洗沉淀2次。待乙醇挥发完后，加入适当量的筛选缓冲液重悬沉淀，75℃处理5min，室温放置10min以上，用于后续实验。
(二)细胞培养及转染：
本实施例中的细胞均在CO 2培养箱中用含10％胎牛血清(FBS)及链霉素和青霉素高糖培养基(DMEM)培养，生长达到80-90％汇合度时将细胞传代培养。转染时，用
Figure PCTCN2021083579-appb-000012
(购自Promega)按照说明书进行操作。
(三)荧光成像：
实施例中主要的成像实验是利用Leica SP8共聚焦激光显微镜进行拍摄，使用HCXPL APO 63.0x1.47油镜和HyD检测器，可使用405nm、457nm、476nm、488nm、497nm、514nm、561nm、633nm多种激光进行成像，各Carrot与染料分子形成的复合物使用靠近其最大激发峰所对应波长的激光进行成像。其他，GFP使用488nm激光器；Hoechst和DAPI荧光使用405nm激光器；Rhodamine使用561nm激光器。
(四)基于同源重组方法的重组质粒构建
1.制备线性化载体:选择合适的克隆位点，并对载体进行线性化，可采用酶切或反向PCR扩增制备线性化载体。
2.PCR扩增制备插入片段:通过在插入片段正、反向PCR引物的5’端引入15-25bp(不包括酶切位点)的线性化载体末端同源序列，使得插入片段PCR产物5’和3’末端分别带有与线性化载体两末端对应的完全一致的序列。
3.线性化载体与插入片段浓度测定:将线性化载体和插入片段扩增产物做数个等体积稀释梯度，原始产物和稀释后产物各取1μL进行琼脂糖凝胶电泳，与DNA分子量标准(DNA Marker)比较条带亮度以确定其近似浓度。
4.重组反应
重组反应体系最适载体使用量为0.03pmol；最适载体与插入片段摩尔比为1:2-1:3，即最适插入片段使用量为0.06-0.09pmol。
Figure PCTCN2021083579-appb-000013
X和Y分别是根据公式计算得到线性化载体和插入片段用。体系配制完成后，混匀各组分，置于50℃反应20min。当插入片段＞5kb时，可将孵育温度延长至25min。待反应完成后，建议将反应管置于冰上冷却5min。反应产物可直接进行转化，也可储存于-20℃，待需要时解冻转化。
(五)核酸适配体的功能检测
按照常用实验方法(一)制备Carrot或Carrot突变体核酸适配体分子，将5μM核酸适配体分子与1μM荧光团分子在检测缓冲液(40mM HEPES,pH 7.4,125mM KCl,5mM MgCl 2,5％DMSO)中孵育，利用Synergy Neo2多功能酶标仪检测获取核酸适配体-荧光团分子复合物荧光的最大激发峰和最大发射峰。再利用Synergy Neo2多功能酶标仪检测核酸适配体-荧光团分子复合物在其最大激发和发射条件下的荧光强度，对照样品(不含核酸适配体的1μM荧光团分子)也在相同的条件进行测定，计算荧光强度的比值。如5μM Carrot-1核酸适配体与1μM IV-39荧光团分子形成的复合物的荧光最大激发峰为524nm，最大发射峰为580。利用Synergy Neo2多功能酶标仪检测该复合物在524±10nm激发、580nm±10nm发射条件下的荧光强度为5720，而对照(1μM IV-39荧光团分子)在相同检测条件下的荧光强度为20，那么Carrot-1核酸适配体对IV-39荧光团分子的激活倍数为286倍。
实施例1.Carrot核酸适配体分子的二级结构
利用mFold在线RNA结构分析软件预测分析Carrot核酸适配体通式的二级结构(图1)，并不代表其真实的二级结构。对于其中两种给定N 1、N 14-N 15-N 16和N 23的Carrot-1和Carrot-2(SEQ ID NO：1、2)，它们预测的二级结构为图2。
实施例2.Carrot-IV-39复合物性质鉴定
为了检测Carrot-IV-39复合物的光谱性质，按照常用实验方法(一)制备Carrot-1 (SEQ ID NO：2)RNA。将1μM IV-39与5μM Carrot-1孵育。检测结果显示，Carrot-IV-39复合物的最大激发光为524nm，最大发射光为580nm(图5a)。
为了检测Carrot-1与IV-39结合的结合常数，利用20nM的Carrot-1与不同浓度的IV-39孵育，检测它们的荧光值。检测结果显示Carrot-1与IV-39结合的结合常数为58nM(图5b)。
为了检测Carrot-IV-39复合物对Mg 2+离子的依赖性，将Carrot-1-IV-39复合物置于含不同Mg 2+离子的缓冲液中，检测荧光值。检测结果显示，相对于Pepper599和Corn-DFHO，Carrot-1-IV-39复合物对Mg 2+离子的依赖性更低(图5c)。
实施例3.不同Carrot突变体对IV-39荧光团分子的荧光激活效果。
为了检测不同Carrot突变体对IV-39荧光团分子的荧光激活效果，对Carrot-1序列进行如表1所示的点突变，按照常用实验方法(一)制备含不同碱基突变的Carrot突变体RNA，将1μM IV-39分别与5μM不同的Carrot-1突变体RNA孵育，按照常用实验方法(五)检测它们对IV-39荧光团分子的荧光激活倍数。检测结果显示，部分含单个碱基突变的Carrot-1突变体保留对IV-39的较强的荧光激活效果(>20倍)(表2)。部分含2-5个碱基突变的Carrot-1突变体仍保留对IV-39一定的荧光激活效果(>10倍)(表3)。综上，Carrot-1的很多单碱基和多碱基突变体仍保留激活IV-39荧光团分子的适配体功能。
表2 含单碱基突变的Carrot-1突变体对IV-39的激活效果
突变体 激活倍数 突变体 激活倍数 突变体 激活倍数
Carrot-1 286 G7U 196 C12A 234
A4U 86 U8C 87 C12U 156
A4G 95 A10U 65 A13U 105
A4C 205 A10G 78 A13G 63
U5A 215 A10C 62 A13C 311
U5G 249 A11U 43 G17A 78
U5C 277 A11G 187 C19A 42
G7C 117 A11C 35 C19U 69
G7A 75 C12G 195 A20C 47
注：表2中的Carrot-1是序列为SEQ ID NO：1的核酸适配体；其它的适配体是在Carrot-1序列中与图1的Carrot对应核苷酸位置做的点突变。
表3 含多碱基突变的Carrot-1突变体对IV-39的激活效果
Figure PCTCN2021083579-appb-000014
实施例4.碱基修饰的Carrot对IV-39的激活效果
为了检测经碱基修饰的Carrot对IV-39的激活效果，合成含碱基修饰的Carrot-3(SEQ ID NO：5，该序列
Figure PCTCN2021083579-appb-000015
中粗体带下划线的碱基为脱氧核糖核苷酸碱基)和Carrot-4(SEQ ID NO：6，该序列
Figure PCTCN2021083579-appb-000016
中粗体带下划线的碱基为经2’-F修饰的碱基)(图6a)(由上海吉玛制药技术有限公司合成)。按照常用实验方法(五)检测这些碱基修饰的Cattor对IV-39荧光团分子的荧光激活效果。检测结果显示，经碱基修饰的Carrot-3和Carrot-4依然可以显著激活IV-39荧光团分子的荧光(图6b)。
实施例5.Carrot串联体
为了检测Carrot串联体对IV-39荧光团分子的荧光激活效果，将Carrot-2按照不同的形式进行串联，包含以下三种：
(1)“串联1”方式(图7a)，将Carrot结构上的“头”和“尾”按照“头-尾”连接的方式进行连接，以此获得nCarrot(其中n为任意拷贝的Carrot)。在本实施例中，分别全基因合成F30-2Carrot-2、F30-4Carrot-2、F30-6Carrot-2、F30-8Carrot-2和F30-10Carrot-2的编码cDNA(编码RNA适配体的序列分别为SEQ ID NO：7，SEQ ID NO：8，SEQ ID NO：9，SEQ ID NO：10，SEQ ID NO：11)，PCR扩增后，按照常用实验方法(一)制备核酸适配体RNA，将0.2μM RNA适配体与5μM IV-39孵育后，按照常用实验方法(五)检测荧光强度。检测结果显示，随着n的增加，F30-nCarrot-IV-39的荧光也随之增加(图 7d),说明可以通过“串联1”方式提高Carrot-IV-39复合物的荧光强度。
(2)“串联2”方式(图7b)，将Carrot作为一个结构单元进行串联，以此获得n×Carrot(其中n为可为任意拷贝的Carrot)。在本实施例中，分别全基因合成2×Carrot-2、4×Carrot-2、6×Carrot-2、8×Carrot-2和10×Carrot-2的编码cDNA(编码RNA适配体的序列分别为SEQ ID NO：12，SEQ ID NO：13，SEQ ID NO：14，SEQ ID NO：15，SEQ ID NO：16)，按照常用实验方法(一)制备核酸适配体RNA，将0.2μM RNA适配体与5μM IV-39孵育后，按照常用实验方法(五)检测荧光强度。检测结果显示，随着n的增加，n×Carrot-IV-39的荧光也随之增加(图7e)，说明可以通过“串联2”方式提高Carrot-IV-39复合物的荧光强度。
(3)“串联3”方式(图7c)，是将上述“串联1”与“串联2”结合起来，将“串联1”得到的nCarrot作为一个结构单元按照“串联2”的方式进行串联，以此获得n2×n1Carrot(其中n1和n2为可为任意拷贝的Carrot)。在本实施例中，分别全基因合成2×2Carrot-2、2×4Carrot-2、4×2Carrot-2、4×4Carrot-2的编码cDNA(编码RNA适配体的序列分别为SEQ ID NO：17，SEQ ID NO：18，SEQ ID NO：19，SEQ ID NO：20)，按照常用实验方法(一)制备核酸适配体RNA，将0.2μM RNA适配体与5μM IV-39孵育后，按照常用实验方法(五)检测荧光强度。检测结果显示，这种通过“串联3”方式得到的Carrot串联体-IV-39的荧光强度要显著高于Carrot-2-IV-39(图7f)，说明可以通过“串联3”方式提高Carrot-IV-39复合物的荧光强度。
实施例6.IV-39类似物的性质鉴定
按照常用实验方法(一)制备Carrot-1 RNA适配体分子，利用其检测IV-39类似物与Carrot结合的基本性质，包括荧光光谱、摩尔消光系数、量子产率和荧光激活倍数，检测结果如表4所示，从表中数据可以看出，Carrot-1可以不同程度地激活IV-39类似物的荧光强度。
表4：Carrot-1 RNA适配体分子与不同荧光分子结合的理化性质测定
Figure PCTCN2021083579-appb-000017
Figure PCTCN2021083579-appb-000018
实施例7.Carrot-IV-4复合物用于细菌中RNA的标记
为了检测Carrot-IV-4在细菌中的效果，首先构建表达F30-Carrot-2的细菌表达质粒。全基因合成F30-Carrot-2编码DNA序列，利用引物对其进行扩增，利用引物对pET28a进行扩增去除了启动子和多克隆位点区，将扩增得到的F30-Carrot-2 DNA片段与pET28a线性化载体按照实验方法(四)进行连接，得到的重组质粒命名为pET28a-T7-F30-Carrot-2。
扩增F30-Carrot-2片段所用的引物为：
上游引物(P1)：5’-TAATACGACTCACTATAGGGTTGCCATGTGTATGTGGGA-3’
下游引物(P2)：5’-CAAGGGGTTATGCTATTGCCATGAATGATCC-3’
扩增pET28a载体使其线性化所用的引物为：
上游引物(P3)：5’-TAGCATAACCCCTTGGGGCCTC-3’
下游引物(P4)：5’-TAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCG-3’
将pET28a-T7-F30-Carrot-2重组质粒转化BL21(DE3,Star)大肠杆菌菌株，挑取单克隆37℃培养，在OD 600＝0.2左右时，加入1mM IPTG诱导F30-F30-Carrot-2的表达，4h后收菌，利用含1μM IV-4的PBS(含10mM Mg 2+)溶液重悬。以转化了pET28a空载体的BL21(DE3,Star)大肠杆菌作为对照。结果显示，只有表达了F30-F30-Carrot-2且 在IV-4存在下，细菌才能显示出明亮的橙红色荧光(图8)，表明Carrot-IV-4复合物可以用于细菌中RNA的荧光标记。
实施例8 Carrot-IV-4复合物用于酵母细胞中RNA的标记
为了检测Carrot-IV-4在酵母中的效果，首先构建表达F30-Carrot-2的酵母表达质粒。利用引物对实施例7中的F30-Carrot-2 DNA片段进行扩增，按照实验方法(四)将扩增得到的F30-Carrot-2片段插入到pYES2.1 TOPO TA载体中，得到的重组质粒命名为pYES2.1-F30-Carrot-2。
扩增F30-Carrot-2片段所用的引物为：
上游引物(P5)：5’-GGAATATTAAGCTCGCCCTTTTGCCATGTGTATGTGGG-3’
下游引物(P6)：5’-TGACCTCGAAGCTCGCCCTTGTTGCCATGAATGATCC-3’
将pYES2.1-F30-Carrot-2重组质粒转化BY4741菌株，挑取单克隆30℃培养，在OD 600＝0.1左右时，加入1mM半乳糖诱导F30-Carrot-2的表达，10h后收菌，利用含1μM IV-4的PBS(含10mM Mg 2+)溶液重悬。以未经处理的BY4741菌株为对照。成像结果显示，只有表达了F30-Carrot-2且在IV-4存在下，酵母细胞才能显示出明亮的橙红色荧光(图9)，表明Carrot-IV-4复合物可以用于酵母细胞中RNA的荧光标记。
实施例9 Carrot与IV-39及其类似物用于哺乳动物细胞中RNA的标记
为了检测Carrot与IV-39用于哺乳动物细胞中RNA的标记，以已报道的Pepper和Corn核酸适配体分子(分别结合HBC599和DFHO荧光团分子)作为对照(Chen et al.Nature Biotechnology 2019.37:1287-1293；Song et al.Nature Chemical Biology 2017.13:1187-1194)，构建它们的哺乳动物细胞表达质粒。全基因合成tRNA-Carrot-2(编码的RNA序列为SEQ ID No：4)、tRNA-Pepper(编码的RNA序列为SEQ ID No：22)和tRNA-Corn(编码的RNA序列为SEQ ID No：21)的编码DNA片段，分别引物P7和P8对这些片段进行PCR扩增，利用引物P9和P10对pEGFP-N1载体进行扩增，去除其本身的CMV启动子和多克隆位点区。利用实验方法(四)分别将这些片段插入到去除了启动子和多克隆位点区的pEGFP-N1载体中。再利用P11和P12扩增这些载体使其线性化，利用P13和P14以pLKO.1puro载体为模板扩增U6启动子(SEQ ID No：23)，利用实验方法(四)分别将U6启动子插入到线性化的载体中，得到的质粒命名为pU6-tRNA-Carrot-2、pU6-tRNA-Pepper和pU6-tRNA-Corn，这些质粒分别表达tRNA-Carrot-2、tRNA- Pepper和tRNA-Corn。将pU6-tRNA-Carrot-2、pU6-tRNA-Pepper和pU6-tRNA-Corn质粒转染293T/17细胞，24h后加入0.5μM IV-39，0.5μM HBC599和10μM DFHO分别对tRNA-Carrot-2、tRNA-Pepper和tRNA-Corn进行标记，以不表达相应RNA适配体的细胞作为对照，利用实验方法(三)检测标记效果。结果显示，无论在检测缓冲液中加不加入20mM Mg 2+，tRNA-Carrot-IV-39复合物展现出非常明亮的橙红色荧光(图10a)，荧光强度明显高于tRNA-Pepper-HBC599和tRNA-Corn-DFHO复合物(图10b)，表明Carrot-IV-39可以很好的在哺乳动物细胞中工作。
扩增tRNA-Carrot-2、tRNA-Pepper和tRNA-Corn所用的引物为：
上游引物(P7)：5’-GCCGCCCCCTTCACCTCTAGAGCCCGGATAGCTCAGTCGG-3’
下游引物(P8)：5’-GAGAATTCAAAAAAATGGCGCCCGAACAGGGAC-3’
扩增pEGFP-N1去除启动子和多克隆位点区所用的引物为：
上游引物(P9)：5’-TTTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCA-3’
下游引物(P10)：5’-GGTGAAGGGGGCGGCCGCTCGAGG-3’
扩增载体使其线性化引入U6启动子所用的引物为：
上游引物(P11)：5’-TCTAGAGCCCGGATAGCTCAGTCGGT-3’
下游引物(P12)：5’-GGTGAAGGGGGCGGCCGCTCGAGG-3’
扩增U6启动子所用的引物为：
上游引物(P13)：5’-GCCGCCCCCTTCACCGAGGGCCTATTTCCCATG-3’
下游引物(P14)：5’-TATCCGGGCTCTAGAGTTTCGTCCTTTCCACAAGATATAT-3’
为了检测Carrot与IV-39类似物用于哺乳动物细胞中RNA的标记，构建了表达F30-4Carrot-2的哺乳动物表达质粒。利用本实施例中的引物P15和P16对实施例5中的F30-4Carrot-2片段进行扩增，利用实验方法(四)将这些片段插入到经XbaI和EcoRI酶切的pU6-tRNA-Carrot-2载体中，得到的表达载体命名为pU6-F30-4Carrot-2。
扩增F30-4Carrot-2片段所用的引物为：
上游引物(P15)：5’-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGGA-3’
下游引物(P16)：5’-TGTCTCGAGGTCGAGAATTCAAAAAAATTGCCATGAATGATCCCGAAG-3’
将pU6-F30-4Carrot-2质粒转染293T/17细胞，24h后加入不同的IV-39类似物进行标记，利用实验方法(三)检测标记效果。结果显示，不同的IV-39类似物均可以特异 性标记表达F30-4Carrot-2的细胞，而不标记不表达F30-4Carrot-2的对照细胞(图10c)，表明Carrot与IV-39及其类似物可用于哺乳动物细胞中RNA的标记。
实施例10.基于Carrot的探针构建
为了构建基于Carrot的待检测物探针，将Carrot-1(SEQ ID No：1)通式中的N 14-N 15-N 16核苷酸更换为可以特异性识别结合腺苷(adenosine)和S-腺苷蛋氨酸(SAM)的RNA适配体，这些适配体与Carrot-1之间利用合适的碱基进行连接(图11a)，按照常用实验方法(一)制备探针RNA，将其与IV-39孵育，利用多功能酶标仪分别检测它们在有无腺苷或SAM存在下的荧光强度。检测结果显示，这些探针在靶标存在时的荧光要显著高于没有靶标时荧光(图11b)，说明它们可以分别作为检测腺苷和SAM的探针，对应的探针RNA序列为SEQ ID No：24和SEQ ID No：25。
实施例11.Carrot用于示踪细胞中RNA定位
为了检测Carrot用于示踪细胞中RNA定位，首先构建Carrot与不同RNA融合的嵌合RNA的表达质粒。利用引物P17和P18扩增4Carrot-2片段，利用实验方法(四)将插入到经HindIII和XhoI双酶切的pCDNA3.1 hygro(+)载体，得到pCDNA3.1 hygro(+)-4Carrot-2重组质粒。全基因合成GAPDH和ACTB基因片段(GAPDH和ACTB编码基因序列分别为Genebank:BC009081,BC016045)，分别利用引物对GAPDH和ACTB基因片段进行扩增，将它们插入到经NheI和HindIII双酶切的pCDNA3.1 hygro(+)-4Carrot-2载体中，获得pCDNA3.1 hygro(+)-GAPDH-4Carrot-2和pCDNA3.1 hygro(+)-ACTB-4Carrot-2重组质粒，它们分别编码GAPDH-4Carrot-2和ACTB-4Carrot-2嵌合RNA，它们的序列为SEQ ID No：26和27
扩增4Carrot-2所用的引物为：
上游引物(P17)：5’-TAGCGTTTAAACTTAAGCTTGGAAGATTGTAAACACGCC-3’
下游引物(P18)：5’-ACGGGCCCTCTAGACTCGAGGGAAGTGTCCGCCGGAAGT-3’
扩增GAPDH所用的引物为：
上游引物(P19)：5’-GGAGACCCAAGCTGGCTAGCATGGGGAAGGTGAAGGTCGG-3’
下游引物(P20)：5’-CACGGACACATGGCAAGCTTAACCATGCTCTAGCGAGTGTTACTCCTTGGAGGCCA TGT-3’
扩增ACTB所用的引物为：
上游引物(P21)：5’-GGAGACCCAAGCTGGCTAGCATGGTGACGCTT GCTGAACT-3’
下游引物(P22)：5’-CACGGACACATGGCAAGCTTAACCATGCTCTAGCGAGTGCTAGAAGCATTTGCGGTGGA-3’
构建完成以上质粒后，均经测序鉴定插入的序列完全正确，用转染级质粒抽提试剂盒提取质粒，用于后续转染实验。
将本实施例构建的pCDNA3.1 hygro(+)-GAPDH-4Carrot-2和pCDNA3.1 hygro(+)-ACTB-4Carrot-2重组质粒分别转染COS-7细胞，转染24h后加入1μM IV-4近标记，按照具体实验方法(三)所述荧光成像方法对细胞进行成像。成像结果显示，GAPDH-4Carrot-2和ACTB-4Carrot-2的荧光主要集中在胞浆中(图12)，这与之前的报道是一致的，与荧光标记的原位杂交技术(FISH)结果也是一致的(图12)，以上结果显示Carrot可用于示踪RNA的定位。
实施例12.Carrot用于检测基因组DNA
为了利用Carrot检测基因组DNA，首先构建表达Carrot-2与sgRNA嵌合RNA的重组质粒。全基因合成含着丝粒靶向序列的sgRNA-Carrot-2-1、sgRNA-Carrot-2-2和sgRNA-Carrot-2-3的cDNA，其编码的RNA序列分别为SEQ ID No：28、29和30。利用引物P23和P24扩增上述嵌合RNA的cDNA，利用引物P25和P26扩增psgRNA质粒(Shao et al.Nucleic acids research 2016.44:e86)，利用实验方法(四)将扩增得到的cDNA与线性化的psgRNA载体连接，得到的质粒分别命名为psgRNA-Carrot-2-1、psgRNA-Carrot-2-2和psgRNA-Carrot-2-3(图13a)。利用引物P27和P28以pSLQ1645(dCas9-GFP)(Shao et al.Nucleic acids research 2016.44:e86)为模板扩增dCas9-GFP基因片段，利用实验方法(四)将其插入到经HindIII和XhoI双酶切的pCDNA3.1hygro(+)载体中，得到的质粒命名为pCDNA3.1 hygro(+)-dCas9-GFP。
扩增Carrot与sgRNA嵌合RNA对应cDNA所用的引物为：
上游引物(P23)：5’-AAAGGACGAAACACCGAATCTGCAAGTGGATATTGTTTGAG-3’
下游引物(P24)：5’-TGATCTAGAAAAAAAGCACCGACTCGGTGCCAC-3’
扩增psgRNA质粒使其线性化的引物为：
上游引物(P25)：5’-TTTTTTTCTAGATCATAATCAGCCATACC-3’
下游引物(P26)：5’-GGTGTTTCGTCCTTTCCACAAG-3’
扩增SpdCas9-GFP所用的引物为：
上游引物(P27)：5’-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCACCATGGCCCC-3’
下游引物(P28)：5’-ACGGGCCCTCTAGACTCGAGTTACTTGTACAGCTCGTCCATGC-3’
将pCDNA3.1 hygro(+)-dCas9-GFP分别与psgRNA-Carrot-2-1、psgRNA-Carrot-2-2和psgRNA-Carrot-2-3重组质粒共转染COS-7细胞，转染24h后，利用1μM IV-4和Hoechst对细胞进行标记，利用荧光显微镜观察Carrot-2-IV-4复合物、GFP和Hoechst的荧光。成像结果显示，Carrot-2-IV-4的荧光在主要集中在细胞核中，且成点状聚集(着丝粒)，与dCas9-GFP的荧光完全吻合(图13b)，说明Carrot可用于对基因DNA的成像。
实施例13.Carrot用于检测RNA-蛋白质间的相互作用
为了利用Carrot检测RNA-蛋白质间的相互作用，以噬菌体衣壳蛋白MCP识别结合序列MS2 RNA为例。全基因合成Carrot-1-MS2片段(编码的RNA序列为SEQ ID No：31)，利用引物P29和P30以其为模板进行扩增，将得到的片段利用实验方法(四)插入到经XbaI和EcoRI双酶切的pU6-tRNA-Carrot-2中，得到的质粒命名为pU6-Carrot-1-MS2.全基因合成tdMCP蛋白编码基因片段(为MCP蛋白的二聚体形式，其编码DNA序列为SEQ ID No：32)和NanoLuc蛋白编码基因片段(其编码DNA序列SEQ ID No：33)，分别利用引物P31和P32、P33和P34分别扩增tdMCP基因片段，利用P35和P36扩增NanoLuc基因片段，利用实验方法(四)将扩增得到的片段插入到经HindIII和XhoI双酶切的pCDNA3.1 hygro(+)载体中，得到的质粒命名为pCDNA3.1 hygro(+)-tdMCP-NanoLuc-tdMCP，其编码的融合蛋白的基因序列为SEQ ID No：34。
扩增Carrot-1-MS2片段所用的引物为：
上游引物(P29)：5’-GGAAAGGACGAAACTCTAGAGGGAAGATTGTAAACAC-3’
下游引物(P30)：5’- TGTCTCGAGGTCGAGAATTCAAAAAAAGGGAAGTGTCCGATGGG-3’
扩增tdMCP所用的引物为：
上游引物(P31)：5’-TAGCGTTTAAACTTAAGCTT ATGCTAGCCGTTAAAATGGC-3’
下游引物(P32)：5’-ACTCCCTCCGCCACCTCCAGAATCCGCGTAGATGC-3’
扩增tdMCP所用的另一套引物为：
上游引物(P33)：5’-ACTCCCTCCCGCCAGAATGCGTTCGCAC-3’
下游引物(P34)：5’-ACGGGCCCTCTAGACTCGAGTTATCCAGAATCCGCGTAG-3’
扩增NanoLuc所用的引物为：
上游引物(P35)：5’-GGTGGCGGAGGGAGTATGGTCTTCACACTCGAAGA-3’
下游引物(P36)：5’-ACTCCCTCCCGCCAGAATGCGTTCGCAC-3’
将pCDNA3.1 hygro(+)-tdMCP-NanoLuc-tdMCP与pU6-Carrot-1-MS2重组质粒共转染293T/17细胞，转染24h后，先加入1μM IV-4荧光团对Carrot进行标记，然后在培养基中加入20μM Furimazine后立即成像。成像结果显示，由于tdMCP-NanoLuc-tdMCP融合蛋白中的tdMCP蛋白会识别结合Carrot-1-MS2嵌合RNA中的MS2 RNA，使得Carrot-IV-4复合物与NanoLuc蛋白间的距离很近，NanoLuc催化底物Furimazine发射的光就会转移给Carrot-IV-4复合物，使得发光能量转移(BRET)的效率较高，进而激发Carrot-IV-4复合物产生明显的橙红色荧光(图14)；作为对照，不含MS2的单独Carrot-1RNA不会与tdMCP-NanoLuc-tdMCP融合蛋白发生相互作用，Carrot-IV-4复合物与NanoLuc蛋白间的距离较远，NanoLuc催化底物Furimazine发射的光就不会转移给Carrot-IV-4复合物，使得发光能量转移(BRET)的效率较低，不能激发Carrot-IV-4复合物产生明显的橙红色荧光(图14)。
实施例14.Carrot用于RNA的提取与纯化的标签
将实施例11中的pCDNA3.1 hygro(+)-GAPDH-4Carrot-2重组质粒转染COS-7细胞，24h后收集细胞，利用40mM HEPES,pH 7.4,125mM KCl,5mM MgCl 2的缓冲液进行重悬(冰上操作)。取Activated Thiol Sepharose 4B(GE Healthcare)经500μL PBS清洗两次后，加入含10mM TCEP(Sigma)的PBS室温孵育1h。利用500μL PBS清洗两次后，加入含马来酰胺的IV-39荧光团分子(Mal-IV-39)于室温反应30min，利用500μL PBS清洗三次。将重悬的细胞破碎后与上述处理过的微珠4℃孵育，30min后4000rpm 离心2min，弃上清，利用预冷的40mM HEPES,pH 7.4,125mM KCl,5mM MgCl 2的缓冲液清洗琼脂糖微珠6次，每次均离心去上清。用DEPC水重选微珠，70℃处理10min，4000rpm离心2min，收集上清。在收集的上清中加入1/10体积的NaAc，2.5倍体积无水乙醇，-80℃冰箱放置20min，4℃条件下14000rpm离心10min，留沉淀，弃上清，利用预冷的70％乙醇溶液清洗沉淀，4℃条件下14000rpm离心10min，留沉淀，弃上清，如此反复一次。将沉淀置于室温下5min，待酒精挥发完后，加入少量体积的DEPC水重悬沉淀。
分别检测细胞破碎后上清液，以及最终高温洗脱后的洗脱液与荧光团IV-39孵育后的荧光，以空白细胞的破碎上清为对照。检测结果显示，洗脱液与IV-39孵育后的荧光显著高于上样前的破碎后上清液(图15)，说明GAPDH-4Carrot-2 RNA得到很好的富集，表明Carrot可以作为一个标签用于RNA的分离与纯化。
实施例15.Ⅳ-39及其类似物的合成
化合物Ⅳ-1：
Figure PCTCN2021083579-appb-000019
化合物1(0.504g,2mmol)、对氰基苯甲醛(0.626g,5mmol)于250ml圆底烧瓶中，加入100ml四氢呋喃溶解，Ar保护条件下加入无水氯化锌(0.545g,4mmol)，体系于80℃条件下油浴加热回流，TLC检测反应完毕，真空除去溶剂，残余物经柱色谱分离得目标产物(0.292g，40％)。 1H NMR(400MHz,DMSO-d 6)δ11.07(s,1H),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).MS(ESI):m/z Calcd.For C 20H 13F 2N 3O 2 365.0976；found 364.0902,[M-H] -.
化合物Ⅳ-2：
Figure PCTCN2021083579-appb-000020
按照化合物Ⅳ-1的合成步骤，(0.475g，65％)。 1H NMR(400MHz,DMSO-d 6)δ11.04(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 C 20H 13F 2N 3O 2 365.0976；found 364.0903,[M-H] -.
化合物Ⅳ-3：
Figure PCTCN2021083579-appb-000021
按照化合物Ⅳ-1的合成步骤，(0.221g，31％)。 1H NMR(400MHz,DMSO-d 6)δ10.94(s,1H),10.11(s,1H),8.07–8.01(m,2H),7.95(d,J＝15.7Hz,1H),7.74(d,J＝8.7Hz,2H),7.01(d,J＝15.7Hz,1H),6.90(s,1H),6.87–6.83(m,2H),3.26(s,3H).MS(ESI):m/z Calcd.For C 19H 14F 2N 2O 3 356.0972；found 355.0901,[M-H] -.
化合物Ⅳ-4：
Figure PCTCN2021083579-appb-000022
按照化合物Ⅳ-1的合成步骤，(0.207g，29％)。 1H NMR(400MHz,DMSO-d 6)δ9.70(s,1H),8.05(d,J＝8.9Hz,2H),7.98-7.89(m,1H),7.30(t,J＝6.2Hz,1H),7.26(d,J＝8.9Hz,1H),7.15(d,J＝15.8Hz,1H),6.97(s,1H),6.87(d,J＝7.5Hz,1H),3.27(s,3H).MS(ESI):m/z Calcd.For C 19H 14F 2N 2O 3 356.0972；found 355.0900,[M-H] -.
化合物Ⅳ-5：
Figure PCTCN2021083579-appb-000023
按照化合物Ⅳ-1的合成步骤，(0.186g，26％)。 1H NMR(400MHz,DMSO-d 6)δ8.16 (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).MS(ESI):m/z Calcd.For C 19H 13F 3N 2O 2 358.0929；found 357.0856,[M-H] -.
化合物Ⅳ-6：
Figure PCTCN2021083579-appb-000024
按照化合物Ⅳ-1的合成步骤，(0.222g，31％)。 1H NMR(400MHz,DMSO-d 6)δ11.02(s,1H),8.09-8.03(m,2H),8.01(d,J＝15.8Hz,1H),7.85(dt,J＝10.6,2.1Hz,1H),7.72(d,J＝7.8Hz,1H),7.51(td,J＝8.0,6.1Hz,1H),7.34(d,J＝15.9Hz,1H),7.28(td,J＝8.7,2.7Hz,1H),7.00(s,1H),3.28(s,3H).MS(ESI):m/z Calcd.For C 19H 13F 3N 2O 2 358.0929；found 357.0857,[M-H] -.
化合物Ⅳ-7：
化合物2：
Figure PCTCN2021083579-appb-000025
化合物Ⅳ-5(0.716g,2.0mmol)、叔丁基二甲基氯硅烷(0.450g,3.0mmol)、咪唑(0.204g,3.0mmol)于100ml圆底烧瓶中，加入50ml干燥二甲基甲酰胺溶解，Ar保护条件下室温搅拌，TLC检测，反应完毕体系倒入150ml水中，二氯甲烷萃取三次，合并有机相，无水硫酸钠干燥，过滤，减压除去溶剂，参与物经柱色谱分离得化合物2(0.927g,98％)。 1H NMR(400MHz,DMSO-d 6)δ8.16(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 25H 28F 3N 2O 2Si 473.2；found 473.2,[M+H] +.
Figure PCTCN2021083579-appb-000026
化合物2(0.473g,1mmol)、劳森试剂(0.808g,2mmol)于250ml三口瓶中，加入100ml甲苯溶解，加入苯胺2滴，Ar保护条件下油浴加热回流，TLC检测，反应完毕减压除去溶剂，残余物溶解于50ml二氯甲烷中，加入四丁基氟化胺(0.313g,1.2mmol)，Ar保护条件下室温搅拌，TLC检测，反应完毕减压除去溶剂，残余物经柱色谱分离得化合物Ⅳ-7(0.209g,56％)。 1H NMR(400MHz,DMSO-d 6)δ8.17(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 19H 13F 3N 2NaOS 397.0598；found 397.0597,[M+Na] +.
化合物Ⅳ-8：
化合物3：
Figure PCTCN2021083579-appb-000027
按照化合物2的合成步骤，(0.932g，99％)。 1H NMR(400MHz,DMSO-d 6)δ8.10(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 26H 28F 2N 3O 2Si 480.2；found 480.2,[M+H] +.
Figure PCTCN2021083579-appb-000028
按照化合物2的合成步骤，(0.332g，49％)。 1H NMR(400MHz,DMSO-d 6)δ11.00(s,1H),8.11(d,J＝8.5Hz,2H),8.07(dd,J＝7.8,2.1Hz,2H),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).HMS(ESI):m/z Calcd.For C 20H 13F 2N 3NaOS 404.0645；found 404.0646,[M+Na] +.
化合物Ⅳ-9：
化合物5：
Figure PCTCN2021083579-appb-000029
3-氟-4-羟基-苯甲醛(0.560g,4.0mmol)于100ml圆底烧瓶中，加入40ml无水乙醇溶解，加入10g无水硫酸钠、5ml 33％甲胺水溶液，Ar保护条件室温搅拌24h，过滤，加压除去有机溶解，残余物溶解于10ml无水乙醇中，加入化合物4(0.790g,5.0mmol)，Ar保护条件下室温搅拌过夜，次日过滤，冷乙醇冲洗三次得化合物5(0.796g，85％)。 1H NMR(400MHz,DMSO-d 6)δ10.52(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 12H 10FN 2O 2 234.2；found 234.2,[M-H] -.
Figure PCTCN2021083579-appb-000030
按照化合物Ⅳ-1的合成步骤，(0.239g，21％)。 1H NMR(400MHz,DMSO-d 6)δ10.52(s,1H),8.71(s,1H),8.19(m,1H),7.84(d,J＝8.0Hz,1H),7.76(m,1H),7.67(d,J＝8.4Hz,1H),7.32(m,1H),6.99(t,J＝8.8Hz,1H),6.89(s,1H),6.78(m,1H),2.34(s,3H).HR-MS(ESI):m/z Calcd.For C 19H 12FN 4O 2 347.0950；found 347.0951,[M-H] -.
化合物Ⅳ-10：
化合物6：
Figure PCTCN2021083579-appb-000031
按照化合物5的合成步骤，(0.812g，91％)。 1H NMR(400MHz,CD 3OD)δ7.28(s,1H),7.19(d,J＝8.0Hz,2H),3.59(t,J＝5.6Hz,3H),3.12(s,3H),1.23(q,J＝5.6Hz,3H).MS(ESI):m/z Calcd.For C 13H 11ClFN 2O 2 281.0；found 281.0,[M-H] -.
Figure PCTCN2021083579-appb-000032
按照化合物Ⅳ-1的合成步骤，(0.239g，21％)。 1H NMR(400MHz,CD 3OD)δ7.84(s,2H),7.28(s,1H),7.19(d,J＝8.0Hz,2H),3.12(s,3H),1.23(q,J＝5.6Hz,3H).HR-MS(ESI):m/z Calcd.For C 19H 12ClFN 5O 2 396.0669；found396.0668,[M-H] -.
化合物Ⅳ-11：
化合物7：
Figure PCTCN2021083579-appb-000033
按照化合物5的合成步骤，(0.812g，91％)。 1H NMR(400MHz,CD 3OD)δ7.28(s,1H),7.19(d,J＝8.0Hz,2H),3.81(s,3H),3.12(s,3H),3.12(s,3H),1.58(m,1H),1.11(d,6H).MS(ESI):m/z Calcd.For C 15H 15BrFN 2O 2 353.0；found 3.0,[M-H] -.
Figure PCTCN2021083579-appb-000034
按照化合物Ⅳ-1的合成步骤，(0.209g，22％)。 1H NMR(400MHz,CD 3OD)δ7.95(d,J＝15.7Hz,1H),7.74(d,J＝8.7Hz,2H),7.28(s,1H),7.19(d,J＝8.0Hz,2H),7.01(d,J＝15.7Hz,1H),6.87–6.83(m,2H),3.12(s,3H),1.58(m,1H),1.11(d,6H).HR-MS(ESI):m/z Calcd.For C 21H 16BrFN 5O 2 468.0477；found468.0478,[M-H] -.
化合物Ⅳ-12：
化合物8：
Figure PCTCN2021083579-appb-000035
按照化合物5的合成步骤，(0.812g，91％)。 1H NMR(400MHz,CD3OD)δ10.52(s,1H),7.76(d,J＝8.5Hz,2H),6.95(s,1H),3.79(t,J＝5.2Hz,2H),3.68-3.56(m,8H),3.55-3.48(m,2H),3.35(s,3H),2.39(s,3H).MS(ESI):m/z Calcd.For C 20H 25FIN 2O 6 535.1；found 535.1,[M-H] -.
Figure PCTCN2021083579-appb-000036
按照化合物Ⅳ-1的合成步骤，(0.156g，18％)。 1H NMR(400MHz,CD 3OD)δ10.52(s,1H),7.98-7.86(m,3H),7.76(d,J＝8.5Hz,2H),7.31(t,J＝8.9Hz,2H),7.18(d,J＝15.9Hz,1H),6.95(s,1H),3.79(t,J＝5.2Hz,2H),3.68-3.56(m,8H),3.55-3.48(m,2H),3.35(s,3H),2.39(s,3H).HR-MS(ESI):m/z Calcd.For C 27H 28ClFIN 2O 6 657.0670；found657.0671,[M-H] -.
化合物Ⅳ-13：
化合物9：
Figure PCTCN2021083579-appb-000037
按照化合物5的合成步骤，(0.732g，93％)。 1H NMR(400MHz,CD 3OD)δ10.52(s,1H),7.76(d,J＝8.5Hz,2H),6.95(s,1H),4.12(s,3H),3.62(s,3H),2.39(s,3H).MS(ESI):m/z Calcd.For C 14H 12Cl 2N 3O 3 340.0；found 340.0,[M-H] -.
Figure PCTCN2021083579-appb-000038
按照化合物Ⅳ-1的合成步骤，(0.156g，18％)。 1H NMR(400MHz,CD 3OD)δ10.52(s,1H),8.07–8.01(m,2H),7.95(d,J＝15.7Hz,1H),7.76(d,J＝8.5Hz,2H),7.01(d,J＝15.7Hz,1H),6.95(s,1H),6.87–6.83(m,2H),4.12(s,3H),3.62(s,3H),2.39(s,3H).HR-MS(ESI):m/z Calcd.For C 21H 15BrCl 2N 3O 3 505.9679；found205.9680,[M-H] -.
化合物Ⅳ-14：
化合物10：
Figure PCTCN2021083579-appb-000039
按照化合物5的合成步骤，(0.732g，93％)。 1H NMR(400MHz,DMSO-d 6)δ10.52(s,1H),8.19(m,1H),7.76(m,1H),6.99(t,J＝8.8Hz,1H),6.89(s,1H),3.89(s,3H),2.34(s,3H).MS(ESI):m/z Calcd.For C 14H 10ClN 2O 2 273.0；found 273.0,[M-H] -.
Figure PCTCN2021083579-appb-000040
按照化合物Ⅳ-1的合成步骤，(0.156g，18％)。 1H NMR(400MHz,DMSO-d 6)δ10.52(s,1H),8.19(m,1H),8.07–8.01(m,2H),7.95(d,J＝15.7Hz,1H),7.76(m,1H),7.01(d,J＝15.7Hz,1H),6.99(t,J＝8.8Hz,1H),6.89(s,1H),6.87–6.83(m,2H),3.89(s,3H),2.34(s,3H).HR-MS(ESI):m/z Calcd.For C 21H 13ClIN 2O 2 486.9716；found 486.9715,[M-H] -.
化合物Ⅳ-15：
化合物11：
Figure PCTCN2021083579-appb-000041
按照化合物5的合成步骤，(0.732g，93％)。 1H NMR(400MHz,CD3OD)δ10.52(s,1H),8.19(m,1H),7.76(m,1H),6.99(t,J＝8.8Hz,1H),6.89(s,1H),3.29(t,J＝4.4Hz,2H),2.78(t,J＝4.4Hz,2H),2.34(s,3H).MS(ESI):m/z Calcd.For C 13H 12BrN 2O 3 323.0；found 323.0,[M-H] -.
Figure PCTCN2021083579-appb-000042
按照化合物Ⅳ-1的合成步骤，(0.156g，18％)。 1H NMR(400MHz,CD3OD)δ10.52(s,1H),8.19(m,1H),7.95(d,J＝15.7Hz,1H),7.76(m,1H),7.49(m,1H),7.40-7.22(m,3H),7.01(d,J＝15.7Hz,1H),6.99(t,J＝8.8Hz,1H),6.89(s,1H),3.29(t,J＝4.4Hz,2H),2.78(t,J＝4.4Hz,2H),2.34(s,3H).HR-MS(ESI):m/z Calcd.For C 20H 15BrClN 2O 3 444.9960；found 444.6991,[M-H] -.
化合物Ⅳ-16：
化合物12：
Figure PCTCN2021083579-appb-000043
按照化合物5的合成步骤，(0.732g，93％)。 1H NMR(400MHz,CD 3OD)δ10.52(s,1H),7.76(d,J＝8.5Hz,2H),6.95(s,1H),2.42(s,3H),2.39(s,3H).MS(ESI):m/z Calcd.For C 12H 9Br 2N 2O 2 370.9；found 370.9,[M-H] -.
Figure PCTCN2021083579-appb-000044
按照化合物Ⅳ-1的合成步骤，(0.156g，18％)。 1H NMR(400MHz,CD 3OD)δ10.52(s,1H),8.07–8.01(m,1H),7.76(d,J＝8.5Hz,2H),7.97(d,J＝15.7Hz,1H),7.01(d,J＝15.7Hz,1H),6.87–6.83(d,J＝8.2Hz,2H),6.95(d,J＝8.2Hz,2H)),2.39(s,3H).HR-MS(ESI):m/z Calcd.For C 19H 12Br 2FN 2O 3 476.9255；found 476.9256,[M-H] -.
化合物Ⅳ-17：
Figure PCTCN2021083579-appb-000045
按照化合物Ⅳ-1的合成步骤，(0.286g，28％)。 1H-NMR(400MHz,DMSO-d 6):δ8.15(d,J＝8.6Hz,2H),7.82(d,J＝15.8Hz,1H),7.31–7.22(m,2H),7.21–7.17(m,1H),7.10 (d,J＝15.8Hz,1H),6.94(s,1H),6.88–6.73(m,3H),3.59(t,J＝5.8Hz,2H),3.51(t,J＝6.0Hz,2H),3.26(s,3H),3.05(s,3H).HR-MS(ESI):m/z Calcd.For C 22H 24N 3O 3 378.1818；found 378.1819,[M+H] +.
化合物Ⅳ-18：
Figure PCTCN2021083579-appb-000046
按照化合物Ⅳ-1的合成步骤，(0.486g，60％)。 1H-NMR(400MHz,DMSO-d 6):δ8.43(d,J＝1.8Hz,1H),8.16(t,J＝8.5Hz,3H),7.93(d,J＝15.9Hz,1H),7.85(dt,J＝7.8,1.4Hz,1H),7.66(t,J＝7.8Hz,1H),7.40(d,J＝15.9Hz,1H),6.98(s,1H),6.86-6.71(m,2H),3.60(t,J＝5.9Hz,2H),3.52(t,J＝5.9Hz,2H),3.28(s,3H),3.06(s,3H).HR-MS(ESI):m/z Calcd.For C 23H 23N 4O 2 387.1821；found 387.1822,[M+H] +.
化合物Ⅳ-19：
Figure PCTCN2021083579-appb-000047
按照化合物Ⅳ-1的合成步骤，(0.286g，38％)。 1H-NMR(400MHz,DMSO-d 6):δ8.17(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),7.34-7.20(m,2H),6.97(s,1H),6.84-6.73(m,2H),3.59(t,J＝5.9Hz,2H),3.52(t,J＝5.9Hz,2H),3.27(s,3H),3.06(s,3H).MS(ESI):m/z Calcd.For C 22H 23N 3O 3F 380.1774；found 380.1775,[M+H] +.
化合物Ⅳ-20：
Figure PCTCN2021083579-appb-000048
按照化合物Ⅳ-1的合成步骤，(0.222g，30％)。 1H NMR(400MHz,DMSO-d 6):8.14(d,J＝8.5Hz,2H),7.84(d,J＝15.7Hz,1H),7.68(d,J＝8.4Hz,2H),6.97(d,J＝15.8Hz,1H),6.88(s,1H),6.84(d,J＝8.5Hz,2H),6.79(d,J＝8.8Hz,2H),3.59(q,J＝5.4Hz,2H),3.51(t,J＝5.9Hz,2H),3.24(s,3H),3.05(s,3H).MS(ESI):m/z Calcd.For C 22H 24N 3O 3 378.1818；found 378.1819,[M+H] +.
化合物Ⅳ-21：
Figure PCTCN2021083579-appb-000049
按照化合物Ⅳ-1的合成步骤，(0.352g，56％)。 1H-NMR(400MHz,DMSO-d 6):δ8.18(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),3.60(t,J＝5.9Hz,2H),3.52(t,J＝5.6Hz,2H),3.28(s,3H),3.06(s,3H).MS(ESI):m/z Calcd.For C 23H 23N 4O 2 387.1821；found 387.1820,[M+H] +.
化合物Ⅳ-22：
Figure PCTCN2021083579-appb-000050
按照化合物Ⅳ-1的合成步骤，(0.252g，32％)。 1H-NMR(400MHz,DMSO-d 6):δ8.16(d,J＝8.5Hz,2H),7.93(t,J＝4.4Hz,2H),7.90(d,J＝6.1Hz,1H),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 22H 23FN 3O 2 380.1774；found 380.1775,[M+H] +.
化合物Ⅳ-23：
化合物13：
Figure PCTCN2021083579-appb-000051
按照化合物Ⅳ-1的合成步骤，(0.252g，32％)。 1H-NMR(400MHz,DMSO-d 6):δ8.43(d,J＝1.8Hz,1H),8.16(t,J＝8.5Hz,3H),7.93(d,J＝15.9Hz,1H),7.85(dt,J＝7.8,1.4Hz,1H),7.66(t,J＝7.8Hz,1H),7.40(d,J＝15.9Hz,1H),6.98(s,1H),6.86-6.71(m,2H),3.60(t,J＝5.9Hz,2H),3.52(t,J＝5.9Hz,2H),3.28(s,3H),3.06(s,3H).MS(ESI):m/z Calcd.For C 23H 23N 4O 2 387.2；found 387.2,[M+H] +.
化合物14：
Figure PCTCN2021083579-appb-000052
化合物14(0.774g,2.0mmol)、碳酸钾(0.276g,2.0mmol)于250ml圆底烧瓶中，加入100ml乙腈溶解，Ar保护条件下加入2ml溴丙烯，油浴加热回流，TLC检测，反应完毕，过滤，减压除去溶剂，残余物经柱色谱分离得化合物18(0.673g,79％)。 1H-NMR(400MHz,DMSO-d 6):δ8.43(d,J＝1.8Hz,1H),8.16(t,J＝8.5Hz,3H),7.93(d,J＝15.9Hz,1H),7.85(dt,J＝7.8,1.4Hz,1H),7.66(t,J＝7.8Hz,1H),7.40(d,J＝15.9Hz,1H),6.98(s,1H),6.86-6.71(m,2H),3.81(s,2H),3.60(t,J＝5.9Hz,2H),3.52(t,J＝5.9Hz,2H),3.41(s,3H),3.06(s,3H).MS(ESI):m/z Calcd.For C 26H 27N 4O 2 427.2；found 427.2,[M+H] +.
Figure PCTCN2021083579-appb-000053
按照化合物Ⅳ-7的合成步骤，(0.152g，72％)。 1H-NMR(400MHz,DMSO-d 6):δ8.43(d,J＝1.8Hz,1H),8.16(t,J＝8.5Hz,3H),7.93(d,J＝15.9Hz,1H),7.85(dt,J＝7.8,1.4Hz,1H),7.66(t,J＝7.8Hz,1H),7.40(d,J＝15.9Hz,1H),6.98(s,1H),6.86-6.71(m,2H),3.81(s,2H),3.60(t,J＝5.9Hz,2H),3.52(t,J＝5.9Hz,2H),3.41(s,3H),3.06(s,3H).MS(ESI):m/z Calcd.For C 26H 27N 4OS 443.1906；found 443.1905,[M+H] +.
化合物Ⅳ-24：
化合物15：
Figure PCTCN2021083579-appb-000054
按照化合物5的合成步骤，(0.692g，82％)。 1H NMR(400MHz,CD 3OD)δ＝8.02(d,J＝2.4Hz,1H),7.44(dd,J＝8.7Hz,J＝2.4Hz,1H),7.09(s,1H),6.51(d,J＝8.7Hz,1H),3.56(t,J HH＝7.6Hz,2H),3.08(s,6H),1.66(m,2H),2.38(s,3H),0.95(t,J＝7.6Hz,3H).MS(ESI):m/z Calcd.For C 15H 21N 4O 273.2；found 273.2,[M+H] +.
化合物16：
Figure PCTCN2021083579-appb-000055
按照化合物Ⅳ-1的合成步骤，(0.452g，34％)。 1H NMR(400MHz,CD 3OD)δ＝8.02(d,J＝2.4Hz,1H),7.95(m,2H),7.68-7.50(m,1H),7.44(dd,J＝8.7Hz,J＝2.4Hz,1H),7.34-7.06(m,2H),7.09(s,1H),7.00(d,J＝15.7Hz,1H),6.51(d,J＝8.7Hz,1H),3.56(t,J HH＝7.6Hz,2H),3.08(s,6H),1.66(m,2H),2.38(s,3H),0.95(t,J＝7.6Hz,3H).MS(ESI):m/z Calcd.For C 22H 24FN 4O 379.2；found 379.2,[M+H] +.
Figure PCTCN2021083579-appb-000056
按照化合物Ⅳ-7的合成步骤，(0.152g，72％)。 1H NMR(400MHz,CD 3OD)δ＝8.02(d,J＝2.4Hz,1H),7.95(m,2H),7.68-7.50(m,1H),7.44(dd,J＝8.7Hz,J＝2.4Hz,1H),7.34-7.06(m,2H),7.09(s,1H),7.00(d,J＝15.7Hz,1H),6.51(d,J＝8.7Hz,1H),3.56(t,J HH＝7.6Hz,2H),3.08(s,6H),1.66(m,2H),2.38(s,3H),0.95(t,J＝7.6Hz,3H).HR-MS(ESI):m/z Calcd.For C 22H 24FN 4S 395.1706；found 395.1705,[M+H] +.
化合物Ⅳ-25:
化合物17：
Figure PCTCN2021083579-appb-000057
按照化合物5的合成步骤，(0.692g，82％)。 1H NMR(400MHz,CD 3OD)δ＝8.02(d,J＝2.4Hz,1H),7.44(dd,J＝8.7Hz,J＝2.4Hz,1H),7.09(s,1H),6.51(d,J＝8.7Hz,1H),4.21(s,2H),3.86(s,2H),3.00(q,J＝4.8Hz,4H),2.45(s,3H),1.22(t,J＝4.8Hz,6H).MS(ESI):m/z Calcd.For C 17H 23N 4O 3 331.2；found 331.2,[M+H] +.
Figure PCTCN2021083579-appb-000058
按照化合物Ⅳ-1的合成步骤，(0.452g，34％)。 1H NMR(400MHz,CD 3OD)δ＝8.02(d,J＝2.4Hz,1H),7.44(dd,J＝8.7Hz,J＝2.4Hz,1H),7.31(t,J＝8.9Hz,2H),7.18(d,J＝15.9Hz,1H),7.09(s,1H),6.95(s,1H),6.83-6.72(m,2H),6.51(d,J＝8.7Hz,1H),4.21(s,2H),3.86(s,2H),3.00(q,J＝4.8Hz,4H),2.45(s,3H),1.22(t,J＝4.8Hz,6H).MS(ESI):m/z Calcd.For C 24H 26ClN 4O 3 453.1693；found 453.1694,[M+H] +.
化合物Ⅳ-26:
化合物18：
Figure PCTCN2021083579-appb-000059
按照化合物5的合成步骤，(0.892g，80％)。 1H NMR(400MHz,CD 3OD)δ8.41(d,J＝1.5Hz,1H),7.97(d,J＝1.5Hz,1H),7.31(s,1H),5.86(s,1H),3.46(t,J＝6.6Hz,4H),3.15(s,3H),2.32(s,3H),1.61(m,4H),1.32(m,12H),0.89(t,6H).MS(ESI):m/z Calcd.For C 22H 36N 5O 386.3；found 386.3,[M+H] +.
Figure PCTCN2021083579-appb-000060
按照化合物Ⅳ-1的合成步骤，(0.452g，34％)。 1H NMR(400MHz,CD 3OD)δ8.41(d,J＝1.5Hz,1H),7.97(d,J＝1.5Hz,1H),7.85(d,J＝15.7Hz,1H),7.49(m,1H),7.40-7.22(m,3H),7.31(s,1H),7.01(d,J＝15.7Hz,1H),5.86(s,1H),3.46(t,J＝6.6Hz,4H),3.15(s,3H),2.32(s,3H),1.61(m,4H),1.32(m,12H),0.89(t,6H).MS(ESI):m/z Calcd.For C 29H 39ClN 5O 508.2843；found 508.2842,[M+H] +.
化合物Ⅳ-27:
化合物19：
Figure PCTCN2021083579-appb-000061
按照化合物5的合成步骤，(0.812g，81％)。 1H NMR(400MHz,CDCl 3)δ7.93(s,2H),7.31(s,1H),4.24(t,J＝6.8Hz,2H),3.44(s,3H)，2.82(s,2H),2.43(s,3H).MS(ESI):m/z Calcd.For C 14H 17N 6O 285.1；found 285.1,[M+H] +.
Figure PCTCN2021083579-appb-000062
按照化合物Ⅳ-1的合成步骤，(0.312g，26％)。 1H NMR(400MHz,CDCl 3)δ＝8.02(d,J＝15.7Hz,1H),7.93(m,3H),7.68-7.50(m,1H),7.31(s,1H),7.24-7.06(m,2H),7.01(d,J＝15.7Hz,1H),4.24(t,J＝6.8Hz,2H),3.44(s,3H)，2.82(s,2H),2.43(s,3H).MS(ESI):m/z Calcd.For C 21H 20lN 6O 499.0743；found 499.0742,[M+H] +.
化合物Ⅳ-28:
化合物20：
Figure PCTCN2021083579-appb-000063
按照化合物5的合成步骤，(0.572g，80％)。1H NMR(400MHz,CDCl3)δ7.60(s,2H),7.16(s,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),2.39(s,3H).MS(ESI):m/z Calcd.For C16H20N3O2 286.2；found 286.2,[M+H]+.
Figure PCTCN2021083579-appb-000064
按照化合物Ⅳ-1的合成步骤，(0.333g，26％)。 1H NMR(400MHz,CDCl 3)δ8.75(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＝16.0Hz,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 22H 22FN 4O 2 393.1727；found 393.1726,[M+H] +.
化合物Ⅳ-29:
化合物21：
Figure PCTCN2021083579-appb-000065
化合物Ⅳ-21(0.792g,2.0mmol)、对甲苯磺酰氯(0.476g,2.5mmol)和三乙胺(0.303g,3.0mmol)于250ml圆底烧瓶中，加入100ml干燥二氯甲烷溶解，Ar保护条件下室温搅拌，TLC检测反应完毕，体系倒入200ml水中，二氯甲烷萃取三次，合并有机相，无水硫酸钠干燥，过滤，减压除去溶解，残余物经柱色谱分离得化合物27(0.822g，76％)。 1H-NMR(400MHz,DMSO-d 6):δ8.18(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),6.86-6.72(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 30H 29N 4O 4S 541.2；found 541.2,[M+H] +.
Figure PCTCN2021083579-appb-000066
化合物21(0.541g,1.0mmol)、亚硫酸钠(0.630g,5.0mmol)于100ml圆底烧瓶中，加入20ml无水DMF，Ar保护条件下50℃油浴加热反应24h，TLC检测反应完毕，减压除去溶剂，残余物经反相色谱分离得产物(0.301g，60％)。 1H-NMR(400MHz,DMSO-d 6):δ8.18(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),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 23H 21N 4O 4S 499.1289；found 499.1288,[M-H] -.
化合物Ⅳ-30:
化合物22：
Figure PCTCN2021083579-appb-000067
按照化合物5的合成步骤，(0.572g，80％)。 1H NMR(400MHz,CDCl 3)δ7.60(s,2H),7.04(s,1H),6.44(d,J＝8.26Hz,1H),3.60(t,J＝8.46Hz,2H),3.06(dd,J＝18.05,9.48Hz,2H),4.57(q,J＝9.25,2H),2.93(s,3H),2.42(s,3H).MS(ESI):m/z Calcd.For C 16H 17F 3N 3O 324.1；found 324.1,[M+H] +.
Figure PCTCN2021083579-appb-000068
按照化合物Ⅳ-1的合成步骤，(0.433g，21％)。 1H NMR(400MHz,CDCl 3)δ8.75(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),7.11(dd,J＝2.5,8.4Hz,1H),7.04(s,1H),6.95(d,J＝16.0Hz,1H),6.44(d,J＝8.26Hz,1H),3.60(t,J＝8.46Hz,2H),3.06(dd,J＝18.05,9.48Hz,2H),4.57(q,J＝9.25,2H),2.93(s,3H).MS(ESI):m/z Calcd.For C 22H 19BrF 3N 4O 491.0694；found 491.0693,[M+H] +.
化合物Ⅳ-31:
Figure PCTCN2021083579-appb-000069
化合物Ⅳ-21(0.386g,1.0mmol)、化合物31(0.265g,1.2mmol)、EDCI(0.382g,2.0mmol)、DMAP(0.183g,1.5mmol)于100ml圆底烧瓶中，加入30ml无水DMF溶解，Ar保护条件下室温搅拌，TLC检测，反应完毕，减压除去溶剂，残余物经柱色谱分离得产物(0.490g,83％)。 1H-NMR(400MHz,DMSO-d 6):δ8.18(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 32H 39N 4O 7S 591.2819；found 591.2820,[M+H] +.
化合物Ⅳ-32:
化合物31：
Figure PCTCN2021083579-appb-000070
按照化合物5的合成步骤，(0.612g，87％)。 1H NMR(400MHz,CDCl 3)δ8.15(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 18H 25N 4O 3 345.2；found 345.2,[M+H] +.
Figure PCTCN2021083579-appb-000071
按照化合物Ⅳ-1的合成步骤，(0.422g，36％)。 1H NMR(400MHz,CDCl 3)δ8.15(d,J＝9.0Hz,2H),8.14(d,J＝9.0Hz,2H),7.95(d,J＝16.0Hz,1H),7.82(d,J＝8.4Hz,2H),7.32(d,J＝8.4Hz,2H),7.21(s,1H),7.01(d,J＝16.0Hz,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).
MS(ESI):m/z Calcd.For C 25H 28BrN 4O 3 511.1345；found 511.1344,[M+H] +.
化合物Ⅳ-33:
化合物25：
Figure PCTCN2021083579-appb-000072
按照化合物27的合成步骤，(0.912g，89％)。 1H-NMR(400MHz,DMSO-d 6):δ8.17(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),7.34-7.20(m,2H),7.18(d,J＝8.2Hz,2H),6.97(s,1H),6.84-6.73(m,2H),6.47(d,J＝8.2Hz,2H),4.06(t,J＝6.1Hz,2H),3.49(t,J＝6.1Hz,2H),3.27(s,3H),3.06(s,3H),2.77(s,3H),2.31(s,3H).MS(ESI):m/z Calcd.For C 29H 29FN 3O 4S 534.2；found 534.2,[M+H] +.
Figure PCTCN2021083579-appb-000073
化合物25(0.534g,1.0mmol)于100ml圆底烧瓶中，加入35ml DMF溶解，小心加入NaN3(0.195g,3.0mmol)，Ar保护条件下50℃油浴加热过夜，次日体系冷却至室温，倒入100ml水中，DCM萃取三次，合并有机相，饱和食盐水洗涤两次，合并有机相，无水硫酸钠干燥，过滤，减压除去溶剂，残余物无需纯化直接用于下一步反应。
残余物溶于30ml THF中，加入Ph 3P(0.524g,2.0mmol)，2ml水，Ar保护条件下室温搅拌过夜，次日加压除去溶剂，残余物经柱色谱分离的目标产物(0.301g,79％)。 1H-NMR(400MHz,DMSO-d 6):δ8.17(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),7.34-7.20(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 22H 24FN 4O 379.1934；found 379.1935,[M+H] +.
化合物Ⅳ-34:
Figure PCTCN2021083579-appb-000074
化合物25(0.534g,1.0mmol)于100ml圆底烧瓶中，加入50ml乙醇溶解，加入33％的二甲胺溶液，Ar保护条件下90℃油浴加热回流，TLC检测，反应完毕，体系恢复至室温，减压除去溶剂，残余物经柱色谱分离的目标产物(0.276g,68％)。 1H-NMR(400MHz,DMSO-d 6):δ8.17(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),7.34-7.20(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 24H 28FN 4O 407.2247；found 407.2246,[M+H] +.
化合物Ⅳ-35:
化合物26：
Figure PCTCN2021083579-appb-000075
按照化合物Ⅳ-1的合成步骤，(0.732g，39％)。 1H NMR(400MHz,CDCl 3)δ8.19(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 20H 15N 4O 3 359.1；found 359.1,[M+H] +.
Figure PCTCN2021083579-appb-000076
化合物26(0.718g,2.0mmol)、氯化亚锡(0.758g,4.0mmol)于250ml圆底烧瓶中，加入100ml乙酸乙酯溶解，Ar保护条件下室温搅拌，TLC检测，反应完毕，体系倒入150ml水中，乙酸乙酯萃取三次，合并有机相，无水硫酸钠干燥，过量，减压除去溶剂，残余物经柱色谱分离纯化得目标产物(0.586g，89％)。 1H NMR(400MHz,CDCl 3)δ8.19(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 20H 17N 4O 329.1402；found 329.1403,[M+H] +.
化合物Ⅳ-36:
Figure PCTCN2021083579-appb-000077
按照化合物18的合成步骤，(0.322g，79％)。 1H-NMR(400MHz,DMSO-d 6):δ8.16(d,J＝8.5Hz,2H),7.93(t,J＝4.4Hz,2H),7.90(d,J＝6.1Hz,1H),7.31(t,J＝8.9Hz,2H),7.18(d,J＝15.9Hz,1H),6.95(s,1H),6.83-6.72(m,2H),4.21(s,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 25H 25FN 3O 2 418.1931；found 418.1932,[M+H] +.
化合物Ⅳ-37：
Figure PCTCN2021083579-appb-000078
按照化合物Ⅳ-1的合成步骤，(0.275g，75％)。 1H NMR(400MHz,DMSO-d 6)δ11.00(s,1H),8.08-8.04(m,2H),8.02(d,J＝15.9Hz,1H),7.92-7.87(m,2H),7.49-7.45(m,2H),7.26(d,J＝15.9Hz,1H),6.98(s,1H),3.29(s,3H).MS(ESI):m/z Calcd.For C 19H 13F 2N 3O 2 339.0951；found 339.0950,[M-H] -.
化合物Ⅳ-38：
Figure PCTCN2021083579-appb-000079
按照化合物Ⅳ-1的合成步骤，(0.327g，55％)。 1H NMR(400MHz,DMSO-d 6)δ9.70(s,1H),8.05(d,J＝8.9Hz,2H),7.98-7.89(m,1H),7.30(t,J＝6.2Hz,1H),7.26(d,J＝8.9Hz,1H),7.15(d,J＝15.8Hz,1H),6.97(s,1H),6.87(d,J＝7.5Hz,1H),3.27(s,3H).MS(ESI):m/z Calcd.For C 18H 12F 2N 3O 2 340.0903；found 340.090,[M-H] -.
化合物Ⅳ-39：
Figure PCTCN2021083579-appb-000080
按照化合物Ⅳ-1的合成步骤，(0.342g，46％)。 1H-NMR(400MHz,DMSO-d 6):δ＝8.21(d,2H,J＝8.8Hz),8.00(d,1H,J＝16Hz),7.85(d,2H,J＝8.0Hz),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),3.85(t,2H,J＝5.6Hz),3.60(t,2H,J＝5.6Hz),3.10(s,3H).MS(ESI):m/z Calcd.For C 22H 24N 3O 2 362.1869；found 362.1868,[M+H] +.
化合物Ⅳ-40：
Figure PCTCN2021083579-appb-000081
按照化合物Ⅳ-1的合成步骤，(0.412g，46％)。 1H-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 21H 21lN 6O 373.1777；found 373.1778,[M+H] +.
应该理解，本说明书各实施例中的用量、反应条件等除非特别注明均为近似值，可根据实际情况略作改变而获得类似结果。除专门定义外，本文所使用的所有专业与科学用语与本领域技术人员所理解的含意相同。本文提及的所有文献都引入本申请作为参考。本说明书中描述的是作为示范用的优选实施方案，本领域技术人员可采用与本文所述相似的方法及材料实施本发明获得相同或相似的结果，对本发明所作的各种改动或修改仍属于本申请所附权利要求书限定的范围内。

Claims (19)

  1. 一种核酸适配体分子，包含下述核苷酸序列(a)、核苷酸序列(b)或核苷酸序列(c)：
    核苷酸序列(a)为N 1AGAUUGUAAACAN 14-N 15-N 16GACACUN 23，其中N 1、N 14、N 15、N 16和N 23代表长度大于或等于1个核苷酸的片段，并且N 1与N 23核苷酸序列中至少有一对碱基形成互补配对，N 14与N 16核苷酸序列中至少有一对碱基形成互补配对；
    与核苷酸序列(a)具有至少70％，72％，77％，83％，88％，94％或100％同一性的核苷酸序列(b)；
    在核苷酸序列(a)中不包括N 1、N 14、N 15、N 16和N 23的位置，经过一个或几个核苷酸的取代、缺失和/或添加，且具有适配体功能的由核苷酸序列(a)衍生的核苷酸序列(c)。
  2. 根据权利要求1所述的核酸适配体分子，其中核苷酸序列(c)是在核苷酸序列(a)中不包括N 1、N 14、N 15、N 16和N 23的位置，经过5个、4个、3个、2个或1个核苷酸的取代、缺失和/或添加而得到的核酸适配体分子。
  3. 根据权利要求1所述的核酸适配体分子，其中核苷酸序列(a)中的N 1与N 23互补配对时，N 1核苷酸序列的方向为5’-3’，N 23核苷酸序列的方向为3’-5’；N 14与N 16互补配对时，N 14核苷酸序列的方向为5’-3’，N 16核苷酸序列的方向为3’-5’。
  4. 根据权利要求3所述的核酸适配体分子，其中当N 1与N 23中的至少一条片段的长度大于或等于5个核苷酸碱基时，则N 1与N 23核苷酸序列中至少有两对核苷酸碱基形成互补配对；当N 14与N 16中的至少一条片段的长度大于或等于5个核苷酸碱基时，则N 14与N 16核苷酸序列中至少有两对碱基形成互补配对。
  5. 根据权利要求1所述的核酸适配体分子，其中对核苷酸序列(a)的核苷酸取代选自下组中的一种：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/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、A4C/U5C/C12A/A13C、A4C/U5C/G7U/A11G/A13C、A4C/U5C/G7U/C12A/A13C、A4C/U5A/A11G/C12A/A13C、 A4C/U5C/A11G/C12A/A13C。
  6. 根据权利要求1所述的核酸适配体分子，其中核苷酸序列(a)中的N 1与N 23处的核苷酸序列为F30或tRNA脚手架RNA序列。
  7. 根据权利要求1所述的核酸适配体分子，其中的适配体分子是RNA分子或经碱基修饰的RNA分子。
  8. 根据权利要求1所述的核酸适配体分子，其中的适配体分子是DNA-RNA杂交分子或经碱基修饰的DNA-RNA分子。
  9. 根据权利要求1所述的核酸适配体分子，其中的N 14-N 15-N 16包含一个可以识别靶标分子的核苷酸序列。
  10. 根据权利要求9所述的核酸适配体分子，其中的靶标分子包括：蛋白质，核酸，脂质分子，碳水化合物，激素，细胞因子，趋化因子，代谢物，金属离子。
  11. 根据权利要求9所述的核酸适配体分子，其中的N 14-N 15-N 16为能够识别S-腺苷蛋氨酸和腺苷分子的核苷酸序列。
  12. 根据权利要求1所述的核酸适配体分子，所述的适配体功能是指核酸适配体能提高荧光团分子在合适波长激发光下的荧光强度至少2倍，至少5-10倍，至少20-50倍，至少100-200倍或者至少200倍以上。
  13. 根据权利要求1所述的核酸适配体分子，其中还包含能够结合多个荧光团分子的串联体，所述串联体通过适当长度的间隔序列连在一起，个数能够是2、3、4、5、6、7、8或者更多，所述串联体的核苷酸选自序列SEQ ID No：7、8、9、10，11、12、13、14、15、16、17、18、19和20。
  14. 根据权利要求1所述的核酸适配体分子，其中的核酸适配体分子具有序列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或31。
  15. 一种核酸适配体分子与荧光团分子的复合物，其中所述的核酸适配体分子为权利要求1所述核酸适配体分子，所述的荧光团分子具有下述式(I)所述的结构：
    Figure PCTCN2021083579-appb-100001
    其中：Ar 1、Ar 2独立地为六元芳基、六元芳杂基；D-为HO-或N(X 1)(X 2)-，X 1、X 2 各自独立地选自氢、烷基和改性烷基；X 1，X 2任选相互连接，与N原子一起形成脂杂环；当D-为N(X 1)(X 2)-，Ar 1为苯基时，X 1，X 2独立地与苯环形成饱和或不饱和的酯杂环；当D-为HO-，Ar 1为苯基时，与HO-相邻的至少一个氢原子被卤素取代；Y为O、S；R 1为氢、烷基和改性烷基；R 2为氢原子、卤原子、-OH、-CN；所述烷基各自独立地为C 1-C 10直链或支链烷基；
    所述改性烷基各自独立地为烷基的任意碳原子被选自卤原子、-OH、-CO-、-O-、-CN、-SO 3H、伯氨基、仲氨基、叔氨基的一种或多种基团置换所得的基团，所述改性烷基具有1-10个碳原子，其中碳碳单键任选独立地被碳碳双键或碳碳三键置换；所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换。
  16. 根据权利要求15所述的复合物，所述荧光团分子选自下式化合物：
    Figure PCTCN2021083579-appb-100002
    Figure PCTCN2021083579-appb-100003
  17. 根据权利要求15所述的复合物，其中复合物中的适配体分子包含核苷酸序列 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或31。
  18. 一种试剂盒，包含权利要求1所述的核酸适配体分子、权利要求15所述的复合物、表达载体或宿主细胞中的至少一种，其中，
    所述表达载体包含转录权利要求1所述的核酸适配体分子的DNA分子；
    所述宿主细胞包含所述表达载体。
  19. 一种权利要求15所述的复合物用于体外或体内目标核酸分子的检测或标记、细胞外或细胞内靶标分子的检测或标记、对基因组DNA进行成像、检测RNA与蛋白质的相互作用、检测基因组DNA以及提纯与纯化RNA。
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