WO2020221238A1 - 一种适配体核酸分子及其复合物和应用 - Google Patents

一种适配体核酸分子及其复合物和应用 Download PDF

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WO2020221238A1
WO2020221238A1 PCT/CN2020/087415 CN2020087415W WO2020221238A1 WO 2020221238 A1 WO2020221238 A1 WO 2020221238A1 CN 2020087415 W CN2020087415 W CN 2020087415W WO 2020221238 A1 WO2020221238 A1 WO 2020221238A1
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nucleic acid
molecule
pepper
rna
acid aptamer
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PCT/CN2020/087415
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English (en)
French (fr)
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杨弋
朱麟勇
陈显军
张大生
苏倪
林秋宁
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华东理工大学
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Priority to US17/607,102 priority Critical patent/US20230002769A1/en
Priority to JP2021563697A priority patent/JP2022530160A/ja
Priority to EP20798707.4A priority patent/EP3964583A4/en
Publication of WO2020221238A1 publication Critical patent/WO2020221238A1/zh

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Definitions

  • This application relates to an aptamer nucleic acid molecule, a complex containing the 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 application can specifically bind a small fluorophore molecule, and significantly increase its fluorescence intensity under excitation by light of a suitable wavelength.
  • RNA Among all biological macromolecules, RNA exhibits the most diverse biological functions. In the central law of biology, RNA acts as a transmitter of genetic material (messenger RNA), a template for protein synthesis (ribosomal RNA), and an amino acid transporter (transfer RNA), forming a series of physiological processes, and ultimately achieving gene transcription and expression. In the past few decades, scientists have gradually discovered the vital functions of RNA in a variety of life activities, including many RNA-protein complexes, such as telomerase, splicing enzyme, ribozyme, and riboswitch.
  • RNA-protein complexes such as telomerase, splicing enzyme, ribozyme, and riboswitch.
  • RNAs such as short-strand interfering RNA (siRNA), small microRNA (microRNA), and long-chain non-coding RNA (lncRNA)
  • siRNA short-strand interfering RNA
  • microRNA small microRNA
  • lncRNA long-chain non-coding RNA
  • Real-time monitoring of RNA transport and metabolism in cells is essential for studying the relationship between RNA localization and gene expression and cell regulation.
  • scientists have identified several mechanisms that can lead to different subcellular locations of RNA, such as active transport, passive diffusion, and anchoring.
  • active transport In many polar cells, especially nerve cells, the spatially specific expression of mRNA is closely related to neuronal plasticity, learning and memory. Therefore, once the RNA regulation process is damaged, it will cause neuronal dysfunction and neurological diseases.
  • RNA fluorescence in situ hybridization technology is a method that has been widely used for a long time to study the level and distribution of RNA in cells. It is a technology that uses molecular hybridization to fluorescently label specific RNA molecules for imaging. However, its operation is more complicated and contains an elution step, which can only be used for the study of immobilized cells, that is, dead cells, and cannot be used to monitor the dynamic change process of RNA in living cells in real time.
  • Molecular beacon technology is the earliest living cell RNA imaging technology developed. It uses a stem-loop double-labeled oligonucleotide probe that forms a hairpin structure at the 5'and 3'ends.
  • the fluorescent group When it binds to the target RNA, the fluorescent group is quenched by a quenching group labeled at one end. The effect is eliminated, the fluorescent group produces fluorescence, or the FRET of the fluorescent groups at both ends disappears.
  • molecular beacons have low fluorescence signals, difficulty in entering cells, easy degradation, serious non-specific accumulation in the nucleus, susceptibility to RNA secondary structure, and the need to customize oligonucleotide probes for each RNA, etc. Disadvantages, these shortcomings limit the wide application of this technology.
  • MCP-FPs can specifically recognize mRNA molecules fused with multiple copies of MS2 sequences, and monitor the synthesis and distribution of mRNA in real time by detecting fluorescent protein signals (Ozawa et al. Nature Methods 2007.4:413-419). However, because the MCP-FPs that are not bound to mRNA molecules will produce high background fluorescence, the signal-to-noise ratio of this method is very low.
  • the scientists added a nuclear localization signal to the MCP-FPs fusion protein so that GFP-MS2, which was not bound to the mRNA molecule, was located in the nucleus, which reduced the non-specific fluorescence in the cytoplasm to a certain extent and increased the signal-to-noise ratio of detection.
  • RNA-binding protein-fluorescent protein technology to detect cellular RNA
  • scientists have been looking for a GFP-like RNA fluorescent tag for RNA imaging.
  • the scientists constructed a fluorophore-quencher combination.
  • the fluorophore aptamer binds to the fluorophore
  • the quencher cannot quench the fluorescent signal of the fluorophore.
  • the aptamer- The fluorophore-quencher complex is fluorescent.
  • the aptamer of the fluorophore is not present, the fluorescence signal of the fluorophore will be quenched by the quencher.
  • IMAGE intracellular multi aptamer genetic
  • the research team replaced a stem-loop structure in "Spinach” with a nucleic acid aptamer that specifically binds to cell metabolites, and developed a tool based on the Spinach-DFHBI complex that can detect cell metabolites (Paige et al. Science) 2012.335:1194). So far, this method has been successfully used to monitor and analyze the dynamic changes of RNA in bacteria, yeast and mammalian cells. Subsequently, the research group also developed the Corn-DFHO complex to detect the activity of the RNA polymerase III promoter in mammalian cells (Song et al. Nature Chemical Biology 2017.13: 1187-1194).
  • the aptamer-fluorophore complex has a weak binding ability, and its dissociation constant (kd) is tens to hundreds of nM; (2) ) The fluorescent signal of the aptamer-fluorophore complex is unstable and is easily quenched, making its fluorescent signal unsuitable for detection (Han et al. Journal of the American Chemical Society 2013.135:19033-19038); (3) So far, the spectrum is only green and yellow, and there is no longer wavelength spectrum for imaging RNA in living animals (Song et al.
  • RNA labeling technologies have their own obvious shortcomings.
  • MCP-FPs labeling technology has unbound background fluorescence and low signal-to-noise ratio.
  • the RNA labeling technology based on the complex composed of aptamer-fluorophore-quencher currently only realizes the labeling of RNA in bacteria, and has not yet realized the labeling of RNA in mammalian cells.
  • the RNA labeling technology based on single fluorophore-nucleic acid aptamer seems to be a perfect RNA labeling technology, but it is limited by the current fluorophore (DFHBI, DFHBI-1T, DFHO) and nucleic acid aptamer complexes.
  • the application 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.
  • a nucleic acid aptamer molecule of the present application comprising the following nucleotide sequence (a), (b) or (c):
  • N 1 CCAAUCGUGGCGUGUCGN 19 -N 20 -N 21 ACUGGCGCCGN 32 (called the general Pepper structure), where N 1 , N 19 , N 20 , N 21 and N 32 represent length greater than or equal to 1 At least one pair of bases in the nucleotide sequence of N 1 and N 32 form a complementary pair, and at least one pair of bases in the nucleotide sequence of N 19 and N 21 form a complementary pair;
  • the nucleotide sequence of the Pepper structure defined by the nucleotide sequence (b) and the nucleotide sequence (a) has at least 75%, 76%, 78%, 80%, 82%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the nucleotide sequence (c) does not include N 1 , N 19 , N 20 , N 21 and N 32 in the nucleotide sequence of the Pepper structure defined by the nucleotide sequence (a) Position, aptamer obtained by substitution, deletion and/or addition of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides molecular.
  • the nucleotide sequence (c) does not include the positions of N 1 , N 19 , N 20 , N 21 and N 32 in the nucleotide sequence defined in (a).
  • a nucleic acid aptamer molecule obtained by the substitution of 6, 5, 4, 3, 2 or 1 nucleotides.
  • N 1 and N 32 in the nucleotide sequence (a) are complementary paired, the direction of the N 1 nucleotide sequence is 5'-3', and the direction of the N 32 nucleotide sequence is 3'-5'; when N 19 is complementary to N 21 , the direction of the N 19 nucleotide sequence is 5'-3', and the direction of the N 21 nucleotide sequence is 3'-5'.
  • N 1 and N 32 in the nucleotide sequence (a) when the length of at least one of N 1 and N 32 in the nucleotide sequence (a) is greater than or equal to 5 nucleotide bases, then N 1 and N 32 nucleosides At least two pairs of bases in the acid sequence form a complementary pair; when at least one fragment of N 19 and N 21 is greater than or equal to 5 nucleotide bases in length, then at least N 19 and N 21 are There are two pairs of bases forming complementary pairs.
  • the nucleotide substitution to the Pepper structure of the general formula is selected from one of the following group: C3A, C3U, A4U, A4G, A4C, A5G, A5C, U6A, U6G, U6C, C7A, C7U, G8C , U9A, G11A, G11U, C12G, C12A, C12U, G13C, U14A, U14G, C17U, G18U, G18C, C27G, C27U, G28U, C29G, C29U, C30A, C30U, C2G/G31C, C2U/G31AU, C2G , G10A/C30U, G10C/C30G, G10U/C30A, C2G/G31C/C3A, C2G/G31C/A4C, C2G/G31C/A5C, C2G/G31C/G8C, C2G/
  • the nucleotide substitution to the Pepper structure of the general formula is selected from one of the following group: C3A, C3U, A4C, A5C, C7U, G8C, U9A, C12G, C12U, U14G, C27U, C29G, C30U , C2G/G31C, C2U/G31A, C2A/G31U, G10A/C30U, G10C/C30G, G10U/C30A, C2G/G31C/C3A, C2G/G31C/A4C, C2G/G31C/A5C, C2G/GC2C, G31C/G8C /G31C/C12U, C2G/G31C/U14G, C2G/G31C/C27U, C2G/G31C/C29G, C2G/G31C/C30U, C2G/G31C/G10A/C30U,
  • the nucleotide substitution to the Pepper structure of the general formula is selected from one of the following group: C3A, C3U, A4C, A5C, C7U, G8C, U9A, C12G, C12U, U14G, C27U, C29G, C30U , C2G/G31C, C2U/G31A, C2A/G31U, G10A/C30U, G10C/C30G, G10U/C30A, C2G/G31C/C3A, C2G/G31C/A4C, C2G/G31C/A5C, C2G/GC2C, G31C/G8C /G31C/C12U, C2G/G31C/U14G, C2G/G31C/C27U, C2G/G31C/C29G, C2G/G31C/C30U, C2G/G31C/G10A/C30U,
  • the nucleotide sequence at N 1 and N 32 in the nucleotide sequence (a) is F30 or tRNA scaffold RNA sequence.
  • the nucleic acid aptamer molecule is an RNA molecule or a base-modified RNA molecule.
  • the nucleic acid aptamer molecule is a DNA-RNA hybrid molecule or a base-modified DNA-RNA molecule.
  • N 19 -N 20 -N 21 in the nucleotide sequence (a) includes a nucleotide sequence that can recognize the target molecule.
  • the target molecules include, but are not limited to: proteins, nucleic acids, lipid molecules, carbohydrates, hormones, cytokines, chemokines, metabolites, metal ions.
  • N 19 -N 20 -N 21 in the nucleotide sequence (a) is a nucleotide sequence that can recognize GTP 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, at least 20-50 times, at least 100 times. -200 times or at least 500-1000 times higher.
  • the nucleic acid aptamer molecule may also include a concatemer that can bind multiple fluorophore molecules, the concatemers are connected together by a spacer sequence of appropriate length, the spacer sequence has 2, 3, Length of 4, 5, 6, 7, 8 or more nucleotide fragments.
  • the nucleotides of the concatemer can be selected from, but not limited to, the sequence SEQ ID No: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19.
  • the nucleic acid aptamer molecule has the sequence SEQ ID No: 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 21, 22, or 23.
  • This application also provides a complex of a nucleic acid aptamer molecule and a fluorophore molecule, wherein the nucleic acid aptamer molecule is any one of the aforementioned nucleic acid aptamer molecules, and the fluorophore molecule has the following formula (I) The structure described:
  • D- is X1O- or N(X2)(X3)-;
  • X1, X2, and X3 are each independently selected from hydrogen, linear or branched alkyl groups with 1-10 carbons and modified alkyl groups, X2 X3 is optionally connected to each other as a saturated or unsaturated ring;
  • R- is selected from hydrogen, cyano, carboxy, amide, ester, hydroxyl, straight or branched chain alkyl or modified alkyl with 1-10 carbons ;
  • Ar1 and Ar2 are each independently selected from a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic aryl group, a monocyclic heteroaryl group or a combination of one or two of 2-3 Aromatic subunits of ring structure;
  • the hydrogen atoms in Ar1 and Ar2 can be independently replaced by F, Cl, Br, I, hydroxyl, nitro, aldehyde, carboxy, cyano, sulfonic, sulfate, phosphoric, amino, primary, and secondary Amino, 1-10 carbon linear or branched alkyl and modified alkyl substitution;
  • nucleic acid aptamer molecule and the fluorophore molecule in the complex are respectively present in separate solutions, or the nucleic acid aptamer molecule and the fluorophore molecule are in the same solution.
  • the aromatic ring contained in the fluorophore molecule is selected from the following structures (II-1) to (II-15):
  • the fluorophore molecule is selected from compounds of the following formula:
  • the fluorophore molecule in the complex is selected from III-1, III-2, III-3, III-4, III-5, III-6, III-7, III-8 , III-9, III-10, III-11, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19, III-20 and III -twenty one.
  • the aptamer molecule in the complex comprises the nucleotide sequence SEQ ID No: 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31.
  • the application also provides any one of the above-mentioned complexes for the detection or labeling of target nucleic acid molecules in vitro or in vivo.
  • the application also provides any one of the above-mentioned complexes for the detection or labeling of extracellular or intracellular target molecules.
  • the present application also provides any one of the above-mentioned complexes for imaging genomic DNA.
  • the present application also provides any one of the above-mentioned complexes for detecting the relationship between mRNA and protein content in cells.
  • the application also provides a DNA molecule that transcribes any of the aforementioned nucleic acid aptamer molecules.
  • the application also provides an expression vector, which contains the above-mentioned DNA molecule.
  • the application also provides a host cell, which comprises the above-mentioned expression vector.
  • the application also provides a kit comprising any one of the aforementioned nucleic acid aptamer molecules and/or any one of the aforementioned expression vectors and/or any one of the aforementioned host cells and/or any one of the aforementioned complexes.
  • This application also provides a method for detecting target molecules, including the steps:
  • the fluorescence of the complex is detected.
  • This application also provides a method for detecting genomic DNA, which includes imaging genomic DNA with any one of the above-mentioned complexes.
  • the present application also provides a method for extracting and purifying RNA, including extracting and purifying RNA by using any one of the aforementioned complexes.
  • the inventor designed a new nucleic acid aptamer molecule and synthesized a new fluorophore molecule to form a new fluorophore-nucleic acid aptamer complex.
  • the aptamer molecules combined with fluorophore molecules can significantly increase the fluorescence intensity of the fluorophore molecules under excitation light at a suitable wavelength. They overcome the shortcomings of the previous fluorophore-nucleic acid aptamer complex and can be effectively used in living cells Real-time labeling of RNA/DNA.
  • the nucleic acid aptamer of the present application has a strong affinity for fluorophore molecules, and exhibits different fluorescence spectra and good light and temperature stability.
  • nucleic acid aptamer-fluorophore molecular complexes can perform real-time labeling and imaging of RNA/DNA in prokaryotic and eukaryotic cells, detect protein-RNA interactions, explore the relationship between mRNA content and protein in cells, or be used for RNA Extraction and purification of tags and other functions.
  • Figure 1 The secondary structure prediction of nucleic acid aptamer molecules.
  • A is the predicted general structure of Pepper, including N 1 and N 32 that can form a stem structure, and N 19 , N 20 and N 21 that can form a stem-loop structure.
  • B is the predicted structure of Pepper-1. The base sequences of N 1 and N 32 are shown in the dashed box corresponding to stem 1 in the figure, and the base sequences of N 19 , N 20 and N 21 are shown in the stem loop. The corresponding dashed box is shown.
  • Figure 4 Characterization of F30-Pepper-1-III-3 complex.
  • A Fluorescence excitation and emission spectra of F30-Pepper-1-III-3 complex;
  • B Absorption spectra of F30-Pepper-1-III-3 complex and III-3;
  • C F30-Pepper -1-III-3 complex oligomerization identification; "ruler” is a single-stranded DNA standard, used to calibrate the size of the aptamer.
  • FIG. 5 The activation effect of Pepper with different base modifications on III-3.
  • A Schematic diagram of the secondary structure of Pepper-3 aptamer containing deoxyribonucleotides (marked in dark color in the figure);
  • B Pepper-4 with 2'F modification (marked in dark color in the figure) Schematic diagram of the secondary structure of the aptamer;
  • C the activation effect of Pepper with different modifications on III-3.
  • Control is to replace Pepper-3 or Pepper-4 aptamer with buffer.
  • FIG. 6 The activation effect of different Pepper series on III-3.
  • A Obtain the Pepper series according to the "series 1” method;
  • B Obtain the Pepper series according to the “series 2” method;
  • C obtain the Pepper series according to the “series 3” method;
  • D According to the “series 1” ”Method to obtain the activation effect of Pepper series on III-3;
  • E Different according to the “series 2” method to obtain the activation effect of Pepper series on III-3;
  • Figure 9 The labeling effect of Pepper, III-3 and their analogs used in the labeling of RNA in mammalian cells.
  • A Compare the effects of F30-Pepper-1-III-3, F30-Broccoli-DFHBI-1T and tRNA-Corn-DFHO on labeling RNA in mammalian cells;
  • B the statistical results of the fluorescence in (A);
  • C The effect of F30-8 Pepper-5 and III-3 analogues on labeling RNA in mammalian cells.
  • FIG. 10 Probe construction based on Pepper-1.
  • A Schematic diagram of probe construction, wherein the stem-loop structure can recognize adenosine or GTP;
  • B the detection effect of adenosine probe;
  • C the detection effect of GTP probe.
  • Pepper is used to track RNA localization in cells.
  • A Pepper is used to detect the location of GAPDH mRNA;
  • B Pepper is used to detect the location of TMED2 mRNA.
  • FIG. 13 Pepper is used to detect genomic DNA.
  • A Schematic diagram of dCas9 and different chimeric sgRNAs;
  • B genomic DNA imaging results of dCas9 and different chimeric sgRNAs;
  • C statistical results of bright particles in each cell in (B).
  • Pepper is used for super-resolution imaging of RNA.
  • A Co-localization of 4Pepper-9-MS2RNA and tdMCP-BFP-H2B protein;
  • B Wide-field and SIM imaging results of the middle layer of the nucleus;
  • C Wide-field and SIM imaging results of the top layer of the nucleus;
  • Figure 15 Pepper's label for RNA extraction and purification.
  • the "ruler” is a single-stranded DNA standard used to calibrate the size of the aptamer.
  • nucleotide and “nucleotide base” are used interchangeably to indicate the same meaning.
  • the "aptamer molecule” described in the present application is also called “aptamer molecule”.
  • the nucleic acid aptamer molecule comprises (a) a nucleotide sequence of N 1 CCAAUCGUGGCGUGUCGN 19 -N 20 -N 21 ACUGGCGCCGN 32 (corresponding to the Pepper structure of FIG. 1A); or (b) and (a) 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 32 forms a reverse complementary pair, that is, the direction of the nucleotide sequence of N 1 is 5'-3' , The direction of the N 32 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 32 is greater than or equal to 5, At least two pairs of bases are required to form complementary pairs.
  • at least one pair of bases in the N 19 and N 21 nucleotide sequences form a reverse complementary pair, that is, the direction of the N 19 nucleotide sequence is 5'-3', and the direction of the N 21 nucleotide sequence is 3'- 5'.
  • N 20 is a nucleotide base of any length and any composition; or (c) 1-7 nucleotide substitutions, deletions and/or additions at any position of the nucleotide sequence (a) .
  • the nucleic acid aptamer molecule includes a substitution to the nucleotide of the general formula Pepper structure, and the substitution is selected from one of the following group: C3A, C3U, A4U, A4G, A4C, A5G, A5C, U6A, U6G, U6C, C7A , C7U, G8C, U9A, G11A, G11U, C12G, C12A, C12U, G13C, U14A, U14G, C17U, G18U, G18C, C27G, C27U, G28U, C29G, C29U, C30A, C30U, C2G/G31C, C2U/G31A, , C2A/G31U, G10A/C30U, G10C/C30G, G10U/C30A, C2G/G31C/C3A, C2G/G31C/A4C, C2G/G31C/A5
  • C3A indicates that the third cytosine nucleotide C of Pepper is replaced with adenine nucleotide A.
  • C2G/G31C means that the second C of Pepper is replaced with G, and the 31st G is replaced with C, which is Pepper(C2G/G31C) in Table 1.
  • Table 1 The general structure of Pepper aptamers with 7, 6, 5, 4, 3, 2 or 1 nucleotide substitutions
  • 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 non-pairing regions (loops) ( Figure 1).
  • the nucleic acid aptamer molecule described in this application contains a secondary structure as predicted in FIG. 1.
  • the secondary structure contains 2 loop structures, 2 stem structures and a stem-loop structure.
  • Stem 1 is used to stabilize the molecular structure of the entire nucleic acid aptamer and can be replaced with any length that can form a stem structure.
  • the 5'end or 3'end of the stem 1 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 a 5S RNA sequence (Genebank: NR_023377.1); in another preferred embodiment of the present application, the 5'end of the nucleic acid aptamer molecule End fusion GAPDH RNA sequence (Genebank: BC009081).
  • the stem-loop structure in Figure 1 stabilizes the molecular structure of the entire nucleic acid aptamer, and can be replaced with other nucleotide base pairs of any length and composition that can form a stem-loop structure.
  • the aptamer molecule described in the present application may also include other nucleotide sequences inserted into the N 19 -N 20 -N 21 position, and the inserted nucleotide sequence replaces the stem-loop structure in Figure 1A.
  • 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 improves the fluorescence of the fluorophore molecule under excitation light of a suitable wavelength.
  • the target molecule may be a small molecule, a signal molecule on the cell surface, and the like.
  • the stem-loop structure can be replaced with an RNA sequence that recognizes the target molecule for extracellular or intracellular detection of the target molecule.
  • the stem-loop structure of the aptamer molecule can be combined with GTP molecules; in another preferred embodiment of the present application, the stem-loop structure can be combined with adenosine molecules.
  • the nucleic acid aptamer molecule is preferably SEQ ID NO: 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 21, 22, or 23, or their mutation sequences that can combine with fluorophore molecules to significantly increase their fluorescence under appropriate wavelength excitation light.
  • the nucleic acid aptamer molecule described in the present application may also include a nucleotide sequence that increases its stability.
  • F30 scaffold RNA sequence 2
  • tRNA scaffold RNA sequence 3
  • its connection with the nucleic acid aptamer molecule is shown in Figure 3.
  • nucleic acid aptamer molecule is an RNA molecule, or a DNA-RNA hybrid molecule in which part of the nucleotides is replaced with deoxyribonucleotides.
  • the nucleotides can be in the form of their D and L enantiomers, as well as their derivatives, including but not limited to 2'-F, 2'-amino, 2'-methoxy, 5'-iodo , 5'-bromo-modified polynucleotide.
  • Nucleic acids contain various modified nucleotides.
  • Identity describes the relatedness between two nucleotide sequences in this application.
  • N 1 , N 19 , N 20 , N 21 , and N 32 in the sequence (a) are not included in the calculation of the identity of the nucleotide sequence of the two aptamers in this application.
  • the degree of identity between two nucleotide sequences uses the Needle 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 EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62).
  • the sequence of Pepper (C3A) in Table 1 of this application is N 1 C A AAUCGUGGCGUGUCGN 19 -N 20 -N 21 ACUGGCGCCGN 32
  • the sequence of Pepper (C3U) is N 1 C U AAUCGUGGCGUGUCGN 19 -N 20 -N 21 ACUGGCGCCGN 32
  • the nucleotide bases N 1 , N 19 -N 20 -N 21 and N 32 should not be included, so their sequence identity alignment result is 96.3 % (With a difference of 1 nucleotide).
  • fluorophore molecule described in this application is also called “fluorophore” or “fluorescent molecule”.
  • fluorophore molecules in this application 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 when the fluorophore is not bound to a specific aptamer, the quantum yield of the fluorophore is less than 0.1, more preferably less than 0.01, and most preferably less than 0.001; when the fluorophore is bound by a specific aptamer Later, 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 application is preferably able to enter the cytoplasm or periplasm through the cell membrane or cell wall through active transport or passive diffusion.
  • 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 described in the present application can specifically bind to a fluorophore and significantly increase its fluorescence value under excitation at a specific wavelength.
  • “Increase fluorescence signal”, “increase fluorescence”, “increase fluorescence intensity”, and “increase fluorescence intensity” in this application 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 increase in quantum yield is at least 2 times; in another preferred embodiment of the present application, the increase in quantum yield is at least 5-10 times; in another more preferred embodiment of the present application In another more preferred embodiment of the present application, the quantum yield is increased by at least 100-200 times; in another more preferred embodiment of the present application, the quantum yield is increased by at least 20-50 times;
  • the increase in quantum yield is at least 500-1000 times; in another more preferred embodiment of the present application, the increase in quantum yield is at least 1000-10000 times; in another more preferred embodiment of the present application, the increase in quantum yield is greater than 10000 times;
  • the light source used to excite the fluorophore to generate the fluorescent signal 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 through other Fluorophore acquisition, such as the donor fluorophore of FERT, or the donor luminophore of BRET.
  • the target molecule described in this application 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 can be a molecule related to a disease or pathogen infection.
  • the inserted nucleotide sequence replaces the stem-loop structure of N 19 , N 20 , and N 21 in FIG. 1, and the core
  • the nucleotide 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 sequence will promote the combination of aptamer molecules and fluorophore molecules, significantly increase the fluorescence of fluorophore molecules under appropriate wavelength excitation light, and 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 the multivalent nucleic acid aptamers in this application.
  • the RNA aptamers that have been reported to bind target molecules include but are not limited to: T4 RNA polymerase aptamers, HIV reverse transcriptase aptamers, and phage R17 capsid protein aptamers.
  • the target molecule is adenosine, and its corresponding probe sequence for identifying the target molecule is as SEQ ID NO: 21 (shown in Figure 10A); a preferred implementation of the present application In the scheme, the target molecule is GTP, and the corresponding probe sequence for identifying the target molecule is as SEQ ID NO: 22 (shown in FIG. 10A).
  • 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.
  • Target RNA molecule in this application 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, long non-coding RNA, 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 N 19 -N 20 -N 21 of the RNA aptamer molecule of the present application.
  • SgRNA in this application refers to a single guide RNA (single guide RNA, sgRNA) formed by transforming tracrRNA and crRNA in the CRISPR/Cas9 system, and its 5'end about 20 nt sequence targets DNA through base pair complementation The site prompts the Cas9 protein to induce a DNA double-strand break at this site.
  • sgRNA single guide RNA
  • the nucleic acid aptamer molecule described in the present application 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 Pepper structures in series can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. There can be many forms of concatenation.
  • the concatenation form is "series 1", as shown in Figure 6A, and the preferred nucleotide sequence is SEQ ID NO: 8, 9, 10 , 11 or 12; wherein 2Pepper-5 represents a series body 1 with two Pepper-5 structures; in another preferred embodiment of this application, the series form is "series 2", as shown in Figure 6B, preferably The nucleotide sequence of is SEQ ID NO: 13, 14, 15 or 16; 2xPepper-6 represents the concatemer 2 with two Pepper-6 structures; in another preferred embodiment of the present application, the concatenated form As shown in Figure 6C, the preferred nucleotide sequence is SEQ ID NO: 17, 18 or 19; 2x2Pepper-5 means concatenation 3 with 4 Pepper-5 structures; no matter what Form, the interval sequence between the series can be replaced.
  • the monomeric aptamer described in this application refers to an aptamer containing only one Pepper structure, that is, an aptamer containing two stem structures, two loop structures and one stem loop structure (Figure 1A) body.
  • An aptamer in the form of a polymer refers to an aptamer containing more than one Pepper structure, including but not limited to the aptamers formed in several tandem forms shown in FIG. 6.
  • the aptamer-fluorophore complex of the present application includes one nucleic acid aptamer molecule and one or more fluorophore molecules.
  • the molecular complex comprising one nucleic acid molecule and one fluorophore molecule is F30-Pepper-2-III-3, F30-Pepper-2-III-7, F30-Pepper- 2-III-6, F30-Pepper-2-III-8, F30-Pepper-2-III-4, F30-Pepper-2-III-15, F30-Pepper-2-III-18 and F30-Pepper- 2-III-21.
  • the nucleic acid molecule of the concatemer and multiple fluorophore molecules form a complex, for example, F30-8Pepper-5 containing 8 aptamer units formed in a “tandem 1” manner is combined with A complex formed by 8 phosphor molecules III-3 8Pepper-5-8 ⁇ (III-3), 8Pepper-5-8 ⁇ (III-7), 8Pepper-5-8 ⁇ (III-6), 8Pepper- 5-8 ⁇ (III-8), 8Pepper-5-8 ⁇ (III-4), 8Pepper-5-8 ⁇ (III-15), 8Pepper-5-8 ⁇ (III-18) and 8Pepper-5- 8 ⁇ (III-21).
  • the molecular complex can exist in the form of two separate solutions in vitro, or in the same solution, or in the cell.
  • the aptamer function in the present application refers to the ability to significantly increase the fluorescence intensity of the fluorophore molecule 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.
  • the increase in fluorescence intensity is at least 2 times (fluorescence intensity is detected according to experimental method (5)); in another preferred embodiment of this application, the increase in fluorescence intensity is at least 5-10 In another more preferred embodiment of this application, the increase in fluorescence intensity is at least 20-50 times; in another more preferred embodiment of this application, the increase in fluorescence intensity is at least 100-200 times; in this application, another In a more preferred embodiment, the increase in fluorescence intensity is at least 500-1000 times; in another more preferred embodiment of this application, the increase in fluorescence intensity is at least 1000-10000 times; in another more preferred embodiment of this application , The increase in fluorescence intensity is greater than 10,000 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 32 , and N 19 and N 21 Complementary pairing of the nucleotides with another fragment can form a stem structure. If N 1 and N 32 are single nucleotides, N 1 and N 32 need to be completely complementary to form a stem structure (as shown in Figure 1).
  • the DNA molecule includes a DNA sequence that can encode the nucleic acid aptamer molecule of the present application.
  • the DNA molecule comprises the nucleotide sequence R 1 CCAATCGTGGCGTGTCGR 19 -R 20 -R 21 ACTGGCGCCGN 32 and a nucleotide sequence having at least 70% identity.
  • R 1 encodes N 1 in the general Pepper structure
  • R 19 encodes N 19 in the General Pepper structure
  • R 20 encodes N 20 in the General Pepper structure
  • R 21 encodes N 21 and R in the General Pepper structure.
  • 32- coded N 32 in the Pepper structure 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 the U6 promoter; in another specific embodiment of the application, the DNA molecule includes the CMV promoter.
  • the DNA molecule includes the DNA molecule and 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) and a transmembrane emp24 domain containing protein 2 (TMED2) (sequence of chimeric RNA) Respectively SEQ ID No: 24, 25).
  • the DNA molecule encoding the target RNA includes DNA sequences encoding mCherry and TagBFP (the sequences of the chimeric RNA are SEQ ID Nos: 26 and 27, respectively).
  • promoter includes eukaryotic and prokaryotic promoters in this application.
  • 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 process the transcription process in time.
  • one or more suitable promoters can be used.
  • 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 aptamers described in this application. Some operator sequences in bacteria can be combined with promoter sequences to form inducible promoters. At this time, specific inducers need to be added to induce transcription of DNA molecules. For example, the lac operator needs to add lactose or lactose analogue (IPTG) to induce its expression, and other operators include trp, pro, etc.
  • IPTG lactose or lactose analogue
  • 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.
  • Promoting transcription in eukaryotic cells also requires suitable promoters, including but not limited to ⁇ -globulin promoter, CAG promoter, GAPDH promoter, ⁇ -actin promoter, actin promoter, Cstf2t promoter , SV40 promoter, PGK promoter, MMTV promoter, adenovirus Ela promoter, CMV promoter, etc.
  • suitable promoters including but not limited to ⁇ -globulin promoter, CAG promoter, GAPDH promoter, ⁇ -actin promoter, actin promoter, Cstf2t promoter , SV40 promoter, PGK promoter, MMTV promoter, adenovirus Ela promoter, CMV promoter, etc.
  • the termination of transcription in eukaryotic cells depends on specific cleavage sites in the RNA sequence. Similarly, RNA polymerase transcribes genes differently, and its transcription terminator is also very different. However, screening for suitable 3'transcription terminator regions is
  • the "expression system” of the present application also called “expression vector”, includes a DNA molecule integrated with an expression nucleic acid aptamer.
  • the expression system of the present application can be a plasmid or a virus particle.
  • the "expression vector" recombinant virus can be obtained by transfecting the plasmid into the 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 bacteria containing Cos plasmid DNA; yeast containing yeast vectors; mammals infected with viruses Animal cells (such as adenovirus, adeno-associated virus, retrovirus); insect cells infected with viruses (such as baculovirus); 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.
  • the constructed DNA molecules are cloned into the vector system, they can be easily transferred into host cells.
  • methods include but are not limited to transformation, transduction, conjugation, immobilization, electrotransduction, etc.
  • expression plasmids pET28a-T7-F30-Pepper-2, pLKO.1-F30-Pepper-2 and pYES2.1-F30- containing DNA molecules encoding F30-Pepper-2 RNA are provided.
  • Pepper-2 In another specific embodiment of the present application, an expression plasmid pLKO.1-F30-8Pepper-5 containing a DNA molecule encoding F30-8Pepper-5RNA is provided.
  • an expression plasmid pCDNA3.1hygro(+)- containing DNA molecules encoding BFP-4Pepper-7, mCherry-4Pepper-7, GAPDH-4Pepper-7 and TMED2-4Pepper-7 is provided.
  • an expression plasmid psgRNA- containing DNA molecules encoding sgRNA-Pepper-8 (loop1), sgRNA-Pepper-8 (tetraloop), sgRNA-Pepper-8 (loop1 and tetraloop) is provided.
  • an expression plasmid pLKO.1-4Pepper-9-MS2 containing a DNA molecule encoding 4Pepper-9-MS2 is provided.
  • This application also provides expression vectors that integrate DNA molecules encoding nucleic acid aptamers, but the coding DNA sequence of the target RNA molecule is missing.
  • the coding DNA sequence gap of the target RNA molecule allows users to select the DNA sequence of the target RNA molecule to be detected.
  • the coding DNA sequence corresponding to GAPDH mRNA use standard recombinant DNA technology to insert the DNA sequence into the expression vector of this application, and introduce the obtained expression vector into the host cell (transfection, transformation, infection, etc.) to detect the target The content and distribution of RNA.
  • “Host cells” in this application include but are not limited to bacteria, yeast, mammalian cells, insect cells, plant cells, zebrafish cells, fruit fly cells, and nematode cells.
  • the host cells are more preferably cultured in vitro cells or whole in vivo living tissues.
  • the host cells in this application include 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 described in the present application includes one or more nucleic acid aptamer molecules of the present application, wherein the nucleic acid aptamer molecules are anchored at discrete positions on the array surface, and the array surface is composed of a solid support, including but not limited to Glass, metal, ceramics, etc.
  • the nucleic acid aptamer molecule described in the present application 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, and affinity The aptamer molecule is 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 is fused
  • 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 exposure to the appropriate excitation light wavelength. Within a certain range, the higher the concentration of target molecules, the higher the fluorescence intensity.
  • the kit of the present application includes the nucleic acid aptamer molecules and/or fluorophore molecules described in the present application, and corresponding instructions; or an expression system and/or fluorophore molecules for expressing the nucleic acid aptamer molecules, and Corresponding instructions; or containing host cells and/or fluorophore molecules expressing nucleic acid aptamer molecule expression systems, 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 pCDNA3.1hygro(+) 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 pYES2.1TOPO 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.
  • EcoRI, BamHI, BglII, HindIII, NdeI, XhoI, SacI, XbaI, SpeI and other restriction enzymes, T4 ligase, T4 phosphorylase (T4PNK), T7 RNA polymerase were purchased from Fermentas, and the corresponding corresponding The buffer, etc.
  • the Hieff Clone TM One Step cloning kit used in the examples was purchased from Shanghai Yisheng Biotechnology Co., Ltd.
  • inorganic salt chemical reagents are 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 minipump 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).
  • a primer containing a T7 promoter is used to amplify the cDNA corresponding to the RNA to be detected, and the T7 RNA polymerase (purchased from Fermentas) is used to transcribe the recovered double-stranded cDNA as a template to obtain RNA.
  • 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.
  • 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 to cultivate.
  • FBS fetal bovine serum
  • DMEM penicillin high glucose medium
  • the main imaging experiment in the examples is to use the Leica SP8 confocal laser microscope to shoot, use the HCXPL APO 63.0x1.47 oil lens and HyD detector.
  • a 488nm laser was used in order to photograph the fluorescence of the Pepepr-III-3 complex.
  • 405nm and 561nm lasers were used respectively.
  • 458nm, 458nm, 488nm, 488nm, 488nm, 561nm, 561nm laser In order to photograph the fluorescence of Broccoli-DFHBI-1T and Corn-DFHO, a 488nm laser was used respectively.
  • Preparation of linearized vector select a suitable cloning site and linearize the vector.
  • the linearized vector can be prepared by restriction digestion or inverse 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 for linearized vector and insert. After the preparation of the system is complete, mix the components and place them at 50°C for 20 minutes. When inserts> 5kb, the incubation temperature can be extended to 25min. After the reaction is complete, it is recommended to place the reaction tube on ice to cool for 5 minutes. The reaction product can be converted directly, or stored at -20°C, and thawed for conversion when needed.
  • nucleic acid aptamer molecules Prepare Pepper or Pepper mutant nucleic acid aptamer molecules according to the common experimental method (1), and mix 5 ⁇ M nucleic acid aptamer molecules and 1 ⁇ M fluorophore molecules in detection buffer (40mM HEPES, pH 7.4, 125mM KCl, 5mM MgCl 2 , 5 %DMSO), and use the Synergy Neo2 multifunctional microplate reader to detect the maximum excitation peak and maximum emission peak of the fluorescence of the nucleic acid aptamer-fluorophore molecular complex.
  • detection buffer 40mM HEPES, pH 7.4, 125mM KCl, 5mM MgCl 2 , 5 %DMSO
  • the Synergy Neo2 multifunctional microplate reader to detect the fluorescence intensity of the nucleic acid aptamer-fluorophore molecule complex under its maximum excitation and emission conditions, and the control sample (1 ⁇ M fluorophore molecule without nucleic acid aptamer) is also the same Measure under the conditions and calculate the ratio of fluorescence intensity.
  • the fluorescence maximum excitation peak of the complex formed by 5 ⁇ M F30-Pepper-2 nucleic acid aptamer and 1 ⁇ M III-3 fluorophore molecule is 485nm, and the maximum emission peak is 530.
  • Synergy Neo2 multifunctional microplate reader to detect the fluorescence intensity of the complex under 485 ⁇ 10nm excitation and 530nm ⁇ 10nm emission conditions is 36000, while the fluorescence intensity of the control (1 ⁇ M III-3 fluorophore molecule) under the same detection conditions is 10. Then the F30-Pepper-2 nucleic acid aptamer activates the III-3 fluorophore molecule by 3600 times.
  • Pepper contains 2 stem structures, 2 loop structures and 1 stem loop structure (Figure 1A).
  • Figure 1B the secondary structure predicted by Pepper-1 (SEQ ID NO: 1) is Figure 1B.
  • F30-Pepper-1 SEQ ID NO: 2 RNA was prepared according to the common experimental method (1). Incubate 1 ⁇ M III-3 with 5 ⁇ M F30-Pepper-1. The test results show that the maximum excitation light of the F30-Pepper-1-III-3 complex is 485nm and the maximum emission light is 530nm ( Figure 4A).
  • F30-Pepper-1 was identified by Native PAGE, and the nucleic acid aptamer F30-Broccoli (SEQ ID NO: 4) and F30-2dBroccoli (SEQ ID NO: 5) (Filonov et al. Journal of the American Chemical Society 2014.136: 16299-16308; Filonov et al. Chemistry&biology 2015.22:649-660) as a control.
  • F30-Pepper-1-III-3 complex was placed in a different pH environment for 60 minutes to detect the fluorescence value. Take F30-Broccoli-DFHBI-1T complex as control.
  • the test results show that the F30-Pepper-1-III-3 complex maintains a high fluorescence signal in the range of pH 5-9, while the fluorescence of F30-Broccoli-DFHBI-1T decreases rapidly with the decrease of pH ( Figure 4F) shows that F30-Pepper-1-III-3 complex has better pH stability.
  • the F30-Broccoli-DFHBI-1T complex is in LiCl
  • the fluorescence in the buffer is only one hundredth of that in the KCl buffer ( Figure 4G).
  • the fluorescence of the F30-Pepper-1-III-3 complex does not depend on the presence of K + ions ( Figure 4G), indicating that there is no G-quadruplex structure in the Pepper structure.
  • the Pepper-1 sequence in F30-Pepper-1 was subjected to point mutations as shown in Table 1, and prepared according to common experimental methods (1) containing different bases
  • 1 ⁇ M III-3 was incubated with 5 ⁇ M different F30-Pepper-1 mutant RNA, and according to the common experimental method (5) their fluorescence activation multiples of III-3 fluorophore molecules.
  • the test results showed that most of the F30-Pepper-1 mutants containing single-base mutations retain a strong fluorescence activation effect (>10 times) on III-3 (Table 2).
  • F30-Pepper-1 in Table 2 is a nucleic acid aptamer with the sequence SEQ ID NO: 2; other aptamers are in the Pepper-1 sequence of F30-Pepper-1 and correspond to Pepper in Figure 1A. Point mutation at the nucleotide position.
  • the base-modified Pepper-3 (SEQ ID NO:6) was synthesized.
  • the sequence GGCCCCCAAUCGUGGCGUGUCGG CCUGCUUCGGCAGG CACUGGCGCCGGGGCC underlined bases is deoxyribonucleotide base Base) and Pepper-4 (SEQ ID NO: 7, the sequence GGCCCCCCAAUCGUGGCGUGUCGG C CUGCUUCGGC
  • a GGCACUGGCGCCGGGGGCC in the underlined base is the base modified by 2'-F) (synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.), they The bases containing the stem-loop structure were replaced with deoxyribonucleotides (the bases shaded in Figure 5A) and some of the bases were 2'-F modified (the bases shaded in Figure 5B).
  • Pepper In order to detect the activation effect of Pepper series body on III-3 fluorescence, Pepper is connected in series according to different forms, including the following three types:
  • nPepper where n is an arbitrary copy Pepper.
  • the cDNA encoding F30-2Pepper-5, F30-4Pepper-5, F30-8Pepper-5, F30-16Pepper-5 and F30-32Pepper-2 are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12).
  • RNA After PCR amplification, prepare nucleic acid aptamers according to common experimental methods (1) For RNA, after incubating 0.1 ⁇ M RNA aptamer with 10 ⁇ M III-3, detect the fluorescence intensity according to the common experimental method (5). The test results showed that with the increase of n, the fluorescence of nPepper-III-3 also increased ( Figure 6D). When n>8, with the increase of n, the fluorescence of nPepper-III-3 does not increase in equal multiples, but it is still much higher than that of Pepper-III-3 ( Figure 6D), indicating that the "series 1" can be used Ways to increase the fluorescence intensity of Pepper-III-3 complex.
  • the coding cDNAs of 2x2Pepper-5, 4x2Pepper-5, and 8x2Pepper-5 are respectively synthesized by the whole gene (the sequence of the coding RNA aptamer is SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19)
  • the sequence of the coding RNA aptamer is SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19
  • F30-Pepper-1RNA aptamer molecules Prepare F30-Pepper-1RNA aptamer molecules according to common experimental methods (1), and use them to detect the basic properties of III-3 analogs and Pepper binding, including fluorescence spectrum, molar extinction coefficient, quantum yield, fluorescence activation multiple and binding Constant (Kd), the test results are shown in Table 4. From the data in the table, it can be seen that F30-Pepper-1 can activate the fluorescence intensity of III-3 analogs to varying degrees.
  • a bacterial expression plasmid expressing F30-Pepper-1 was first constructed.
  • the primers were used to amplify F30-Pepper-1 in Example 2, and the primers were used to amplify pET28a.
  • the promoter and multiple cloning site regions were removed, and the amplified F30-Pepper-1 DNA fragment was linearized with pET28a
  • the vector was connected according to the experimental method (4), and the obtained recombinant plasmid was named pET28a-T7-F30-Pepper-1.
  • the primers used to amplify the F30-Pepper-1 fragment are:
  • Upstream primer (P1) 5’-TCGATCCCGCGAAATTAATACGACTCACTATAGGGTTGCCA
  • the primers used to amplify the pET28a vector to linearize it are:
  • Upstream primer 5’-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG-3’
  • a yeast expression plasmid expressing F30-Pepper-1 was first constructed.
  • the F30-Pepper-1 DNA fragment in Example 2 was amplified using primers, and the amplified F30-Pepper-1 fragment was inserted into the pYES2.1TOPO TA vector according to experimental method (4), and the resulting recombinant plasmid was named pYES2.1-F30-Pepper-1.
  • the primers used to amplify the F30-Pepper-1 fragment are:
  • Upstream primer (P5): 5’-GGAATATTAAGCTCGCCCTTTTGCCATGTGTATGTGGG-3’
  • the pYES2.1-F30-Pepper-1 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-Pepper-1.
  • the bacteria were harvested after 10 hours. Resuspend in PBS containing 2 ⁇ M III-3.
  • the untreated BY4741 strain was used as a control.
  • the results show that only when F30-Pepper-1 is expressed and in the presence of III-3, yeast cells can show bright yellow-green fluorescence (Figure 8), indicating that Pepper-III-3 complex can be used for RNA in yeast cells Fluorescent labeling.
  • Example 9 Pepper, III-3 and their analogs are used for labeling of RNA in mammalian cells
  • Primers P7 and P8 were used to amplify F30-Pepper-1 and F30-Broccoli in Example 2, and primers P9 and P10 were used to amplify the tRNA-Corn cDNA fragment synthesized by the whole gene (the encoded RNA sequence is SEQ ID No: 20) , Using experimental method (four) to insert these fragments into pLKO.1puro vector.
  • the resulting expression vectors are named pLKO.1-F30-Pepper-1, pLKO.1-F30-Broccoli and pLKO.1-tRNA-Corn, and these plasmids express F30-Pepper-1, F30-Broccoli and tRNA-Corn RNA, respectively .
  • the primers used to amplify F30-Pepper-1 and F30-Broccoli are:
  • Upstream primer (P7) 5’-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGG-3’;
  • the primers used to amplify tRNA-Corn are:
  • Downstream primer (P10) 5'-TGTCTCGAGGTCGAGAATTCAAAAAAATGGCGCCCGAACAGGGACTTGCGAGCTCAGGATCCTTCCGTTTCGCACTGG-3'.
  • a mammalian expression plasmid expressing F30-8Pepper-5 was constructed.
  • the primers P7 and P8 in this example were used to amplify the F30-8Pepper-5 fragment in Example 5, and these fragments were inserted into the pLKO.1puro vector using experimental method (4).
  • the resulting expression vector was named pLKO.1-F30-8Pepper-5.
  • the pLKO.1-F30-8Pepper-5 plasmid was transfected into 293T/17 cells. After 24 hours, different III-3 analogues were added for labeling, and the labeling effect was tested by experimental method (3). The results show that different III-3 analogs can specifically label cells expressing F30-8Pepper-5, but not control cells that do not express F30-8Pepper-5 ( Figure 9C), indicating that Pepper and III-3 and their Analogs can be used to label RNA in mammalian cells.
  • RNA aptamers In order to construct a Pepper-based analyte probe, the nucleotides at the stem-loop structure in the Pepper-1 (SEQ ID No: 2) structure were replaced with those that can specifically recognize and bind adenosine and guanosine (GTP). ) RNA aptamers, these aptamers and Pepper-1 are connected with bases of different length and composition ( Figure 10A). According to the common experimental method (1), prepare probe RNA and combine it with III-3 Incubate, and use a multifunctional microplate reader to detect their fluorescence intensity in the presence or absence of adenosine or GTP.
  • the test results show that for the adenosine probe, when the base pair between the adenosine aptamer and Pepper-1 is the base pair of connection 2 in Figure 10B, the activation factor can reach 88 times, and the corresponding probe RNA
  • the sequence is SEQ ID No: 21.
  • the activation factor is 10 times, and the corresponding probe RNA sequence is SEQ ID No: 22.
  • Example 11 Pepper is used to track RNA localization in cells
  • RNA expression plasmid in which Pepper is fused with different RNAs is first constructed.
  • the cDNA of 4Pepper-7 was synthesized by the whole gene (the sequence encoding the RNA aptamer is SEQ ID No: 23), and the 4Pepper-7 gene fragment was amplified using primers, and inserted into the HindIII and XhoI pairs by homologous recombination.
  • the pCDNA3.1hygro(+) vector was digested to obtain the pCDNA3.1hygro(+)-4Pepper-7 recombinant plasmid.
  • the primers used to amplify 4Pepper-7 are:
  • the primers used to amplify GAPDH are:
  • the primers used to amplify TMED2 are:
  • Upstream primer 5’-GGAGACCCAAGCTGGCTAGCATGGTGACGCTTGCTGAACT-3’
  • Downstream primer 5’-GGATCCTCCGTGGGAAGCTTAACCATGCTCTAGCGAGTTAAACAACTCTCCGGACTTC-3’
  • the plasmid was extracted with a transfection-grade plasmid extraction kit for subsequent transfection experiments.
  • the recombinant plasmids pCDNA3.1hygro(+)-GAPDH-4Pepper-7 and pCDNA3.1hygro(+)-TMED2-4Pepper-7 constructed in this example were co-transfected with pCDNA3.1hygro(+)-BFP into COS-7 cells, respectively , 24h after transfection, the cells were imaged according to the fluorescence imaging method described in the specific experimental method (3).
  • the imaging results show that the fluorescence of GAPDH-4Pepper-7-III-3 is mainly concentrated in the cytoplasm, while the fluorescence of TMED2-4Pepper-7-III-3 can observe the phenomenon of endoplasmic reticulum enrichment, which is different from the previous
  • the report is consistent, and the results of fluorescent-labeled in situ hybridization (FISH) are also consistent ( Figure 11).
  • FISH fluorescent-labeled in situ hybridization
  • Example 12 Pepper is used to detect the relationship between mRNA and protein content in cells
  • Pepper In order to use Pepper to detect mRNA translation in cells, it is first necessary to construct mRNA expression plasmids fused with different Peppers. Use primers to amplify mCherry and TagBFP gene fragments using mCherry2-N1 (Addgene: 54517) and EasyFusion T2A-H2B-TagBFP (Addgene: 113086) as templates, and insert them into the pCDNA3.1hygro (double digested with NheI and HindIII).
  • +)-GAPDH-4Pepper-7 vector obtain pCDNA3.1hygro(+)-mCherry-4Pepper-7 and pCDNA3.1hygro(+)-TagBFP-4Pepper-7 recombinant plasmids, which encode mCherry-4Pepper-7 and TagBFP, respectively -4Pepper-7, its RNA sequence is SEQ ID No: 26 and 27 respectively.
  • the primers used to amplify mCherry are:
  • the primers used to amplify TagBFP are:
  • Upstream primer (P19): 5’-GGAGACCCAAGCTGGCTAGCATGAGCGAGCTGATTAAGGA-3’
  • Downstream primer 5’-GGATCCTCCGTGGGAAGCTTCTCCCAAACCATGCTCTAGCGAGTGTTAATTGAGCTTGTGCCCCA-3’
  • Recombinant plasmids pCDNA3.1hygro(+)-BFP-4Pepper-7 and pCDNA3.1hygro(+)-mCherry-4Pepper-7 were transfected into COS-7 cells, 24h later, pCDNA3 was transfected with 0.2 ⁇ M III-3 marker .1hygro(+)-BFP-4Pepper-7 and pCDNA3.1hygro(+)-mCherry-4Pepper-7 recombinant plasmid cells, using flow cytometry to detect mRNA (4Pepper-7-III-3) fluorescence and protein ( The fluorescence of BFP and mCherry) is fitted to the fluorescence of mRNA and protein by Mie equation to obtain R 2 . The test results show that the translation efficiency of different mRNAs is significantly different ( Figure 12), indicating that Pepper can be used to detect the relationship between mRNA and protein content.
  • Example 13 Pepper is used to detect genomic DNA
  • a recombinant plasmid expressing chimeric RNA of Pepper-8 and sgRNA was first constructed. Full-gene synthesis of sgRNA-Pepper-8 (loop1), sgRNA-Pepper-8 (tetraloop) and sgRNA-Pepper-8 (loop1 and tetraloop) cDNAs containing centromere targeting sequences, the encoded RNA sequences are respectively SEQ ID No: 28, 29, and 30.
  • Use primers P21 and P22 to amplify the cDNA of the above chimeric RNA use primers P23 and P24 to amplify the psgRNA plasmid (Shao et al.
  • the primers used to amplify the cDNA corresponding to Pepper and sgRNA chimeric RNA are:
  • the primers for amplifying the psgRNA plasmid to linearize it are:
  • the primers used to amplify SpdCas9-GFP are:
  • Upstream primer 5’-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCACCATGGCCCC-3’
  • Example 14 Pepper is used for super-resolution imaging of RNA
  • Pepper In order to use Pepper for super-resolution imaging of RNA, first construct a plasmid that anchors RNA to the nucleus.
  • the primers used to amplify the 4Pepper-9-MS2 DNA fragment are:
  • Upstream primer 5’-GGAAAGGACGAAACTCTAGAGGGGCCCCCCAATCGTGG-3’
  • the primers used to amplify the tdMCP gene fragment are:
  • Upstream primer 5’-GAACCGTCAGATCCGCTAGCCACCATGGGCTACCCCTACGACGTGCCCG-3’
  • the primers used to amplify the tagBFP gene fragment are:
  • Upstream primer 5’-CTACGCGGATTCTGGAGGCGGTGGATCCATGAGCGAGCTGATTAAGGAG-3’
  • the primers used to amplify H2B gene fragments are:
  • Upstream primer 5’-CAAGCTCAATAGATCTATGCCTGAACCGGCAAAATCC-3’
  • the pLKO.1-4Pepper-9-MS2 and pH2B-tdPP7-tagBFP recombinant plasmids were co-transfected into COS-7 cells. After 24 hours, they were labeled with III-21 fluorophore molecules, and then detected by Zeiss Elyra PS.1 super-resolution fluorescence microscope. Fluorescence distribution of Pepper-III-21 complex, excitation light uses 561 long-pass filter, lens is Zeiss Plan-Apochromat 63 ⁇ (NA,1.4)Oil DIC M27, CMOS size is 1024x1024 pixels, picture uses ZEN 2011Black(Zeiss) Software for processing.
  • the recombinant plasmids pCDNA3.1hygro(+)-TagBFP-4Pepper-7 and pCDNA3.1hygro(+)-mCherry-4Pepper-7 in Example 12 were transfected into COS- 7 cells, 24 hours later, the cells were collected and the total RNA of the cells was extracted using the Easystep Super Total RNA Extraction Kit (Promega). The extracted total RNA was dissolved in a buffer containing 40 mM HEPES, pH 7.4, 125 mM KCl, 5 mM MgCl 2 and incubated at 70°C for 10 minutes, and then placed at room temperature for more than 30 minutes.
  • the recovered RNA was identified by electrophoresis, and the gel after the run was incubated with a buffer containing 5 ⁇ M III-3 in 40 mM HEPES, pH 7.4, 125 mM KCl, and 5 mM MgCl 2 for 30 min, and the fluorescence of 4Pepper-III-3 in the gel was detected.
  • the imaging results show that there are 3 RNA bands in the gel showing obvious Pepper-III-3 fluorescence signals, TagBFP-4Pepper, and mCherry-4Pepper ( Figure 15), indicating that Pepper can be used as a label for RNA isolation And purification.
  • 6-(N-methyl-N-hydroxyethyl)amino-pyrazine-3-aldehyde 6-(N-methyl-N-hydroxyethyl)amino-pyrazine-3-aldehyde:
  • 6-methylamine-benzo[b]thiophene-2-carbaldehyde 6-methylamine-benzo[b]thiophene-2-carbaldehyde:
  • 6-Bromobenzo[b]thiophene-2-carbaldehyde (0.42g, 1.7mmol), dimethylethylamine (40% aqueous solution, 1g, 8.9mmol), CuI (13.9mg, 0.073mmol), K 3 PO 4 ⁇ H 2 O (155.4mg, 0.73mmol), methylamine (33% aqueous solution, 1g) in a 100ml pressure-resistant bottle, heated in an oil bath at 60°C for 12h under sealed conditions, cooled the system to room temperature, added 50ml water, and extracted with DCM ( 3 ⁇ 100 ml), the organic phases were combined, dried over Na 2 SO 4 , the organic solvent was removed under reduced pressure, and the residue was separated and purified by column chromatography (0.23 g, 68%).

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Abstract

提供一种适配体核酸分子，包含该适配体核酸分子和荧光团小分子的复合物，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，以及包含该适配体核酸分子的试剂盒。该适配体核酸分子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

一种适配体核酸分子及其复合物和应用 技术领域
本申请涉及一种适配体核酸分子，包含该适配体核酸分子的复合物，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。本申请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
在所有的生物大分子中，RNA展示出了种类最为繁多的生物学功能。在生物的中心法则中，RNA作为遗传物质的传递者(信使RNA)、蛋白质合成的模板(核糖体RNA)和氨基酸运输工具(转移RNA)，组成了一连串的生理过程，最终实现基因的转录与表达。过去的几十年里，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，近些年来一些非编码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。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。目前有科学家们已经鉴定出了几种机理可以导致RNA的不同亚细胞定位，如主动运输、被动扩散，锚定等。在很多极性细胞特别是神经细胞中，mRNA的空间特异性表达与神经元的可塑性、学习和记忆都密切相关。因此，RNA的这些调控过程一旦被损坏就会引起神经元功能紊乱和神经系统疾病。
RNA荧光原位杂交技术是长期以来被广泛用于来研究RNA在细胞内水平与分布的方法，它是通过分子杂交对特异RNA分子进行荧光标记，进而进行成像的技术。然而其操作较复杂且含有洗脱步骤，只能用于固定化细胞亦即死细胞的研究，不能用于实时监测活细胞中RNA的动态变化过程。分子信标技术是最早发展起来的活细胞RNA成像技术。它是利用在5’和3’末端自身形成发夹结构的茎环双标记寡核苷酸探针，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。然而分子信标存在着荧光信号偏低、进细胞困难、易降解、较严重的细胞核内非特异性聚集、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，这些缺点限制了该技术的广泛应用。
目前用于活细胞RNA成像的方法主要是利用MCP-FPs系统，MCP-FPs可以特异性识别结合融合了多拷贝MS2序列的mRNA分子，通过检测荧光蛋白的信号实时监测mRNA的合成及分布(Ozawa et al.Nature Methods 2007.4:413-419)。但由于未结合mRNA分子的MCP-FPs会产生很高的本底荧光，使得该方法的信噪比很低。随后，科学家们将MCP-FPs 融合蛋白加上核定位信号，使得没有与mRNA分子结合的GFP-MS2定位细胞核中，一定程度降低了细胞浆中的非特异荧光，增加了检测的信噪比。
除了RNA结合蛋白-荧光蛋白技术检测细胞RNA外，科学家们一直在寻找一个类似GFP的RNA荧光标签用于RNA成像。科学家们构建了一个荧光团-淬灭剂结合体，当荧光团的适配体(Aptamer)与荧光团结合时，淬灭剂就不能将荧光团的荧光信号淬灭，此时适配体-荧光团-淬灭剂构成的复合物是有荧光的。当荧光团的适配体不存在时，荧光团的荧光信号就会被淬灭剂淬灭。基于这样的原理，科学家们实现了对细菌中mRNA的成像(Arora et al.Nucleic Acids Research 2015.21:e144)。此外，人们还开发了一个称为IMAGE(intracellular multi aptamer genetic)的标签，它由两种不同的适配体-小分子复合物构成。当小分子结合到RNA序列中的适配体上时，相邻的两个小分子所携带的荧光团就会发生荧光共振能量转移(FRET)现象，通过检测荧光信号的变化就可以检测细胞中RNA情况。然而这两种方法目前均没有在哺乳动物细胞中实现对RNA的实时监测。2011年S.Jaffrey课题组获得了一个称为“Spinach”的核酸适配体，它可以特异性结合一个荧光团(3,5-difluoro-4-hydroxybenzyli-dene imidazolinone，DFHBI)，使得其荧光得到显著增加(Paige et al.Science 2011.333:642-646；Strack et al.Nature Methods 2013.10:1219-1224)。“Spinach”的突变体“Spinach2”有着更好的稳定稳定性，它提供了一个很好的工具用于遗传编码标记活细胞中的RNA。该课题组将”Spinach”中的一个茎环结构换成可以特异性结合细胞代谢物的核酸适配体，开发了基于Spinach-DFHBI复合物的可以检测细胞代谢物的工具(Paige et al.Science 2012.335:1194)。到目前为止，该方法已被成功用于分监测和分析细菌、酵母和哺乳动物细胞中的RNA动态变化。随后，该课题组还开发了Corn-DFHO复合物用于检测哺乳动物细胞RNA聚合酶III启动子的活性检测(Song et al.Nature Chemical Biology 2017.13:1187-1194)。然而，该方法还存在以下缺点大大限制了它的广泛应用：(1)适配体-荧光团复合物的结合能力较弱，其解离常数(kd)为几十到几百nM；(2)适配体-荧光团形成的复合物的荧光信号不稳定，极容易发生淬灭，使得其荧光信号不宜被检测(Han et al.Journal of the American Chemical Society 2013.135:19033-19038)；(3)目前为止，光谱只有绿色和黄色，缺乏更长波长的光谱对活体动物体内的RNA进行成像(Song et al.Journal of the American Chemical Society 2014.136:1198-1201)；(4)Corn为二聚体，可能会干扰靶标RNA的功能；(5)目前还没有其他的适配体-荧光团复合物，不能同时监测细胞中的多条RNA。
综上所述，目前使用的RNA标记技术都存在各自的明显的缺点。MCP-FPs标记技术存在着非结合的本底荧光强，信噪比低。基于适配体-荧光团-淬灭剂构成的复合物的RNA标记技术目前只在细菌中实现对RNA的标记，还没有实现在哺乳动物细胞中标记RNA。基于单荧光团-核酸适配体的RNA标记技术看起来是非常完美的RNA标记技术，然而受限于当前的 荧光团(DFHBI，DFHBI-1T，DFHO)与核酸适配体形成的复合体的性质不理想，该技术也没有被广泛使用。因此，科研界和产业界一直都需要更加有效的荧光团-核酸适配体复合物，能克服此前荧光团-核酸适配体复合物的缺点，用于活细胞中的RNA或DNA的实时标记。
技术解决方案
本申请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。
本申请提供
本申请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)、(b)或(c)：
(a):核苷酸序列N 1CCAAUCGUGGCGUGUCGN 19-N 20-N 21ACUGGCGCCGN 32(称为通式Pepper结构)，其中N 1、N 19、N 20、N 21和N 32代表长度大于或等于1个的核苷酸片段，并且N 1与N 32核苷酸序列中至少有一对碱基形成互补配对，N 19与N 21核苷酸序列中至少有一对碱基形成互补配对；
(b):与(a)限定的核苷酸序列具有至少70％同一性的核苷酸序列；
(c):在(a)限定的核苷酸序列中不包括N 1、N 19、N 20、N 21和N 32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(a)衍生的核酸适配体分子。
在一些实施例中，所述核苷酸序列(b)与核苷酸序列(a)限定的通式Pepper结构核苷酸序列具有至少75％，76％，78％，80％，82％，85％，87％，90％，93％，95％，96％，97％，98％，99％或100％同一性。在一些实施例中，核苷酸序列(c)是在核苷酸序列(a)限定的通式Pepper结构核苷酸序列中不包括N 1、N 19、N 20、N 21和N 32的位置，经过10个、9个、8个、7个、6个、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。在一些实施例中，所述核苷酸序列(c)是在(a)限定的核苷酸序列中不包括N 1、N 19、N 20、N 21和N 32的位置，经过7个、6个、5个、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。
在一些实施例中，所述核苷酸序列(a)中的N 1与N 32互补配对时，N 1核苷酸序列的方向为5’-3’，N 32核苷酸序列的方向为3’-5’；N 19与N 21互补配对时，N 19核苷酸序列的方向为5’-3’，N 21核苷酸序列的方向为3’-5’。
在一些实施例中，当所述核苷酸序列(a)中的N 1与N 32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 32核苷酸序列中至少有两对碱基形成互补配对；当N 19与N 21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 19与N 21核苷酸序列中至少有两对碱基形成互补配对。
在一些实施例中，对通式Pepper结构的核苷酸取代选自下组中的一种：C3A、C3U、A4U、A4G、A4C、A5G、A5C、U6A、U6G、U6C、C7A、C7U、G8C、U9A、G11A、G11U、 C12G、C12A、C12U、G13C、U14A、U14G、C17U、G18U、G18C、C27G、C27U、G28U、C29G、C29U、C30A、C30U、C2G/G31C、C2U/G31A、C2A/G31U、G10A/C30U、G10C/C30G、G10U/C30A、C2G/G31C/C3A、C2G/G31C/A4C、C2G/G31C/A5C、C2G/G31C/G8C、C2G/G31C/C12U、C2G/G31C/U14G、C2G/G31C/C27U、C2G/G31C/C29G、C2G/G31C/C30U、C2G/G31C/G10A/C30U、C2G/G31C/G10C/C30G、C2G/G31C/G10U/C30A、C2U/G31A/G10A/C30U、C2U/G31A/G10C/C30G、C2U/G31A/G10U/C30A、C2A/G31U/G10A/C30U、C2A/G31U/G10C/C30G、C2A/G31U/G10U/C30A、C2G/G31C/G10C/C30G/C3A、C2G/G31C/G10C/C30G/A4C、C2G/G31C/G10C/C30G/A5C、C2G/G31C/G10C/C30G/G8C、C2G/G31C/G10C/C30G/C12U、C2G/G31C/G10C/C30G/U14G、C2G/G31C/G10C/C30G/C27U、C2G/G31C/G10C/C30G/C29G、C2G/G31C/G10A/C30U/U6G/C27U、C2G/G31C/G10C/C30G/U6G/C27U、C2G/G31C/G10U/C30A/U9A/U14G/C27U和C2A/G31U/G10U/C30A/U9A/U14G/C27U。
在一些实施例中，对通式Pepper结构的核苷酸取代选自下组中的一种：C3A、C3U、A4C、A5C、C7U、G8C、U9A、C12G、C12U、U14G、C27U、C29G、C30U、C2G/G31C、C2U/G31A、C2A/G31U、G10A/C30U、G10C/C30G、G10U/C30A、C2G/G31C/C3A、C2G/G31C/A4C、C2G/G31C/A5C、C2G/G31C/G8C、C2G/G31C/C12U、C2G/G31C/U14G、C2G/G31C/C27U、C2G/G31C/C29G、C2G/G31C/C30U、C2G/G31C/G10A/C30U、C2G/G31C/G10C/C30G、C2G/G31C/G10U/C30A、C2U/G31A/G10A/C30U、C2U/G31A/G10C/C30G、C2U/G31A/G10U/C30A、C2A/G31U/G10A/C30U、C2A/G31U/G10C/C30G、C2A/G31U/G10U/C30A、C2G/G31C/G10C/C30G/C3A、C2G/G31C/G10C/C30G/A4C、C2G/G31C/G10C/C30G/A5C、C2G/G31C/G10C/C30G/G8C、C2G/G31C/G10C/C30G/C12U、C2G/G31C/G10C/C30G/U14G、C2G/G31C/G10C/C30G/C27U、C2G/G31C/G10C/C30G/C29G、C2G/G31C/G10A/C30U/U6G/C27U和C2G/G31C/G10C/C30G/U6G/C27U。
在一些实施例中，对通式Pepper结构的核苷酸取代选自下组中的一种：C3A、C3U、A4C、A5C、C7U、G8C、U9A、C12G、C12U、U14G、C27U、C29G、C30U、C2G/G31C、C2U/G31A、C2A/G31U、G10A/C30U、G10C/C30G、G10U/C30A、C2G/G31C/C3A、C2G/G31C/A4C、C2G/G31C/A5C、C2G/G31C/G8C、C2G/G31C/C12U、C2G/G31C/U14G、C2G/G31C/C27U、C2G/G31C/C29G、C2G/G31C/C30U、C2G/G31C/G10A/C30U、C2G/G31C/G10C/C30G、C2G/G31C/G10U/C30A、C2U/G31A/G10A/C30U、C2U/G31A/G10C/C30G、C2U/G31A/G10U/C30A、C2A/G31U/G10A/C30U、C2A/G31U/G10C/C30G和C2A/G31U/G10U/C30A。
在一些实施例中，所述核苷酸序列(a)中的N 1与N 32处的核苷酸序列为F30或tRNA脚 手架RNA序列。
在一些实施例中，所述核酸适配体分子是RNA分子或经碱基修饰的RNA分子。
在一些实施例中，所述核酸适配体分子是DNA-RNA杂交分子或经碱基修饰的DNA-RNA分子。
在一些实施例中，所述核苷酸序列(a)中的N 19-N 20-N 21包含一个可以识别靶标分子的核苷酸序列。
在一些实施例中，所述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。
在一些实施例中，所述核苷酸序列(a)中的N 19-N 20-N 21为可以识别GTP和腺苷分子的核苷酸序列。
在一些实施例中，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倍或者提高至少500-1000倍。
在一些实施例中，所述核酸适配体分子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，所述间隔序列具有2、3、4、5、6、7、8或者更多个核苷酸片段的长度。所述串联体的核苷酸可选自但不限于序列SEQ ID No：8、9、10，11、12、13、14、15、16、17、18和19。
在一些实施例中，所述核酸适配体分子具有序列SEQ ID No：1、2、3、6、7、8、9、10、11、12、13、14、15、16、17、18、19、21、22或23。
本申请还提供一种核酸适配体分子与荧光团分子的复合物，其中所述核酸适配体分子为上述任意一种核酸适配体分子，所述荧光团分子具有下述式(I)所述的结构：
Figure PCTCN2020087415-appb-000001
其中：D-为X1O-或N(X2)(X3)-；X1、X2、X3各自独立地选自氢、1-10个碳的直链或支链烷基和改性烷基，X2、X3任选相互连接为饱和或不饱和的环；R-选自氢、氰基、羧基、酰胺基、酯基、羟基、1-10个碳的直链或支链烷基或改性烷基；Ar1、Ar2各自独立地选自单环芳亚基、单环杂芳亚基，或由单环芳基、单环杂芳基中的一种或两种稠合组成的具有2-3个环结构的芳香亚基；
其中：Ar1、Ar2中的氢原子可以独立地被F、Cl、Br、I、羟基、硝基、醛基、羧基、氰基、磺酸基、硫酸基、磷酸基、氨基、伯氨基、仲氨基、1-10个碳的直链或支链烷基和改性烷基取代；
其中：以上所述改性烷基为烷基的任意碳原子被选自F、Cl、Br、I、-O-、-OH、-CO-、-NO2、-CN、-S-、-SO2-、-(S＝O)-、叠氮基、亚苯基、伯氨基、仲氨基、叔氨基、季铵盐基、环氧乙烷、琥珀酸酯、异氰酸酯、异硫氰酸酯、酰氯、磺酰氯、饱和或不饱和的单环或双环亚环羟基、桥联酯杂环中的至少一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，所述复合物中的所述核酸适配体分子与所述荧光团分子分别存在于单独的溶液中，或者所述核酸适配体分子与所述荧光团分子在同一溶液中。
在一些实施例中，所述改性烷基含有选自-OH、-O-、乙二醇单元、单糖单元、二糖单元、-O-CO-、-NH-CO-、-SO 2-O-、-SO-、Me 2N-、Et 2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、-NO 2和氰基中的至少一种基团。
在一些实施例中，所述荧光团分子含有的芳香环选自下式(Ⅱ-1)～(Ⅱ-15)中的结构：
Figure PCTCN2020087415-appb-000002
在一些实施例中，荧光团分子选自下式化合物：
Figure PCTCN2020087415-appb-000003
Figure PCTCN2020087415-appb-000004
在一些实施例中，所述复合物中的所述荧光团分子选自III-1、III-2、III-3、III-4、III-5、III-6、III-7、III-8、III-9、III-10、III-11、III-12、III-13、III-14、III-15、III-16、III-17、III-18、III-19、III-20和III-21。
在一些实施例中，所述复合物中的所述适配体分子包含核苷酸序列SEQ ID No：1、2、3、6、7、8、9、10、11、12、13、14、15、16、17、18、19、21、22、23、24、25、26、27、28、29、30或31。
本申请还提供一种上述任意一种复合物用于体外或体内目标核酸分子的检测或标记。
本申请还提供一种上述任意一种复合物用于细胞外或细胞内靶标分子的检测或标记。
本申请还提供一种上述任意一种复合物用于对基因组DNA进行成像。
本申请还提供一种上述任意一种复合物用于检测细胞中mRNA与蛋白质含量的关系。
本申请还提供一种DNA分子，其转录上述任意一种核酸适配体分子。
本申请还提供一种表达载体，其包含上述DNA分子。
本申请还提供一种宿主细胞，其包含上述表达载体。
本申请还提供一种试剂盒，包含上述任意一种核酸适配体分子和/或上述任意一种表达载体和/或上述任意一种宿主细胞和/或上述任意一种复合物。
本申请还提供一种检测靶标分子的方法，包括步骤：
在包含靶标分子的溶液中加入上述任意一种复合物；
用合适波长的光激发复合物；
检测复合物的荧光。
本申请还提供一种检测基因组DNA的方法，包含上述任意一种复合物对基因组DNA进行成像。
本申请还提供一种提取与纯化RNA的方法，包含利用上述任意一种复合物提取与纯化RNA。
有益效果
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，以组成全新的荧光团-核酸适配体复合物。所述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。本申请的核酸适配体对荧光团分子具有很强的亲和力，并展现出了不同的荧光光谱和很好的光与温度稳定性。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，检测蛋白质-RNA相互作用，探究细胞中mRNA含量与蛋白质间关系，或用于RNA的提取与纯化的标签等作用。
附图说明
图1.核酸适配体分子的二级结构预测。(A)为预测的Pepper的通用结构，包括可以形成茎结构的N 1和N 32，可以形成茎环结构的N 19、N 20和N 21。(B)为预测的Pepper-1的结构，N 1和N 32的碱基序列如图中茎1所对应的虚线框所示，N 19、N 20和N 21的碱基序列如茎环所对应的虚线框所示。
图2.F30-Pepper-1的二级结构预测。
图3.tRNA-Pepper-2的二级结构预测。
图4.F30-Pepper-1-III-3复合物性质鉴定。(A)F30-Pepper-1-III-3复合物的荧光激发光谱和发射光谱；(B)F30-Pepper-1-III-3复合物和III-3的吸收光谱；(C)F30-Pepper-1-III-3复合物的寡聚化鉴定；“标尺”为单链DNA标准，用于标定适配体的大小。(D)F30-Pepper-1与III-3结合的解离常数测定；(E)F30-Pepper-1-III-3复合物温度稳定性测定；(F)F30-Pepper-1-III-3复合物的pH稳定性测定；(G)F30-Pepper-1-III-3复合物对K +的依赖性测定。
图5.不同碱基修饰的Pepper对III-3的激活效果。(A)含脱氧核糖核苷酸(图中深色颜色标注)的Pepper-3适配体的二级结构示意图；(B)含2’F修饰(图中深色颜色标注)的Pepper-4适配体的二级结构示意图；(C)含不同修饰的Pepper对III-3的激活效果。“对照”是利用缓冲液替换Pepper-3或Pepper-4适配体。
图6.不同Pepper串联体对III-3的激活效果。(A)按照“串联1”方式获得Pepper串联体；(B)按照“串联2”方式获得Pepper串联体；(C)按照“串联3”方式获得Pepper串联体；(D)不同按照“串联1”方式获得Pepper串联体对III-3的激活效果；(E)不同按照“串联2”方式获得Pepper串联体对III-3的激活效果；(F)不同按照“串联3”方式获得Pepper串联体对III-3的激活效果。
图7.F30-Pepper-1-III-3复合物用于细菌中RNA的标记效果；
图8.F30-Pepper-1-III-3复合物用于酵母细胞中RNA的标记效果；
图9.Pepper与III-3及其类似物用于哺乳动物细胞中RNA的标记的标记效果。(A)比较 F30-Pepper-1-III-3、F30-Broccoli-DFHBI-1T和tRNA-Corn-DFHO在哺乳动物细胞中标记RNA的效果；(B)对(A)图荧光的统计结果；(C)F30-8Pepper-5与III-3类似物在哺乳动物细胞中标记RNA的效果。
图10.基于Pepper-1的探针构建。(A)探针构建示意图，其中的茎-环结构能识别腺苷或者能识别GTP；(B)腺苷探针的检测效果；(C)GTP探针的检测效果。
图11.Pepper用于示踪细胞中RNA定位。(A)Pepper用于检测GAPDH mRNA的定位；(B)Pepper用于检测TMED2mRNA的定位。
图12.Pepper用于探究细胞中mRNA与蛋白质间的关系。(A)BFP蛋白质与其RNA表达的流式细胞仪分析结果；(B)mCherry蛋白质与其RNA表达的流式细胞仪分析结果。
图13.Pepper用于检测基因组DNA。(A)dCas9和不同嵌合sgRNA示意图；(B)dCas9和不同嵌合sgRNA对基因组DNA成像结果；(C)对(B)中每个细胞中的亮点颗粒进行统计结果。
图14.Pepper用于RNA的超分辨成像。(A)4Pepper-9-MS2RNA与tdMCP-BFP-H2B蛋白的共定位情况；(B)细胞核中层的宽场和SIM成像结果；(C)细胞核顶层的宽场和SIM成像结果；
图15.Pepper用于RNA的提取与纯化的标签。“标尺”为单链DNA标准，用于标定适配体的大小。
本发明的实施方式
本申请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。
以下，关于本申请的一些术语进行详细解释。
核酸适配体分子
本申请所述的“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 1CCAAUCGUGGCGUGUCGN 19-N 20-N 21ACUGGCGCCGN 32(对应于图1A的通式Pepper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 32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 32核苷酸序列的方向为3’-5’。当N 1与N 32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 32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 19与N 21核苷酸序列中至少有一对碱基形成反向互补配对，即N 19核苷酸序列的方向为5’-3’，N 21核苷酸序列的方向为3’-5’。当N 19与N 21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 19与N 21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 20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1-7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Pepper结构的核苷酸的取代，该取代选自下组中的一种：C3A、C3U、A4U、A4G、A4C、A5G、A5C、U6A、U6G、U6C、C7A、C7U、G8C、U9A、G11A、G11U、C12G、C12A、C12U、G13C、U14A、U14G、C17U、G18U、G18C、C27G、C27U、G28U、C29G、C29U、C30A、C30U、C2G/G31C、C2U/G31A、C2A/G31U、G10A/C30U、G10C/C30G、G10U/C30A、C2G/G31C/C3A、C2G/G31C/A4C、C2G/G31C/A5C、C2G/G31C/G8C、C2G/G31C/C12U、C2G/G31C/U14G、C2G/G31C/C27U、C2G/G31C/C29G、C2G/G31C/C30U、C2G/G31C/G10A/C30U、C2G/G31C/G10C/C30G、C2G/G31C/G10U/C30A、C2U/G31A/G10A/C30U、C2U/G31A/G10C/C30G、C2U/G31A/G10U/C30A、C2A/G31U/G10A/C30U、C2A/G31U/G10C/C30G、C2A/G31U/G10U/C30A、C2G/G31C/G10C/C30G/C3A、C2G/G31C/G10C/C30G/A4C、C2G/G31C/G10C/C30G/A5C、C2G/G31C/G10C/C30G/G8C、C2G/G31C/G10C/C30G/C12U、C2G/G31C/G10C/C30G/U14G、C2G/G31C/G10C/C30G/C27U、C2G/G31C/G10C/C30G/C29G、C2G/G31C/G10A/C30U/U6G/C27U、C2G/G31C/G10C/C30G/U6G/C27U、C2G/G31C/G10U/C30A/U9A/U14G/C27U和C2A/G31U/G10U/C30A/U9A/U14G/C27U(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1A中的位置。
上述的突变表示在通式Pepper结构的适配体核苷酸序列的相应位点发生核苷酸替换，如C3A表示Pepper的第3位胞嘧啶核苷酸C被取代为腺嘌呤核苷酸A，也即表1中的Pepper(C3A)；C2G/G31C表示Pepper的第2位C被取代为G，同时第31位G被取代为C，也即表1中的Pepper(C2G/G31C)。
表1：Pepper通式结构经过7个、6个、5个、4个、3个、2个或1个核苷酸取代的适配体结构
Figure PCTCN2020087415-appb-000005
Figure PCTCN2020087415-appb-000006
Figure PCTCN2020087415-appb-000007
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)。本申请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。该二级结构含有2个环结构、2个茎结构和一个茎-环结构，其中茎1是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结构的任意长度任意组成的核苷酸碱基对。所述茎1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。在本申请一优选的实施方案中，核酸适配体分子的5’端融合5S RNA序列(Genebank:NR_023377.1)；在本申请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碱基对。本申请所述的适配体分子还可包含插入到N 19-N 20-N 21位置的其他核苷酸序列，该插入的核苷酸序列替换图1A中的茎-环结构。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。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。在本申请一优选的实施 方案中，适配体分子的茎-环结构可以结合GTP分子；在本申请另一优选的实施方案中，茎-环结构可以结合腺苷分子。
本申请优选的实施方案中，所述核酸适配体分子优选为SEQ ID NO：1，2，3，6，7，8，9，10，11，12，13，14，15，16，17，18，19，21，22或23，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。
本申请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。在本申请的一优选的实施方案中，采用F30脚手架RNA(序列2)，其与所述核酸适配体分子的连接方式如图2所示；在在本申请的另一优选的实施方案中，采用tRNA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。
本申请中所述的“核酸适配体分子”是一种RNA分子，或者部分核苷酸被替换成脱氧核糖核苷酸的DNA-RNA杂交分子。其中的核苷酸可以是它们的D和L对映体形式，同时也包含它们的衍生物，包括但不限于2’-F，2’-氨基，2’-甲氧基，5’-iodo，5’-溴-修饰的多聚核苷酸。核酸包含各种修饰的核苷酸。
同一性
“同一性”在本申请中描述两个核苷酸序列之间的相关性。本申请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 19、N 20、N 21、N 32。就本申请而言，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)。
如本申请表1中Pepper(C3A)的序列为N 1C AAAUCGUGGCGUGUCGN 19-N 20-N 21ACUGGCGCCGN 32，Pepper(C3U)的序列为N 1C UAAUCGUGGCGUGUCGN 19-N 20-N 21ACUGGCGCCGN 32，对它们同一性比对的时候，按照本申请的定义，应不包含 N 1、N 19-N 20-N 21和N 32 的核苷酸碱基，因此它们的序列同一性比对结果为96.3％(相差1个核苷酸)。
荧光团分子
本申请所述的“荧光团分子”也称为“荧光团”或“荧光分子”。“荧光团分子”在本申请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。本申请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。在本申请的实施方式中，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。
本申请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。“提高荧光信号”、“荧光增加”、“提高荧光强度”、“增加荧光强度”在本申请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。在本申请一优选实施方式中，量子产率的增加至少是2倍；在本申请另一优选实施方式中，量子产率的增加至少是5-10倍；在本申请另一更优选实施方式中，量子产率的增加至少是20-50倍；在本申请另一更优选实施方式中，量子产率的增加至少是100-200倍；在本申请另一更优选实施方式中，量子产率的增加至少是500-1000倍；在本申请另一更优选实施方式中，量子产率的增加至少是1000-10000倍；在本申请另一更优选实施方式中，量子产率的增加大于10000倍；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FERT的供体荧光团，或BRET的供体发光团。
靶标分子
本申请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。
通过在本申请所述的适配体分子，如图1所示的结构中，该插入的核苷酸序列替换了图1中的N 19、N 20、N 21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，它们可以被整合本申请中的多价核酸适配体中。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。
在本申请的一个优选的实施方案中，靶标分子为腺苷(adenosine)，其对应的识别靶标分子的探针序列如SEQ ID NO：21(图10A所示)；本申请的一个优选的实施方案中，靶标分子为GTP，其对应的识别靶标分子的探针序列如SEQ ID NO：22(图10A所示)。
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。本申请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”在本申请中包括任意的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，长链非编码RNA，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可以融合在本申请RNA适配体分子的5’端或3’端或 N 19-N 20-N 21的位置 。
“sgRNA”在本申请中指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对互补来靶向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
本申请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Pepper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，在本申请一优选的实施方案中，串联的形式为“串联1”，如图6A所示，优选的核苷酸序列为SEQ ID NO：8、9、10、11或12；其中的2Pepper-5表示具有2个Pepper-5结构的串联体1；在本申请另一优选的实施方案中，串联的形式为“串联2”，如图6B所示，优选的核苷酸序列为SEQ ID NO：13、14、15或16；其中的2xPepper-6表示具有2个Pepper-6结构的串联体2；在本申请另一优选的实施方案中，串联的形式为“串联3”，如图6C所示，优选的核苷酸序列为SEQ ID NO：17、18或19；其中的2x2Pepper-5表示具有4个Pepper-5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。
本申请所述的单体形式的适配体是指仅含有1个Pepper结构的适配体，也就是含2个茎结构，2个环结构和1个茎环结构(图1A)的适配体。
多聚体形式的适配体是指含有1个以上Pepper结构的适配体，包含但不限于图6所示的 几种串联形式构成的适配体。
适配体-荧光团复合物
本申请的适配体-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。在本申请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F30-Pepper-2-III-3、F30-Pepper-2-III-7、F30-Pepper-2-III-6、F30-Pepper-2-III-8、F30-Pepper-2-III-4、F30-Pepper-2-III-15、F30-Pepper-2-III-18和F30-Pepper-2-III-21。
在本申请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8个适配体单元的F30-8Pepper-5与8个荧光体分子III-3形成的复合物8Pepper-5-8×(III-3)、8Pepper-5-8×(III-7)、8Pepper-5-8×(III-6)、8Pepper-5-8×(III-8)、8Pepper-5-8×(III-4)、8Pepper-5-8×(III-15)、8Pepper-5-8×(III-18)和8Pepper-5-8×(III-21)。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
本申请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。在本申请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)；在本申请另一优选实施方式中，荧光强度的增加至少是5-10倍；在本申请另一更优选实施方式中，荧光强度的增加至少是20-50倍；在本申请另一更优选实施方式中，荧光强度的增加至少是100-200倍；在本申请另一更优选实施方式中，荧光强度的增加至少是500-1000倍；在本申请另一更优选实施方式中，荧光强度的增加至少是1000-10000倍；在本申请另一更优选实施方式中，荧光强度的增加大于10000倍。
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 32，以及N 19与N 21中其中一段序列的至少50％的核苷酸与另一片段发生发生互补配对即可形成茎结构。如果N 1与N 32是单个核苷酸，则需要N 1与N 32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申请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 1CCAATCGTGGCGTGTCGR 19-R 20-R 21ACTGGCGCCGN 32，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Pepper结构中的N 1，R 19编码通式Pepper结构中 的N 19，R 20编码通式Pepper结构中的N 20，R 21编码通式Pepper结构中的N 21，R 32编码通式Pepper结构中的N 32。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。在本申请的一具体实施方式中，DNA分子包含U6启动子；在本申请的另一具体实施方式中，DNA分子包含CMV启动子。DNA分子包含所述DNA分子进一步还可包含编码任意目标核酸分子的DNA序列。在本申请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)和跨膜emp24域含有蛋白质2(TMED2)的DNA序列(嵌合RNA的序列分别为SEQ ID No：24、25)。在本申请的另一具体实施方式中，编码目标RNA的DNA分子包含编码mCherry和TagBFP的DNA序列(嵌合RNA的序列分别为SEQ ID No：26、27)。
启动子
“启动子”在本申请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。比如当用于标记的话，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处理转录过程。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，均可用于转录本申请所述的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’转录终止子区域是本领域人源日常的实验技能就能实现。
表达系统
本申请的“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。本申请的表达系统，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申请所述的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)；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。
在本申请的一具体实施方式中，提供含编码F30-Pepper-2RNA的DNA分子的表达质粒pET28a-T7-F30-Pepper-2、pLKO.1-F30-Pepper-2和pYES2.1-F30-Pepper-2。在本申请的另一具体实施方式中，提供含编码F30-8Pepper-5RNA的DNA分子的表达质粒pLKO.1-F30-8Pepper-5。在本申请的另一具体实施方式中，提供含编码BFP-4Pepper-7、mCherry-4Pepper-7、GAPDH-4Pepper-7和TMED2-4Pepper-7的DNA分子的表达质粒pCDNA3.1hygro(+)-BFP-4Pepper-7、pCDNA3.1hygro(+)-mCherry-4Pepper-7、pCDNA3.1hygro(+)-GAPDH-4Pepper-7和pCDNA3.1hygro(+)-TMED2-4Pepper-7。在本申请的另一具体实施方式中，提供含编码sgRNA-Pepper-8(loop1)、sgRNA-Pepper-8(tetraloop)、sgRNA-Pepper-8(loop1和tetraloop)的DNA分子的表达质粒psgRNA-Pepper-8(loop1)、psgRNA-Pepper-8(tetraloop)和psgRNA-Pepper-8(loop1和tetraloop)、。在本申请的另一具体实施方式中，提供含编码4Pepper-9-MS2的DNA分子的表达质粒pLKO.1-4Pepper-9-MS2。
本申请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序列插入本申请的这种表达载体中，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”在本申请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。本申请中的宿主细胞，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
本申请所述的检测阵列包含一个或多个本申请的核酸适配体分子，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。将本申请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
本申请的试剂盒，包含本申请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。
以下用实施例对本申请作进一步阐述。这些实施例仅仅用于举例说明，而不对本申请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申请。
实施例中所用的pCDNA3.1hygro(+)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，pYES2.1TOPO 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(T4PNK)、T7RNA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 TMOne 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T7RNA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 PCTCN2020087415-appb-000008
(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。为了拍摄Pepepr-III-3复合物荧光，使用488nm激光器。为了拍摄BFP和mCherry荧光，分别使用405nm和561nm激光器。为了拍摄Pepper-III-7、Pepper-III-6、Pepper-III-8、Pepper-III-4、Pepper-III-15、Pepper-III-18和Pepper-III-21的荧光，分别使用458nm、458nm、488nm、488nm、488nm、561nm、561nm的激光器。为了拍摄Broccoli-DFHBI-1T和Corn-DFHO的荧光，分别使用488nm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 PCTCN2020087415-appb-000009
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Pepper或Pepper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 F30-Pepper-2核酸适配体与1μM III-3荧光团分子形成的复合物的荧光最大激发峰为485nm，最大发射峰为530。利用Synergy Neo2多功能酶标仪检测该复合物在485±10nm激发、530nm±10nm发射条件下的荧光强度为36000，而对照(1μM III-3荧光团分子)在相同检测条件下的荧光强度为10，那么F30-Pepper-2核酸适配体对III-3荧光团分子的激活倍数为3600倍。
实施例1.Pepper核酸适配体分子的二级结构
利用mFold在线RNA结构分析软件分析Pepper核酸适配体的二级结构。Pepper包含2个茎结构，2个环结构和1个茎环结构(图1A)。对于其中一种茎1和茎环的序列，Pepper-1(SEQ ID NO：1)预测的二级结构为图1B。
实施例2.Pepper-III-3复合物性质鉴定
为了检测Pepper-III-3复合物的光谱性质，按照常用实验方法(一)制备F30-Pepper-1(SEQ ID NO：2)RNA。将1μM III-3与5μM F30-Pepper-1孵育。检测结果显示，F30-Pepper-1-III-3复合物的最大激发光为485nm，最大发射光为530nm(图4A)。为了检测F30-Pepper-1-III-3复合物的光吸收与III-3荧光团分子自身光吸收的区别，将5μM III-3与25μM F30-Pepper-1孵育，或者单独的5μM III-3，分别检测F30-Pepper-1-III-3复合物和III-3的光吸收。检测结果显示，F30-Pepper-1-III-3复合物的最大光吸收相对III-3自身发生了很大的红移，最大吸收光为484nm(图4B)。
为了检测Pepper是以单体的形式还是多聚体的形式与III-3结合，对F30-Pepper-1进行Native PAGE鉴定，用已知的单体形式的核酸适配体F30-Broccoli(SEQ ID NO：4)和F30-2dBroccoli(SEQ ID NO：5)(Filonov et al.Journal of the American Chemical Society 2014.136:16299-16308；Filonov et al.Chemistry&biology 2015.22:649-660)为对照。荧光成像结果与SYBR Gold(通用核酸染料，购自Invitrogen)染色结果对比可以发现，F30-Pepper-1位于 100bp左右，与F30-Broccoli类似，这与其实际大小103bp是一致的。因此，结果说明F30-Pepper-1是以单体的形式与III-3结合的(图4C)。
为了检测Pepper与III-3结合的结合常数，利用2nM的F30-Pepper-1与不同浓度的III-3孵育，检测它们的荧光值。检测结果显示F30-Pepper-1与III-3结合的结合常数为3.5nM(图4D)。
为了检测Pepper的温度稳定性，将10μM III-3与1μM F30-Pepper-1孵育，然后置于不同的温度下放置5min，检测荧光值。以10μM DFHBI-1T与1μM F30-Broccoli孵育为对照。检测结果显示，F30-Pepper-1的Tm值为55℃，显著高于F30-Broccoli的48℃(图4E)，说明F30-Pepper-1有着更好的温度稳定性。
为了检测Pepper-III-3复合物在不同pH下的稳定性，将F30-Pepper-1-III-3复合物置于不同pH环境下60min，检测荧光值。以F30-Broccoli-DFHBI-1T复合物为对照。检测结果显示，F30-Pepper-1-III-3复合物在pH5-9的范围内均保持很高的荧光信号，而F30-Broccoli-DFHBI-1T的荧光则随着pH的下降而迅速下降(图4F)，说明F30-Pepper-1-III-3复合物有着更好的pH稳定性。
为了检测Pepper-III-3复合物对K +离子的依赖性，分别将1μM F30-Pepper-1和5μM III-3在含100mM KCl或100mM LiCl的缓冲液中孵育，70℃处理5min后置于室温15min以上，检测不同条件下的荧光值。以F30-Broccoli-DFHBI-1T复合物为对照。此前的文献报道显示，Broccoli的结构中含G四联体，而G四联体结构的稳定非常依赖于K +离子的存在，这与实验结果一致，F30-Broccoli-DFHBI-1T复合物在LiCl缓冲液中的荧光只有在KCl缓冲液中的几百份之一(图4G)。而相比之下，F30-Pepper-1-III-3复合物的荧光不依赖于K +离子的存在(图4G)，表明Pepper结构中不存在G四联体结构。
实施例3.不同Pepper突变体对III-3荧光团分子的荧光激活效果。
为了检测不同Pepper突变体对III-3荧光团分子的荧光激活效果，对F30-Pepper-1中Pepper-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Pepper突变体RNA，将1μM III-3分别与5μM不同的F30-Pepper-1突变体RNA孵育，按照常用实验方法(五)它们对III-3荧光团分子的荧光激活倍数。检测结果显示，大部分含单个碱基突变的F30-Pepper-1突变体保留对III-3的较强的荧光激活效果(>10倍)(表2)。部分含2-7个碱基突变的F30-Pepper-1突变体仍保留对III-3的较强的荧光激活效果(>100倍)(表3)。综上，Pepper的很多单碱基和多碱基突变体仍保留激活III-3荧光团分子的适配体功能。
表2含单碱基突变的Pepper突变体对III-3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
F30-Pepper-1 3600 G10A 847 G18C 1028
C3G 360 G10C 856 A22U 87
C3A 2484 G11U 1512 A22G 687
C3U 2016 G11A 1526 A22C 147
A4U 1836 G11C 325 C23G 65
A4G 2160 C12G 2125 C23A 547
A4C 2772 C12A 458 G26U 532
A5G 1800 C12U 2268 C27G 1875
A5C 2628 G13U 587 C27A 186
U6A 1872 G13A 792 C27U 3158
U6G 1980 G13C 1758 G28U 873
U6C 2088 U14A 1524 G28A 42
C7A 1044 U14G 3152 G28C 145
C7U 2268 G15C 15 C29G 2145
G8C 3168 U16A 28 C29U 1437
G8A 324 U16G 125 C29A 18
U9A 2124 C17A 52 C30G 145
U9C 72 C17U 1268 C30U 2587
G10U 900 G18U 1024 C30A 1596
注：表2中的F30-Pepper-1是序列为SEQ ID NO：2的核酸适配体；其它的适配体是在F30-Pepper-1的Pepper-1序列中与图1A的Pepper对应核苷酸位置做的点突变。
表3含多碱基突变的Pepper突变体对III-3的激活效果
Figure PCTCN2020087415-appb-000010
Figure PCTCN2020087415-appb-000011
实施例4.碱基修饰的Pepper对III-3的激活效果
为了检测经碱基修饰的Pepper对III-3的激活效果，合成含碱基修饰的Pepper-3(SEQ ID NO：6，该序列GGCCCCCAAUCGUGGCGUGUCGG CCUGCUUCGGCAGGCACUGGCGCCGGGGCC中带下划线的碱基为脱氧核糖核苷酸碱基)和Pepper-4(SEQ ID NO：7，该序列GGCCCCCCAAUCGUGGCGUGUCGG CCUGCUUCGGC AGGCACUGGCGCCGGGGGCC中带下划线的碱基为经2’-F修饰的碱基)(由上海吉玛制药技术有限公司合成)，它们分别含茎-环结构碱基替换为脱氧核糖核苷酸(图5A中阴影部分的碱基)和部分碱基经2’-F修饰(图5B中阴影部分的碱基)。按照常用实验方法(五)检测这些碱基修饰的Pepper对III-3荧光团分子的荧光激活效果。检测结果显示，经碱基修饰的Pepper-3和Pepper-4依然可以显著激活III-3荧光团分子的荧光(图5C)。
实施例5.Pepper串联体
为了检测Pepper串联体对III-3荧光的激活效果，将Pepper按照不同的形式进行串联， 包含以下三种：
(1)“串联1”方式(图6A)，将Pepper结构上的“头”和“尾”按照“头-尾”连接的方式进行连接，以此获得nPepper(其中n为可为任意拷贝的Pepper)。在本实施例中，分别全基因合成F30-2Pepper-5、F30-4Pepper-5、F30-8Pepper-5、F30-16Pepper-5和F30-32Pepper-2的编码cDNA(编码RNA适配体的序列分别为SEQ ID NO：8，SEQ ID NO：9，SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12)，PCR扩增后，按照常用实验方法(一)制备核酸适配体RNA，将0.1μM RNA适配体与10μM III-3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，nPepper-III-3的荧光也随之增加(图6D)。当n>8时，随着n的增加，nPepper-III-3的荧光并不是等倍数增加，但依然比Pepper-III-3的荧光要高很多(图6D)，说明可以通过“串联1”方式提高Pepper-III-3复合物的荧光强度。
(2)“串联2”方式(图6B)，将Pepper作为一个结构单元进行串联，以此获得nxPepper(其中n为可为任意拷贝的Pepper)。在本实施例中，分别全基因合成2xPepper-6、4xPepper-6、8xPepper-6和16xPepper-6的编码cDNA(编码RNA适配体的序列分别为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0.1μM RNA适配体与10μM III-3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，nPepper-III-3的荧光也随之增加(图6E)，说明可以通过“串联2”方式提高Pepper-III-3复合物的荧光强度。
(3)“串联3”方式(图6C)，是将上述“串联1”与“串联2”结合起来，将“串联1”得到的nPepper作为一个结构单元按照“串联2”的方式进行串联，以此获得n1xn2Pepper(其中n1和n2为可为任意拷贝的Pepper)。在本实施例中，分别全基因合成2x2Pepper-5、4x2Pepper-5、8x2Pepper-5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)，按照常用实验方法(一)制备核酸适配体RNA，将0.1μM RNA适配体与20μM III-3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Pepper串联体-III-3的荧光强度要显著高于Pepper-III-3(图6F)，说明可以通过“串联3”方式提高Pepper-III-3复合物的荧光强度。
实施例6.III-3类似物的性质鉴定
按照常用实验方法(一)制备F30-Pepper-1RNA适配体分子，利用其检测III-3类似物与Pepper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和结合常数(Kd)，检测结果如表4所示，从表中数据可以看出，F30-Pepper-1可以不同程度地激活III-3类似物的荧光强度。
表4：F30-Pepper-1RNA适配体分子与不同荧光分子结合的理化性质测定
Figure PCTCN2020087415-appb-000012
Figure PCTCN2020087415-appb-000013
实施例7.Pepper-III-3复合物用于细菌中RNA的标记
为了检测Pepper-III-3在细菌中的效果，首先构建表达F30-Pepper-1的细菌表达质粒。利用引物对实施例2中的F30-Pepper-1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-Pepper-1DNA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-Pepper-1。
扩增F30-Pepper-1片段所用的引物为：
上游引物(P1)：5’-TCGATCCCGCGAAATTAATACGACTCACTATAGGGTTGCCA
TGTGTATGTGGG-3’
下游引物(P2)：5’-CAAGGGGTTATGCTATTGCCATGAATGATCC-3’(SEQ ID No：)
扩增pET28a载体使其线性化所用的引物为：
上游引物(P3)：5’-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG-3’
下游引物(P4)：5’-ATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACG-3’
将pET28a-T7-F30-Pepper-1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-Pepper-1的表达，4h后收菌，利 用含2μM III-3的PBS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-Pepper-1且在III-3存在下，细菌才能显示出明亮的黄绿色荧光(图7)，表明Pepper-III-3复合物可以用于细菌中RNA的荧光标记。
实施例8.Pepper-III-3复合物用于酵母细胞中RNA的标记
为了检测Pepper-III-3在酵母中的效果，首先构建表达F30-Pepper-1的酵母表达质粒。利用引物对实施例2中的F30-Pepper-1DNA片段进行扩增，按照实验方法(四)将扩增得到的F30-Pepper-1片段插入到pYES2.1TOPO TA载体中，得到的重组质粒命名为pYES2.1-F30-Pepper-1。
扩增F30-Pepper-1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-Pepper-1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-Pepper-1的表达，10h后收菌，利用含2μM III-3的PBS溶液重悬。以未经处理的BY4741菌株为对照。结果显示，只有表达了F30-Pepper-1且在III-3存在下，酵母细胞才能显示出明亮的黄绿色荧光(图8)，表明Pepper-III-3复合物可以用于酵母细胞中RNA的荧光标记。
实施例9.Pepper与III-3及其类似物用于哺乳动物细胞中RNA的标记
为了检测Pepper与III-3用于哺乳动物细胞中RNA的标记，以已报道的Broccoli和Corn核酸适配体分子(分别结合DFHBI-1T和DFHO荧光团分子)作为对照(Filonov et al.Journal of the American Chemical Society 2014.136:16299-16308；Song et al.Nature chemical biology2017.13:1187-1194)，构建它们的哺乳动物细胞表达质粒。分别引物P7和P8扩增实施例2中的F30-Pepper-1和F30-Broccoli，利用引物P9和P10扩增全基因合成的tRNA-Corn cDNA片段(编码的RNA序列为SEQ ID No：20)，利用实验方法(四)将这些片段插入到pLKO.1puro载体中。得到的表达载体命名为pLKO.1-F30-Pepper-1、pLKO.1-F30-Broccoli和pLKO.1-tRNA-Corn，这些质粒分别表达F30-Pepper-1、F30-Broccoli和tRNA-Corn RNA。
将pLKO.1-F30-Pepper-1、pLKO.1-F30-Broccoli和pLKO.1-tRNA-Corn质粒转染293T/17细胞，24h后加入1μM III-3，20μM DFHBI和10μM DFHO分别对F30-Pepper-1、F30-Broccoli和tRNA-Corn进行标记，以不表达相应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，F30-Pepper-1-III-3复合物展现出非常明亮的黄绿色荧光，荧光强度明显高于F30-Broccoli-DFHBI-1T和tRNA-Corn-DFHO复合物(图9A和B)，表明Pepper-III-3可以很好的在哺乳动物细胞中工作。
扩增F30-Pepper-1和F30-Broccoli所用的引物为：
上游引物(P7)：5’-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGG-3’；
下游引物(P8)：5’-TGTCTCGAGGTCGAGAATTCAAAAAAAGTTGCCATGAATGATCC-3’
扩增tRNA-Corn所用的引物为：
上游引物(P9)：5’-GGAAAGGACGAAACTCTAGAGCCCGGATAGCTCAGTCGG-3’
下游引物(P10)：5’-TGTCTCGAGGTCGAGAATTCAAAAAAATGGCGCCCGAACAGGGACTTGCGAGCTCAGGATCCTTCCGTTTCGCACTGG-3’。
为了检测Pepper与III-3类似物用于哺乳动物细胞中RNA的标记，构建了表达F30-8Pepper-5的哺乳动物表达质粒。利用本实施例中的引物P7和P8对实施例5中的F30-8Pepper-5片段进行扩增，利用实验方法(四)将这些片段插入到pLKO.1puro载体中。得到的表达载体命名为pLKO.1-F30-8Pepper-5。
将pLKO.1-F30-8Pepper-5质粒转染293T/17细胞，24h后加入不同的III-3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III-3类似物均可以特异性标记表达F30-8Pepper-5的细胞，而不标记不表达F30-8Pepper-5的对照细胞(图9C)，表明Pepper与III-3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Pepper的探针构建
为了构建基于Pepper的待检测物探针，将Pepper-1(SEQ ID No：2)结构中茎-环结构处的核苷酸更换为可以特异性识别结合腺苷(adenosine)和鸟苷(GTP)的RNA适配体，这些适配体与Pepper-1之间利用不同长度和组成的碱基进行连接(图10A)，按照常用实验方法(一)制备探针RNA，将其与III-3孵育，利用多功能酶标仪分别检测它们在有无腺苷或GTP存在下的荧光强度。检测结果显示，对于腺苷探针，当腺苷适配体与Pepper-1间的连接碱基为图10B中的连接2的碱基对时，激活倍数可达88倍，对应的探针RNA序列为SEQ ID No：21。对于GTP探针，当GTP适配体与Pepper-1间连接碱基为图10B中的连接3的碱基对时，激活倍数为10倍，对应的探针RNA序列为SEQ ID No：22。
实施例11.Pepper用于示踪细胞中RNA定位
为了检测Pepper用于示踪细胞中RNA定位，首先构建Pepper与不同RNA融合的嵌合RNA的表达质粒。全基因合成4Pepper-7的cDNA(其编码RNA适配体的序列为SEQ ID No：23)，利用引物扩增4Pepper-7基因片段，利用同源重组的方法将其插入到经HindIII和XhoI双酶切的pCDNA3.1hygro(+)载体，得到pCDNA3.1hygro(+)-4Pepper-7重组质粒。全基因合成GAPDH和TMED2基因片段(GAPDH和TMED2编码基因序列分别为Genebank:BC009081,BC025957)，分别利用引物对GAPDH和TMED2基因片段进行扩增，将它们插入到经NheI 和HindIII双酶切的pCDNA3.1hygro(+)-4Pepper-7载体中，获得pCDNA3.1hygro(+)-GAPDH-4Pepper-7和pCDNA3.1hygro(+)-TMED2-4Pepper-7重组质粒，它们分别编码GAPDH-4Pepper-7和TMED2-4Pepper-7嵌合RNA，它们的序列为SEQ ID No：24和25。
扩增4Pepper-7所用的引物为：
上游引物(P11)：5’-TAGCGTTTAAACTTAAGCTTCCCACGGAGGATCCCCAATC-3’
下游引物(P12)：5’-ACGGGCCCTCTAGACTCGAGCCCACGGAGGATCCCGGCGCC-3’
扩增GAPDH所用的引物为：
上游引物(P13)：5’-GGAGACCCAAGCTGGCTAGCATGGGGAAGGTGAAGGTCGG-3’
下游引物(P14)：5’-GGATCCTCCGTGGGAAGCTTAACCATGCTCTAGCGAGTGTTACTCCTTGGAGGCCATG T-3’
扩增TMED2所用的引物为：
上游引物(P15)：5’-GGAGACCCAAGCTGGCTAGCATGGTGACGCTTGCTGAACT-3’
下游引物(P16)：5’-GGATCCTCCGTGGGAAGCTTAACCATGCTCTAGCGAGTTAAACAACTCTCCGGACTTC-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pCDNA3.1hygro(+)-GAPDH-4Pepper-7和pCDNA3.1hygro(+)-TMED2-4Pepper-7重组质粒分别与pCDNA3.1hygro(+)-BFP共转染COS-7细胞，转染24h后按照具体实验方法(三)所述荧光成像方法对细胞进行成像。成像结果显示，GAPDH-4Pepper-7-III-3的荧光主要集中在胞浆中，而TMED2-4Pepper-7-III-3的荧光则可以观察到内质网富集的现象，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11)，以上结果显示Pepper可用于示踪RNA的定位。
实施例12.Pepper用于检测细胞中mRNA与蛋白质含量的关系
为了利用Pepper检测细胞中mRNA的翻译，首先需要构建不同Pepper融合的mRNA表达质粒。分别利用引物以mCherry2-N1(Addgene：54517)和EasyFusion T2A-H2B-TagBFP(Addgene：113086)为模板扩增mCherry和TagBFP基因片段，将其插入到经NheI和HindIII双酶切的pCDNA3.1hygro(+)-GAPDH-4Pepper-7载体中，获得pCDNA3.1hygro(+)-mCherry-4Pepper-7和pCDNA3.1hygro(+)-TagBFP-4Pepper-7重组质粒，它们分别编码mCherry-4Pepper-7和TagBFP-4Pepper-7，其RNA序列分别为SEQ ID No：26和27。
扩增mCherry所用的引物为：
上游引物(P17)：5’- GGAGACCCAAGCTGGCTAGCATGGTGAGCAAGGGCGAGGAGG-3’
下游引物(P18)：5’-GGATCCTCCGTGGGAAGCTTAACCATGCTCTAGCGAGTTACTTGTACAGCTCGTCCAT G-3’
扩增TagBFP所用的引物为：
上游引物(P19)：5’-GGAGACCCAAGCTGGCTAGCATGAGCGAGCTGATTAAGGA-3’
下游引物(P20)：5’-GGATCCTCCGTGGGAAGCTTCTCCCAAACCATGCTCTAGCGAGTGTTAATTGAGCTTG TGCCCCA-3’
分别将pCDNA3.1hygro(+)-BFP-4Pepper-7和pCDNA3.1hygro(+)-mCherry-4Pepper-7重组质粒转染COS-7细胞，24h后，利用0.2μM III-3标记转染了pCDNA3.1hygro(+)-BFP-4Pepper-7和pCDNA3.1hygro(+)-mCherry-4Pepper-7重组质粒的细胞，利用流式细胞仪检测mRNA(4Pepper-7-III-3)的荧光以及蛋白质(BFP和mCherry)的荧光，利用米氏方程对mRNA的荧光和蛋白质荧光进行拟合，得到R 2。检测结果显示，不同的mRNA的翻译效率差异显著(图12)，表明可以利用Pepper检测mRNA与蛋白质含量的关系。
实施例13.Pepper用于检测基因组DNA
为了利用Pepper检测基因组DNA，首先构建表达Pepper-8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-Pepper-8(loop1)、sgRNA-Pepper-8(tetraloop)和sgRNA-Pepper-8(loop1和tetraloop)的cDNA，其编码的RNA序列分别为SEQ ID No：28、29和30。利用引物P21和P22扩增上述嵌合RNA的cDNA，利用引物P23和P24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-Pepper-8(loop1)、psgRNA-Pepper-8(loop2)和psgRNA-Pepper-8(loop1和tetraloop)(图13A)。利用引物P25和P26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pCDNA3.1hygro(+)-dCas9-GFP。
扩增Pepper与sgRNA嵌合RNA对应cDNA所用的引物为：
上游引物(P21)：5’-AAAGGACGAAACACCGAATCTGCAAGTGGATATTGTTTGAG-3’
下游引物(P22)：5’-TGATCTAGAAAAAAAGCACCGACTCGGTGCCAC-3’
扩增psgRNA质粒使其线性化的引物为：
上游引物(P23)：5’-TTTTTTTCTAGATCATAATCAGCCATACC-3’
下游引物(P24)：5’-GGTGTTTCGTCCTTTCCACAAG-3’
扩增SpdCas9-GFP所用的引物为：
上游引物(P25)：5’-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCACCATGGCCCC-3’
下游引物(P26)：5’-ACGGGCCCTCTAGACTCGAGTTACTTGTACAGCTCGTCCATGC-3’
将pCDNA3.1hygro(+)-dCas9-GFP分别与psgRNA-Pepper-6(loop1)、psgRNA-Pepper-6(loop2)和psgRNA-Pepper-6(loop1和tetraloop)重组质粒共转染COS-7细胞，转染24h后，利用1μM III-21和Hoechst对细胞进行标记，利用荧光显微镜观察Pepper-8-III-21、GFP和Hoechst的荧光。成像结果显示，Pepper-8-III-21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的荧光几乎完全吻合(图13B)，且点的个数也与单独的sgRNA一致(图13C)，说明Pepper可用于对基因DNA的成像。
实施例14.Pepper用于RNA的超分辨成像
为了将Pepper用于RNA的超分辨成像，首先构建将RNA锚定到细胞核的质粒。全基因合成4Pepper-9-MS2DNA片段(SEQ ID No：31)，利用引物P27和P28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pLKO.1载体中，得到的质粒命名为pLKO.1-4Pepper-9-MS2。利用引物以pCS-H2B-EGFP(Addgene：53744)为模板扩增H2B基因片段，利用引物以pHAGE-Ubc-NLS-HA-tdMCP-GFP(Addgene：40649)为模板扩增tdMCP基因片段，利用引物扩增tagBFP基因片段，利用overlap PCR将tdMCP、tagBFP和H2B基因片段连接起来获得tdMCP-tagBFP-H2B融合片段，利用实验方法(四)将它插入到pmTurquoise2-Golgi(Addgene:36205)，得到的质粒命名为pH2B-tdMCP-tagBFP，它编码细胞核定位的tdMCP-tagBFP。
扩增4Pepper-9-MS2DNA片段所用的引物为：
上游引物(P27)：5’-GGAAAGGACGAAACTCTAGAGGGGCCCCCCAATCGTGG-3’
下游引物(P28)：5’-TGTCTCGAGGTCGAGAATTCAAAAAAAGGGGCCCCCGGCGCCAGTG-3’
扩增tdMCP基因片段所用的引物为：
上游引物(P29)：5’-GAACCGTCAGATCCGCTAGCCACCATGGGCTACCCCTACGACGTGCCCG-3’
下游引物(P30)：5’-TCCAGAATCCGCGTAGATGCCGG-3’
扩增tagBFP基因片段所用的引物为：
上游引物(P31)：5’-CTACGCGGATTCTGGAGGCGGTGGATCCATGAGCGAGCTGATTAAGGAG-3’
下游引物(P32)：5’-AGATCTATTGAGCTTGTGCCCCAGTTTG-3’
扩增H2B基因片段所用的引物为：
上游引物(P33)：5’-CAAGCTCAATAGATCTATGCCTGAACCGGCAAAATCC-3’
下游引物(P34)：5’-GACTGCAGAATTCGAAGCTTACTTGGAGCTGGTGTACTTG-3’。
将pLKO.1-4Pepper-9-MS2和pH2B-tdPP7-tagBFP重组质粒共转染COS-7细胞，24h后利用III-21荧光团分子进行标记，然后利用Zeiss Elyra PS.1超分辨荧光显微镜检测Pepper-III-21复合物的荧光分布，激发光使用561长通滤片，镜头为Zeiss Plan-Apochromat 63×(NA,1.4)Oil DIC M27，CMOS大小为1024x1024像素，图片利用ZEN 2011Black(Zeiss)软件进行处理。成像结果显示，pLKO.1-4Pepper-9-MS2与pH2B-tdMCP-tagBFP共转染的细胞呈现出明显的核孔结构(图14)。该结果表明Pepper可以用于RNA的超分辨成像。
实施例15.Pepper用于RNA的提取与纯化的标签
为了检测将Pepper用于RNA的提取与纯化，将实施例12中的pCDNA3.1hygro(+)-TagBFP-4Pepper-7和pCDNA3.1hygro(+)-mCherry-4Pepper-7重组质粒分别转染COS-7细胞，24h后收集细胞利用Eastep Super总RNA提取试剂盒(Promega)提取细胞的总RNA。将提取的总RNA溶解在含40mM HEPES,pH 7.4,125mM KCl,5mM MgCl 2的缓冲液中，70℃孵育10min后，室温放置30min以上。
500uL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III-3荧光团分子(Mal-III-3)于室温反应30min，利用500μL PBS清洗三次。将经上述处理的总RNA与处理过的微珠室温孵育，30min后4000rpm离心2min，弃上清，利用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。
对回收得到的RNA进行电泳鉴定，将跑完的胶与含5μM III-3的40mM HEPES,pH 7.4,125mM KCl,5mM MgCl 2的缓冲液孵育30min，检测胶中4Pepper-III-3的荧光。成像结果显示，胶中有3条RNA条带显示出明显的Pepper-III-3荧光信号，分别为TagBFP-4Pepper,和mCherry-4Pepper(图15)，表明Pepper可以作为一个标签用于RNA的分离与纯化。
实施例16.III-3及其类似物的合成
化合物Ⅲ-1：
Figure PCTCN2020087415-appb-000014
4-N,N-二甲基-苯甲醛(0.35g，2.3mmol)，4-氰基-苯乙腈(0.4g，2.8mmol)于100ml 圆底烧瓶中，加入40ml无水乙醇溶解，加入二滴哌啶，Ar保护条件下油浴加热回流2h，反应完毕，冷却至室温，大量固体析出，过滤，冷乙醇冲洗滤饼三次，真空烘干的橙色固体(0.60g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝3.05(s,6H),6.83(d,J＝9.2Hz,2H,),7.84-7.94(m,6H),8.02ppm(s,1H).HRMS(ESI-TOF):Calcd.For C 18H 16O 3[M+H] +:274.1344.Found:274.1345.
化合物Ⅲ-2：
Figure PCTCN2020087415-appb-000015
参照化合物Ⅲ-1的合成方法，(0.34g，89％)。 1H NMR(400MHz,DMSO-d 6):δ＝1.23(t，J＝7.60Hz,6H),3.05(t，J＝7.60Hz,4H),6.84(d,J＝9.2Hz,2H,),7.84-7.95(m,6H),8.09ppm(s,1H).HRMS(ESI-TOF):Calcd.For C 20H 20O 3[M+H] +:302.1657.Found:302.1658.
化合物Ⅲ-3：
Figure PCTCN2020087415-appb-000016
参照化合物Ⅲ-1的合成方法，(0.33g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝7.96(s,1H),7.85(d,J＝16.0Hz,6H),6.81(d,J＝8.0Hz,2H),4.77(s,1H),3.55(d,J＝28.0Hz,4H),3.04(s,1H).LR-HRMS(ESI-TOF):Calcd.For C 19H 18N 3O[M+H] +:304.1450.Found:304.1451.
化合物Ⅲ-4：
Figure PCTCN2020087415-appb-000017
化合物Ⅲ-3(0.61g，2.0mmol)于40mL干燥DCM中，加入TEA(0.25g，2.2mmol)，0℃条件下缓慢加入对甲苯磺酰氯的(0.38g，2.0mmol)的10ml DCM溶液，Ar保护条件下缓慢升至室温，反应完毕，加入2mL水淬灭反应，分出有机相，Na 2SO 4干燥，减压条件下除去有机溶剂，残余物不经进一步处理，直接用于下一步。
残余物溶于20ml乙腈中，加入1ml甲胺的甲醇溶液，体系在Ar保护条件下油浴加热回流过夜，反应完毕，加压除去溶剂，体系溶于50ml DCM中，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na 2SO 4干燥，加压除去溶剂，残余物经柱色谱分离得橙红色固体(0.54g,82％)。 1H NMR(400MHz,CDCl 3):δ＝7.88(d,J＝9.0Hz,2H),7.74–7.65(m,4H),7.48(s,1H),6.73(d,J＝9.1Hz,2H),3.60–3.55(m,2H),3.08(s,3H),2.57–2.52(m,2H),2.34(s,6H).LR-MS(ESI-TOF):Calcd.For C 21H 23N 4[M+H] +:331.1923.Found:331.1925.
化合物Ⅲ-5：
Figure PCTCN2020087415-appb-000018
4-羟基-3,5-二氟-苯甲醛(0.32g，2.0mmol)，4-氰基-苯乙腈(0.35g，2.4mmol)于100ml圆底烧瓶中，加入40ml无水乙醇溶解，加入二滴哌啶，Ar保护条件下油浴加热回流2h，反应完毕，冷却至室温，大量固体析出，过滤，冷乙醇冲洗滤饼三次，真空烘干的橙色固体。 1H NMR(400MHz,CDCl 3):δ＝7.80(d,J＝9.0Hz,2H),7.74–7.66(m,4H),7.48(s,1H).LR-MS(ESI-TOF):Calcd.For C 16H 9F 2N 2O[M+H] +:283.0683.Found:283.0684.
化合物Ⅲ-6：
Figure PCTCN2020087415-appb-000019
其中，5-(N-甲基-N-羟乙基)氨基-吡嗪-2-甲醛：
Figure PCTCN2020087415-appb-000020
4-N-甲基-N-羟乙基胺(2.6g，35mmol)，5-氯-吡嗪-2-甲醛(0.50g，3.5mmol)于100ml圆底烧瓶中，加入20ml无水乙腈溶解，加入K 2CO 3(0.71g，5.3mmol)，Ar保护条件下油浴加热回流24h，反应完毕，冷却至室温，过滤，真空除去溶剂，残余物溶于100ml DCM中，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na2SO4干燥，除去有机溶剂，残余物经柱色谱分离得5-(N-甲基-N-羟乙基)-吡嗪-2-醛(0.48g，76％)。 1H NMR(400MHz,CDCl 3):δ9.88(s,1H),8.62(d,J＝1.2Hz,1H),8.14(d,J＝1.1Hz,1H),3.92(m,2H),3.88–3.83(m,2H),3.28(s,3H).LR-MS(ESI-TOF):Calcd.For C 8H 12N 3O 2[M+H] +:182.1.Found:182.1.
化合物Ⅲ-6的制备参照化合物Ⅲ-1的合成方法，(0.36g，96％)。 1H NMR(400MHz,CDCl 3):δ8.39(s,1H),8.30(s,1H),7.80(d,J＝8.5Hz,2H),7.72(d,J＝8.4Hz,2H),7.51(s,1H),3.93(t,J＝4.9Hz,2H),3.88–3.83(m,2H),3.29(s,3H).LR-HRMS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1355.Found:306.1357.
化合物Ⅲ-7：
Figure PCTCN2020087415-appb-000021
参照化合物Ⅲ-4的合成方法，(0.21g，67％)。 1H NMR(400MHz,DMSO-d 6):δ8.37(d,J＝5.2Hz,2H),8.06(s,1H),8.00–7.85(m,4H),3.77(t,J＝6.5Hz,2H),3.20(s,3H),2.56(m,2H),2.23(s,6H).LR-HRMS(ESI-TOF):Calcd.For C 19H 21N 6[M+H] +:333.1828.Found:333.1829.
化合物Ⅲ-8：
Figure PCTCN2020087415-appb-000022
其中，6-(N-甲基-N-羟乙基)氨基-吡嗪-3-醛：
Figure PCTCN2020087415-appb-000023
按照5-(N-甲基-N-羟乙基)氨基-吡嗪-2-醛的合成方法，(0.45g，68％)。 1H NMR(400MHz,CDCl 3):δ＝9.69(s,1H),8.43(d,J＝2.1Hz,1H),7.86(dd,J＝9.0,2.3Hz,1H),6.56(d,J＝9.1Hz,1H),3.86–3.79(m,4H),3.15(s,3H).LR-MS(ESI-TOF):Calcd.For C 9H 13O 2N 2[M+H] +:181.1.Found:181.1.
化合物Ⅲ-8的制备参照化合物Ⅲ-1的合成方法，(0.39g，89％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.54(d,J＝4.0Hz,1H),8.30(dd,J＝9.3,2.5Hz,1H),8.03(s,1H),7.92(d,J＝8.0Hz,2H),7.85(d,J＝8.0Hz,2H),6.84(d,J＝8.0Hz,1H),4.77(t,J＝5.4Hz,1H),3.67(t,J＝5.3Hz,2H),3.60(q,J＝5.4Hz,2H),3.15(s,3H).LR-HRMS(ESI-TOF):Calcd.For C 18H 27N 4O[M+H] +:305.1402.Found:305.1401.
化合物Ⅲ-9：
Figure PCTCN2020087415-appb-000024
参照化合物Ⅲ-4的合成方法，(0.31g,92％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.55(d,J＝4.0Hz,1H),8.31(dd,J＝9.3,2.5Hz,1H),8.05(s,1H),7.93(d,J＝8.0Hz,2H),7.84(d,J＝8.0Hz,2H),6.85(d,J＝8.0Hz,1H),4.78(t,J＝5.4Hz,1H),3.67(t,J＝5.3Hz,2H),3.60(q,J＝5.4Hz,2H),3.17(t,J＝8.0Hz,4H),1.17(t,J＝8.0Hz,6H).LR-HRMS(ESI-TOF):Calcd.For C 22H 26N 5[M+H] +:360.2188.Found:360.2187.
化合物Ⅲ-10：
Figure PCTCN2020087415-appb-000025
其中，4-N,N-二甲基-6醛基-吡啶：
Figure PCTCN2020087415-appb-000026
参照4-N-甲基-N-(2-N’,N’-二甲基-乙基)-苯甲醛的合成方法，(0.31g，49％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.86(d,J＝0.6Hz,1H),8.17(d,J＝2.9Hz,1H),7.83(d,J＝8.9Hz,1H),6.94(dd,J＝8.8,2.9Hz,1H),3.10(s,6H).LR-HRMS(ESI-TOF):Calcd.For C 8H 11N 2O [M+H] +:151.1.Found:151.1.
化合物Ⅲ-10的制备参照化合物Ⅲ-1的合成方法，(0.36g，96％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.86(d,J＝0.6Hz,1H),8.26(s，1H),8.17(d,J＝2.9Hz,1H),7.83(d,J＝8.9Hz,1H),7.46(m,4H),6.94(dd,J＝8.8,2.9Hz,1H),3.10(s,6H).LR-HRMS(ESI-TOF):Calcd.For C 17H 15N 4[M+H] +:275.1297.Found:275.1298.
化合物Ⅲ-11：
Figure PCTCN2020087415-appb-000027
其中，2-(N-甲基-N-羟乙基)氨基-5-醛基-嘧啶：
Figure PCTCN2020087415-appb-000028
参照4-N-甲基-N-(2-N’,N’-二甲基-乙基)-苯甲醛的合成方法，(0.42g，72％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.89(s,1H),8.73(s，2H),3.64(t,J＝8.9Hz,2H),3.45(t,J＝8.8Hz,2H),3.10(s,3H).LR-MS(ESI-TOF):Calcd.For C 8H 12N 3O[M+H] +:182.1.Found:182.1.
化合物Ⅲ-11的制备参照化合物Ⅲ-1的合成方法，(0.36g，96％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.26(s,1H),8.73(s，2H),7.64(m,4H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.11(s,3H).LR-HRMS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1355.Found:306.1356.
5-(N-甲基-N-羟乙基)氨基-2-醛基-嘧啶：
Figure PCTCN2020087415-appb-000029
参照化合物4-N-甲基-N-(2-N’,N’-二甲基-乙基)-苯甲醛的合成方法，(0.42g，72％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.98(s,1H),8.21(s，2H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.12(s,3H).LR-MS(ESI-TOF):Calcd.For C 8H 12N 3O 2[M+H] +:182.1.Found:182.1.
化合物Ⅲ-12：
Figure PCTCN2020087415-appb-000030
其中，1-氰基-1-(4-苯乙腈)-2-2-(5-(N-甲基-N-羟乙基)氨基-)嘧啶-乙烯：
Figure PCTCN2020087415-appb-000031
参照化合物Ⅲ-1的合成方法，(0.56g，89％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.21(s， 2H),7.99(s,1H),7.64(s,4H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.12(s,3H).LR-MS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1.Found:306.1.
化合物Ⅲ-12的制备参照化合物Ⅲ-4的合成方法，(0.36g，96％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.21(s，2H),7.99(s,1H),7.64(s,4H),3.77(t,J＝6.5Hz,2H),3.20(s,3H),2.56(m,2H),2.23(s,6H).LR-HRMS(ESI-TOF):Calcd.For C 19H 21N 6[M+H] +:333.1828.Found:333.1829.
化合物Ⅲ-13：
Figure PCTCN2020087415-appb-000032
其中，2-乙腈-5-氰基-吡啶：
Figure PCTCN2020087415-appb-000033
2-溴甲基-5-氰基吡啶(0.50g，2.5mmol)于100ml圆底烧瓶中，加入50ml THF溶解，Ar保护条件下加入10ml NaCN的2M的水溶液，油浴加热回流12h，反应完毕，体系冷却至室温，DCM萃取(3×100ml)，合并有机相，分别用水、饱和食盐水洗涤(2×100ml)，有机相用NaSO4干燥，减压除去溶剂，残余物经柱色谱纯化分离得2-乙腈-5-氰基吡啶(0.19g，56％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.78(s,1H),7.95(m,1H),7.56(m,1H),4.01(s,2H).LR-MS(ESI-TOF):Calcd.For C 8H 6N 3[M+H] +:144.1.Found:144.1.
化合物Ⅲ-13的制备参照化合物Ⅲ-1的合成方法，(0.45g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.78(s,1H),8.21(s,1H),7.94(m,1H),7.86(d,J＝8.0Hz,2H),7.57(m,1H),6.80(d,J＝8.0Hz,2H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.12(s,3H).LR-MS(ESI-TOF):Calcd.For C 18H 17N 4O[M+H] +:305.1402.Found:305.1403.
化合物Ⅲ-14：
Figure PCTCN2020087415-appb-000034
其中，2-氰基-5-乙腈-吡嗪：
Figure PCTCN2020087415-appb-000035
2-氯-吡嗪-5-乙腈(0.32g,2.0mmol)、CuCN(0.93g，10.0mmol)于100ml圆底烧瓶中，加入30ml干燥DMSO溶解，Ar保护条件下80℃油浴加热12h，反应完毕，体系倒入100ml水中，DCM萃取(4×50ml)，合并有机相，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na2SO4干燥，减压除去有机溶剂，残余物经柱色谱分离得2-氰基-吡嗪-5-乙腈(0.20 g，69％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.60(s,1H),8.48(s,1H),3.92(s,2H).LR-MS(ESI-TOF):Calcd.For C 7H 5N 4[M+H] +:145.1.Found:145.1.
化合物Ⅲ-14的制备参照化合物Ⅲ-1的合成方法，(0.25g，91％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.60(s,1H),8.48(s,1H),8.11(s,1H),7.81(d,J＝8.2Hz,2H),6.84(d,J＝8.2Hz,2H),3.60(t,J＝9.2Hz,2H),3.46(t,J＝9.2Hz,2H),3.12(s,3H).LR-MS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1355.Found:306.1354.
化合物Ⅲ-15：
Figure PCTCN2020087415-appb-000036
参照化合物Ⅲ-1的合成方法，(0.25g，91％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.22(s,1H),8.00(d,J＝9.1Hz,1H),7.77–7.69(m,1H),7.43–7.34(m,1H),6.88(d,J＝9.1Hz,1H),4.81(t,J＝5.2Hz,1H),3.64–3.52(m,3H),3.09(s,1H).LR-HRMS(ESI-TOF):Calcd.For C 19H 18N 3O 2[M+H] +:320.1399.Found:320.1397.
化合物Ⅲ-16：
Figure PCTCN2020087415-appb-000037
参照化合物Ⅲ-1的合成方法，(0.29g，94％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.11(2H,d,J＝10.4Hz),7.99(3H,dd,J＝8.6,3.0Hz),7.54(1H,dd,J＝8.0,8.0Hz),7.44(1H,dd,J＝8.0,8.0Hz),6.88(2H,d,J＝9.2Hz),4.82(1H,bt,t,J＝5.2Hz),3.60(2H,t,J＝5.2Hz),3.56(2H,t,J＝5.2Hz),3.09(3H,s).LR-HRMS(ESI-TOF):Calcd.For C 19H 18N 3OS[M+H] +:336.1171.Found:336.1170.
化合物Ⅲ-17：
Figure PCTCN2020087415-appb-000038
其中，6-甲胺-苯并[b]噻吩-2-甲醛：
Figure PCTCN2020087415-appb-000039
6-溴苯并[b]噻吩-2-甲醛(0.42g，1.7mmol)、二甲基乙胺(40％水溶液，1g，8.9mmol)、CuI(13.9mg，0.073mmol)、K 3PO 4·H 2O(155.4mg，0.73mmol)、甲胺(33％水溶液，1g)于100ml耐压瓶中，密封条件下60℃油浴加热12h，体系冷却至室温，加入50ml水，DCM 萃取(3×100ml)，合并有机相，Na 2SO 4干燥，减压除去有机溶剂，残余物经柱色谱分离纯化(0.23g，68％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.92(1H,s),8.14(1H,s),7.82(1H,d,J＝9.1Hz),7.18(1H,d,J＝2.1Hz),7.01(1H,dd,J＝9.1,2.3Hz),3.05(3H,s).LR-MS(ESI-TOF):Calcd.For C 10H 10NOS[M+H] +:192.0.Found:192.0.
化合物Ⅲ-17的制备参照化合物Ⅲ-1的合成方法，(0.29g，94％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.45(s,1H),7.92(d,J＝8.6Hz,2H),7.85(d,J＝8.3Hz,3H),7.73(dd,J＝8.6,3.9Hz,1H),7.21(d,J＝1.9Hz,1H),7.21(d,J＝1.9Hz,1H),6.96(dd,J＝9.1,2.3Hz,1H),3.05(s,3H).LR-HRMS(ESI-TOF):Calcd.For C 19H 14N 3S[M+H] +:360.1171.Found:360.1173.
化合物Ⅲ-18：
Figure PCTCN2020087415-appb-000040
其中，6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛：
Figure PCTCN2020087415-appb-000041
参照化合物6-甲胺-苯并[b]噻吩-2-甲醛的合成方法，(0.54g，79％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.91(s,1H),8.14(s,1H),7.81(d,J＝5.2Hz,1H),7.17(d,J＝2.0Hz,1H),7.01(dd,J＝2.0,8.8Hz,1H),4.76(t,J＝5.6Hz,1H),3.58(t,J＝4.2Hz,2H),3.52(t,J＝4.2Hz,2H),3.04(s,3H).MS(ESI):m/z Calcd.For C 12H 14NO 2S,[M+H] +:235.1.Found 236.1.
化合物Ⅲ-18的制备参照化合物Ⅲ-1的合成方法，(0.21g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.45(s,1H),7.92(d,J＝8.6Hz,2H),7.85(d,J＝8.3Hz,3H),7.73(dd,J＝8.6,3.9Hz,1H),7.21(d,J＝1.9Hz,1H),7.21(d,J＝1.9Hz,1H),6.96(dd,J＝9.1,2.3Hz,1H),3.63–3.57(m,2H),3.52(t,J＝5.7Hz,2H),3.05(s,3H).LR-HRMS(ESI-TOF):Calcd.For C 21H 19N 3OS[M+H] +:360.1171.Found:360.1173.
化合物Ⅲ-19：
Figure PCTCN2020087415-appb-000042
其中，5-N,N-二甲胺-2-醛基并二噻吩：
Figure PCTCN2020087415-appb-000043
参照6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.54g，79％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.66(s,1H),8.05(s,1H),6.30(s,1H),4.88(bt,1H),3.07(s,6H). MS(ESI):m/z Calcd.For C 9H 12NOS 2[M+H] +:214.0；found 214.0.
化合物Ⅲ-19的制备参照化合物Ⅲ-1的合成方法，(0.31g，90％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.34(s,1H),7.86(d,J＝8.0Hz,2H),7.81(s,1H),7.77(d,J＝8.0Hz,2H),6.32(s,1H),4.88(t,J＝4.0Hz,1H),3.08(s,6H).LR-HRMS(ESI-TOF):Calcd.For C 18H 14N 3S 2[M+H] +:336.0629.Found:336.0630.
化合物Ⅲ-20：
Figure PCTCN2020087415-appb-000044
其中，5-N,N-二乙胺-2-醛基并二噻吩：
Figure PCTCN2020087415-appb-000045
参照6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.44g，75％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.78(s,1H),8.09(s,1H),6.30(s,1H),4.87(bt,1H),3.27(t,J＝8.4Hz,4H),1.26(t,J＝8.4Hz,4H).MS(ESI):m/z Calcd.For C 9H 12NOS 2[M+H] +:214.0；found 214.0.
化合物Ⅲ-20的制备参照化合物Ⅲ-1的合成方法，(0.31g，90％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.34(s,1H),7.86(d,J＝8.0Hz,2H),7.81(s,1H),7.77(d,J＝8.0Hz,2H),6.32(s,1H),4.88(t,J＝4.0Hz,1H),3.27(t,J＝8.4Hz,4H),1.26(t,J＝8.4Hz,4H).LR-HRMS(ESI-TOF):Calcd.For C 20H 18N 3S 2[M+H] +:364.0942.Found:364.0943.
化合物Ⅲ-21：
Figure PCTCN2020087415-appb-000046
其中，5-(N-甲基-N-羟乙基)氨基-2-醛基并二噻吩：
Figure PCTCN2020087415-appb-000047
参照化合物6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.44g，75％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.66(s,1H),8.05(s,1H),6.30(s,1H),4.88(bt,1H),3.64(t,J＝5.6Hz,2H),3.44(t,J＝5.6Hz,2H),3.07(s,3H).MS(ESI):m/z Calcd.For C 10H 12NO 2S 2[M+H] +:241.0；found 242.0.
化合物Ⅲ-21的制备参照化合物Ⅲ-1的合成方法，(0.31g，90％)。 1H NMR(400MHz,DMSO-d 6):δ8.34(s,1H),7.86(d,J＝8.0Hz,2H),7.81(s,1H),7.77(d,J＝8.0Hz,2H),6.32(s,1H),4.88(t,J＝4.0Hz,1H),3.65(q,J＝5.5Hz,2H),3.44(t,J＝5.5Hz,2H),3.34(s,1H),3.08(s,3H).LR-HRMS(ESI-TOF):Calcd.For C 19H 16N 3OS 2[M+H] +:366.0735.Found:366.0736.
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申请获得相同或相似的结果，对本申请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。
Figure PCTCN2020087415-appb-000048
Figure PCTCN2020087415-appb-000049
Figure PCTCN2020087415-appb-000050
Figure PCTCN2020087415-appb-000051
Figure PCTCN2020087415-appb-000052
Figure PCTCN2020087415-appb-000053
Figure PCTCN2020087415-appb-000054
Figure PCTCN2020087415-appb-000055
Figure PCTCN2020087415-appb-000056
Figure PCTCN2020087415-appb-000057
Figure PCTCN2020087415-appb-000058
Figure PCTCN2020087415-appb-000059

Claims (19)

  1. 一种核酸适配体分子，包含下述核苷酸序列(a)、(b)或(c)：
    (a):核苷酸序列N 1CCAAUCGUGGCGUGUCGN 19-N 20-N 21ACUGGCGCCGN 32，其中N 1、N 19、N 20、N 21和N 32代表长度大于或等于1个核苷酸的片段，并且N 1与N 32核苷酸序列中至少有一对碱基形成互补配对，N 19与N 21核苷酸序列中至少有一对碱基形成互补配对；
    (b):与(a)限定的核苷酸序列具有至少70％同一性的核苷酸序列；
    (c):在(a)限定的核苷酸序列中不包括N 1、N 19、N 20、N 21和N 32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(a)衍生的核酸适配体分子。
  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(a)中的N 1与N 32互补配对时，N 1核苷酸序列的方向为5’-3’，N 32核苷酸序列的方向为3’-5’；N 19与N 21互补配对时，N 19核苷酸序列的方向为5’-3’，N 21核苷酸序列的方向为3’-5’。
  3. 根据权利要求2所述的核酸适配体分子，其中当N 1与N 32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 32核苷酸序列中至少有两对核苷酸碱基形成互补配对；当N 19与N 21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 19与N 21核苷酸序列中至少有两对碱基形成互补配对。
  4. 根据权利要求1所述的核酸适配体分子，其中对所述核苷酸序列(a)的核苷酸取代选自下组中的一种：C3A、C3U、A4U、A4G、A4C、A5G、A5C、U6A、U6G、U6C、C7A、C7U、G8C、U9A、G11A、G11U、C12G、C12A、C12U、G13C、U14A、U14G、C17U、G18U、G18C、C27G、C27U、G28U、C29G、C29U、C30A、C30U、C2G/G31C、C2U/G31A、C2A/G31U、G10A/C30U、G10C/C30G、G10U/C30A、C2G/G31C/C3A、C2G/G31C/A4C、C2G/G31C/A5C、C2G/G31C/G8C、C2G/G31C/C12U、C2G/G31C/U14G、C2G/G31C/C27U、C2G/G31C/C29G、C2G/G31C/C30U、C2G/G31C/G10A/C30U、C2G/G31C/G10C/C30G、C2G/G31C/G10U/C30A、C2U/G31A/G10A/C30U、C2U/G31A/G10C/C30G、C2U/G31A/G10U/C30A、C2A/G31U/G10A/C30U、C2A/G31U/G10C/C30G、C2A/G31U/G10U/C30A、C2G/G31C/G10C/C30G/C3A、C2G/G31C/G10C/C30G/A4C、C2G/G31C/G10C/C30G/A5C、C2G/G31C/G10C/C30G/G8C、C2G/G31C/G10C/C30G/C12U、C2G/G31C/G10C/C30G/U14G、C2G/G31C/G10C/C30G/C27U、C2G/G31C/G10C/C30G/C29G、C2G/G31C/G10A/C30U/U6G/C27U、C2G/G31C/G10C/C30G/U6G/C27U、C2G/G31C/G10U/C30A/U9A/U14G/C27U和C2A/G31U/G10U/C30A/U9A/U14G/C27U。
  5. 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 32处的核苷酸序列为F30或tRNA脚手架RNA序列。
  6. 根据权利要求1所述的核酸适配体分子，其中所述核酸适配体分子是RNA分子或经碱基修饰的RNA分子。
  7. 根据权利要求1所述的核酸适配体分子，其中所述核酸适配体分子是DNA-RNA杂交分子或经碱基修饰的DNA-RNA分子。
  8. 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)的N 19-N 20-N 21包含一个可以识别靶标分子的核苷酸序列。
  9. 根据权利要求8所述的核酸适配体分子，其中所述靶标分子为蛋白质、核酸、脂质分子、碳水化合物、激素、细胞因子、趋化因子和代谢物金属离子中的至少一种。
  10. 根据权利要求8或9所述的核酸适配体分子，其中核苷酸序列(a)的N 19-N 20-N 21为可以识别GTP和腺苷分子的核苷酸序列。
  11. 根据权利要求1所述的核酸适配体分子，其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倍或者提高至少500-1000倍。
  12. 根据权利要求1所述的核酸适配体分子，还包含：可以结合多个荧光团分子的串联体，所述串联体通过间隔序列连在一起，所述间隔序列具有2、3、4、5、6、7、8或者更多个核苷酸片段的长度；并且，所述串联体的核苷酸选自序列SEQ ID No：8、9、10，11、12、13、14、15、16、17、18和19。
  13. 根据权利要求1所述的核酸适配体分子，具有序列SEQ ID No：1、2、3、6、7、8、9、10、11、12、13、14、15、16、17、18、19、21、22或23。
  14. 一种核酸适配体分子与荧光团分子的复合物，其中所述核酸适配体分子为权利要求1所述核酸适配体分子，所述荧光团分子具有下述式(I)所述的结构：
    Figure PCTCN2020087415-appb-100001
    其中，D-为X1O-或N(X2)(X3)-；X1、X2、X3各自独立地选自氢、1-10个碳的直链或支链烷基和改性烷基，X2、X3任选相互连接为饱和或不饱和的环；R-选自氢、氰基、羧基、酰胺基、酯基、羟基、1-10个碳的直链或支链烷基或改性烷基；Ar1、Ar2各自独立地选自单环芳亚基、单环杂芳亚基，或由单环芳基、单环杂芳基中的一种或两种稠合组成的具有2-3个环结构的芳香亚基；
    Ar1、Ar2中的氢原子独立地被F、Cl、Br、I、羟基、硝基、醛基、羧基、氰基、磺酸基、硫酸基、磷酸基、氨基、伯氨基、仲氨基、1-10个碳的直链或支链烷基和改性烷基取代；
    所述改性烷基为烷基的任意碳原子被选自F、Cl、Br、I、-O-、-OH、-CO-、-NO2、-CN、-S-、 -SO2-、-(S＝O)-、叠氮基、亚苯基、伯氨基、仲氨基、叔氨基、季铵盐基、环氧乙烷、琥珀酸酯、异氰酸酯、异硫氰酸酯、酰氯、磺酰氯、饱和或不饱和的单环或双环亚环羟基、桥联酯杂环中的至少一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；
    并且，所述复合物中的所述核酸适配体分子与所述荧光团分子分别存在于单独的溶液中，或者，所述核酸适配体分子与所述荧光团分子在同一溶液中。
  15. 根据权利要求14所述的复合物，其中所述改性烷基含有选自-OH、-O-、乙二醇单元、单糖单元、二糖单元、-O-CO-、-NH-CO-、-SO 2-O-、-SO-、Me 2N-、Et 2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、-NO 2和氰基中的至少一种基团；
    所述荧光团分子含有的芳香环选自下式(Ⅱ-1)～(Ⅱ-15)中的结构：
    Figure PCTCN2020087415-appb-100002
  16. 根据权利要求14所述的复合物，其中所述荧光团分子选自下式化合物：
    Figure PCTCN2020087415-appb-100003
    Figure PCTCN2020087415-appb-100004
  17. 根据权利要求14所述的复合物，其中所述适配体分子包含核苷酸序列SEQ ID No：1、2、3、6、7、8、9、10、11、12、13、14、15、16、17、18、19、21、22、23、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14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14所述的复合物在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、基因组DNA成像、检测细胞中mRNA与蛋白质含量、检测基因组DNA，或者，提取及纯化RNA中的应用。
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