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WO2021057816A1 - Molécule d'acide nucléique aptamère - Google Patents

Molécule d'acide nucléique aptamère Download PDF

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WO2021057816A1
WO2021057816A1 PCT/CN2020/117252 CN2020117252W WO2021057816A1 WO 2021057816 A1 WO2021057816 A1 WO 2021057816A1 CN 2020117252 W CN2020117252 W CN 2020117252W WO 2021057816 A1 WO2021057816 A1 WO 2021057816A1
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molecule
nucleic acid
cppepper
rna
nucleotide sequence
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杨弋
陈显军
方梦悦
朱麟勇
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East China University of Science and Technology
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
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    • G01N21/64—Fluorescence; Phosphorescence
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Definitions

  • This application relates to an aptamer nucleic acid molecule, a method for detecting RNA, DNA or other target molecules in or outside the cell, and a kit containing the aptamer.
  • the aptamer of the present 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 cell biology 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-chain interfering RNA (siRNA), small microRNA (microRNA), and long-chain non-coding RNA (lncRNA)
  • siRNA short-chain interfering RNA
  • microRNA small microRNA
  • lncRNA long-chain non-coding RNA
  • Real-time monitoring of RNA transport and metabolic processes in cells is essential for studying the relationship between RNA localization and gene expression and cell regulatory processes.
  • scientists have identified several mechanisms that can lead to different subcellular locations of RNA, such as active transport, passive diffusion, and anchoring.
  • the spatial specific expression of mRNA is closely related to the plasticity, learning and memory of neurons. Therefore, once the RNA regulation process is damaged, it will cause neuronal dysfunction and neurological diseases.
  • Biomacromolecule labeling technology is the key to biomolecular imaging.
  • fluorescent protein technology is one of the most important research tools in contemporary biological science research; in just over ten years, its research has been awarded the Nobel Prize.
  • RNA also has a unique structure, a wide variety of biological functions, and complex temporal and spatial distribution. Compared with proteins, there are more types of RNA, most of which have not yet been identified in terms of structure and function, and are called “dark matter" in the genome. The research on the identification, function and regulation of different types of RNA and its modified forms has now become the international frontier. RNA research also urgently needs a disruptive labeling technology similar to fluorescent protein, which is an extremely useful tool for in-depth study of RNA functional mechanisms.
  • 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 for real-time monitoring of the dynamic change process of RNA in living cells.
  • 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 the 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 that bind to multiple copies of MS2 sequences. Detect the signal of fluorescent protein to monitor the synthesis and distribution of mRNA in real time (Ozawa et al. Nature Methods 2007.4:413-419). However, the MCP-FPs that are not bound to mRNA molecules will produce high background fluorescence, which makes the signal-to-noise ratio of this method very low.
  • the scientists added the MCP-FPs fusion protein to the nuclear localization signal, so that GFP-MS2 that 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 the detection. But there is still some non-specific fluorescence.
  • 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 group replaced a stem-loop structure in "Spinach” with a nucleic acid aptamer that can specifically bind 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 fluorescence signal of the aptamer-fluorophore complex is unstable and is easily quenched, making its fluorescence 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 technology based on single fluorophore-nucleic acid aptamer seems to be a perfect RNA labeling technology, but it is limited by the current complex of fluorophores (DFHBI, DFHBI-1T, DFHO) and nucleic acid aptamer.
  • fluorophores DFHBI, DFHBI-1T, DFHO
  • nucleic acid aptamer The nature is not ideal, and the technology has not been widely used. Therefore, the scientific and industrial circles have always needed more effective fluorophore-aptamer complexes, which can overcome the shortcomings of the previous fluorophore-aptamer complexes for real-time labeling of RNA or DNA in living cells. .
  • 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.
  • nucleic acid aptamer molecule comprising the following nucleotide sequence (a), (b) or (c):
  • N 1 CACUGGCGCCN 12 -N 13 -N 14 CAAUCGUGGCGUGUCGGN 32 (referred to as the general formula cpPepper structure), where N 1 , N 12 , N 13 , N 14 and N 32 represent a 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 12 and N 14 form a complementary pair;
  • nucleotide sequence defined in (a) does not include the positions of N 1 , N 12 , N 13 , N 14 and N 32 , after one or several nucleotide substitutions, deletions and/or additions, A nucleic acid aptamer molecule derived from (a) that has an aptamer function.
  • the nucleotide sequence with the general formula cpPepper structure defined by the nucleotide sequence (b) and nucleotide sequence (a) has at least 75%, 76%, 78%, 80%, 82% , 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences.
  • the nucleotide sequence (c) does not include N 1 , N 12 , N 13 , N in the nucleotide sequence with the general formula cpPepper structure defined by the nucleotide sequence (a).
  • the positions of 14 and N 32 are obtained by substitution, deletion and/or addition of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides Nucleic acid aptamer molecule.
  • the nucleotide sequence (c) does not include the positions of N 1 , N 12 , N 13 , N 14 and N 32 in the nucleotide sequence defined by the nucleotide sequence (a) , A nucleic acid aptamer molecule obtained by the substitution of 7, 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';
  • N 12 and N 14 are complementary paired, the direction of the N 12 nucleotide sequence is 5'-3', and the direction of the N 14 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 12 and N 14 has a length greater than or equal to 5 nucleotide bases, then at least N 12 and N 14 nucleotide sequences There are two pairs of bases forming complementary pairs.
  • the nucleotide substitution of the general formula cpPepper structure defined by the nucleotide sequence (a) is selected from one of the following group: C8G, C8U, G9U, C10G, C10U, C11A, C11U, C15A, C15U, A16U, A16G, A16C, A17G, A17C, U18A, U18G, U18C, C19A, C19U, G20C, U21A, G23A, G23U, C24G, C24A, C24U, G25C, U26A, U26G, CG30C, G30U, C2A/G31U, C2U/G31A, C2G/G31C, C11U/G22A, C11G/G22C, C11A/G22U, C2G/G31C/C15A, C2G/G31C/A16C, C2G/G31C/A17C, C2
  • the nucleotide substitution of the general formula cpPepper structure defined by the nucleotide sequence (a) is selected from one of the following group: C15A, C15U, A16C, A17C, C19U, G20C, U21A, C24G, C24U, U26G, C8U, C10G, C11U, C2A/G31U, C2U/G31A, C2G/G31C, C11U/G22A, C11G/G22C, C11A/G22U, C2G/G31C/C15A, C2G/G31C/A16C, C2G/G31C/A16C G31C/A17C, C2G/G31C/G20C, C2G/G31C/C24U, C2G/G31C/U26G, C2G/G31C/C8U, C2G/G31C/C10G, C2G/G31C/
  • the nucleotide substitution of the general formula cpPepper structure defined by the nucleotide sequence (a) is selected from one of the following group: C15A, C15U, A16C, A17C, C19U, G20C, U21A, C24G, C24U, U26G, C8U, C10G, C11U, C2A/G31U, C2U/G31A, C2G/G31C, C11U/G22A, C11G/G22C, C11A/G22U, C2G/G31C/C15A, C2G/G31C/A16C, C2G/G31C/A16C G31C/A17C, C2G/G31C/G20C, C2G/G31C/C24U, C2G/G31C/U26G, C2G/G31C/C8U, C2G/G31C/C10G, C2G/G31C/
  • the nucleotide sequence at N 1 and N 32 in the nucleotide sequence (a) is F30 or tRNA scaffold RNA sequence.
  • the aptamer molecule is an RNA molecule or a base-modified RNA molecule.
  • the aptamer molecule is a DNA-RNA hybrid molecule or a base-modified DNA-RNA molecule.
  • N 12 -N 13 -N 14 in the nucleotide sequence (a) includes a nucleotide sequence that can recognize the target molecule.
  • the target molecule includes but is not limited to: at least one of protein, nucleic acid, lipid molecule, carbohydrate, hormone, cytokine, chemokine, and metabolite metal ion.
  • N 12 -N 13 -N 14 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-fold. 200 times or at least 500-1000 times higher.
  • the nucleic acid aptamer molecule further includes a tandem body that can bind multiple fluorophores, and the tandem body is connected together by a spacer sequence of an appropriate length, and the spacer sequence has 2, 3, 4, Length of 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: 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the nucleic acid aptamer molecule has the sequence SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 21, 22, 23 or 24.
  • a complex of a nucleic acid aptamer molecule and a fluorophore molecule the nucleic acid aptamer molecule being the nucleic acid aptamer molecule of any one of (1) to (17),
  • the fluorophore molecule has the structure described in the following formula (I):
  • 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 monocyclic aryl groups, monocyclic heteroaryl groups, or one or two condensed components of monocyclic aryl groups and monocyclic heteroaryl groups with 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 acid, sulfuric acid, phosphoric acid, amino, primary amino, and secondary amino groups.
  • nucleic acid aptamer molecule and the fluorophore molecule in the complex are 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 structures in the following formulas (II-1) to (II-15):
  • the fluorophore molecule is selected from compounds of the following formula:
  • the fluorophore molecule 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-21.
  • the aptamer molecule in the complex comprises the nucleotide sequence SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 21, 22, 23 or 24.
  • the above-mentioned complex is also provided for the detection or labeling of target nucleic acid molecules in vitro or in vivo.
  • the above-mentioned complex is also provided for the detection or labeling of extracellular or intracellular target molecules.
  • the above-mentioned complex is also provided for imaging genomic DNA.
  • the above-mentioned complex is also provided for detecting the relationship between mRNA and protein content in a cell.
  • a DNA molecule which transcribes any one of the aforementioned nucleic acid aptamer molecules.
  • an expression vector comprising the above-mentioned DNA molecule.
  • a host cell comprising the above-mentioned expression system.
  • kits 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 The complex.
  • a method for detecting target molecules including the steps:
  • the fluorescence of the complex is detected.
  • a method for detecting genomic DNA including any one of the above-mentioned complexes for imaging genomic DNA.
  • a new nucleic acid aptamer molecule is designed and a new fluorophore molecule is synthesized to form a new fluorophore-nucleic acid aptamer complex.
  • the aptamer molecule can significantly increase the fluorophore after being combined with the fluorophore molecule.
  • the fluorescence intensity of the molecule under the appropriate wavelength of excitation light can be effectively used for real-time labeling of RNA/DNA in living cells.
  • 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.
  • Figure 1 The secondary structure prediction of nucleic acid aptamer molecules.
  • the picture on the left shows the predicted general structure of cpPepper, including N 1 and N 32 that can form a stem structure, and N 12 , N 13 and N 14 that can form a stem-loop structure.
  • the figure on the right shows the predicted structure of cpPepper-1.
  • the base sequences of N 1 and N 32 are shown in the dashed box corresponding to stem 1
  • the base sequences of N 12 , N 13 and N 14 correspond to the stem loop. As shown in the dashed box.
  • FIG. 6 The activation effect of different cpPepper concatemers on III-3.
  • A Obtain the cpPepper cascade according to the "series 1" method;
  • B Obtain the activation effect of the cpPepper cascade on III-3 according to the "series 1” method;
  • C obtain the cpPepper cascade according to the "series 2” method;
  • D Different according to the "series 2” method to obtain the activation effect of the cpPepper series on III-3;
  • E according to the "series 3” method to obtain the cpPepper series;
  • F according to the "series 3” method to obtain the cpPepper series The activation effect on III-3.
  • Figure 9 The labeling effect of cpPepper, III-3 and their analogues used in the labeling of RNA in mammalian cells
  • A The labeling effect of F30-cpPepper-1-III-3 complex used in the labeling of RNA in mammalian cells
  • B The effect of F30-8cpPepper-5 and III-3 analogues in labeling RNA in mammalian cells.
  • FIG. 10 Probe construction based on cpPepper-1.
  • A Detection effect of adenosine probe
  • B Detection effect of GTP probe.
  • cpPepper is used to track RNA localization in cells.
  • (A) cpPepper is used to detect the location of GAPDH mRNA;
  • (B) cpPepper is used to detect the location of TMED2 mRNA.
  • Figure 12 The imaging results of cpPepper used to detect genomic DNA.
  • cpPepper can be used for the detection results of RNA extraction and purification tags.
  • nucleotide and “nucleotide base” are used interchangeably to indicate the same meaning.
  • nucleic acid aptamer molecule described in this application is also referred to as "aptamer molecule".
  • the nucleic acid aptamer molecule comprises (a) the nucleotide sequence is N 1 CACUGGCGCCN 12 -N 13 -N 14 CAAUCGUGGCGUGUCGGN 32 (corresponding to the general formula cpPepper structure of Figure 1); or (b) and (a) described
  • the nucleotide sequence is a sequence with at least 70% identity; wherein at least one pair of bases in the nucleotide sequence of N 1 and N 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 ⁇ 5, at least two pairs are required
  • the bases form complementary pairs.
  • at least one pair of bases in the nucleotide sequence of N 12 and N 14 forms a reverse complementary pair, that is, the direction of the N 12 nucleotide sequence is 5'-3', and the direction of the N 14 nucleotide sequence is 3'- 5'.
  • N 13 is a nucleotide base of any length and composition; or (c) 1-7 nucleotide substitutions, deletions and/or additions at any position of the nucleotide sequence (a) .
  • the nucleic acid aptamer molecule contains a substitution to the nucleotide of the general formula cpPepper structure, and the substitution is selected from one of the following group: C8G, C8U, G9U, C10G, C10U, C11A, C11U, C15A, C15U, A16U, A16G , A16C, A17G, A17C, U18A, U18G, U18C, C19A, C19U, G20C, U21A, G23A, G23U, C24G, C24A, C24U, G25C, U26A, U26G, C29U, G30U, G30C, C2A/G31U, C2U/G31U, , C2G/G31C, C11U/G22A, C11G/G22C, C11A/G22U, C2G/G31C/C15A, C2G/G31C/A16C, C2G/G
  • C15A indicates that the third cytosine nucleotide C of cpPepper is replaced with adenine nucleotide A.
  • C2G/G31C means that the second C of cpPepper is replaced with G, and the 31st G is replaced with C, that is, cpPepper (C2G/G31C) in Table 1.
  • Aptamer molecules are single-stranded nucleic acid molecules that have a secondary structure composed of one or more base pairing regions (stems) and one or more unpaired regions (loops) ( Figure 1).
  • the nucleic acid aptamer molecule 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 other 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 serves to stabilize 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 at positions N 12 -N 13 -N 14 , and the inserted nucleotide sequence replaces the stem-loop structure in FIG. 1.
  • 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 the excitation light of the appropriate wavelength.
  • the target molecule may be a small molecule, a signal molecule on the surface of a cell, or the like. These nucleic acid aptamers are non-covalently bound to specific target molecules.
  • the stem-loop structure can be replaced with an RNA sequence that recognizes the target molecule for detecting the target molecule outside or inside the cell.
  • 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, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 21, 22, 23, or 24, or their mutation sequences that can combine with fluorophore molecules to significantly increase their fluorescence under the appropriate wavelength of 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 scaffolding RNA sequence 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 12 , N 13 , N 14 , and N 32 in the sequence (a) are not included in the calculation of the identity of the nucleotide sequences of the two aptamers in this application.
  • the degree of identity between two nucleotide sequences uses the Needle software package such as EMBOSS (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277).
  • the program is preferably determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in version 3.0.0 or higher.
  • the optional parameters used are gap penalty of 10, gap extension penalty of 0.5 and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). Use the output of Needle marked as "longest identity” (obtained with the -nobrief option) as the percentage identity, and calculate it as follows:
  • the sequence of cpPepper (C15A) and cpPepper (C15U) in Table 1 of this application is N 1 CACUGGCGCCN 12 -N 13 -N 14 A AAUCGUGGCGUGUCGGN 32 and N 1 CACUGGCGCCN 12 -N 13 -N 14 U AAUCGUGGCGUGUCGGN 32 , for their identity
  • the nucleotide bases of N 1 , N 12 -N 13 -N 14 and N 32 should not be included, so their sequence identity comparison result is 96.3% (a difference of 1 Nucleotides).
  • fluorophore molecule described in this application is also referred to as “fluorophore” or “fluorescent molecule”.
  • fluorophore molecules are a type of fluorophore molecules that can be conditionally activated. They show lower quantum yield in the absence of nucleic acid aptamers.
  • the quantum yield of the fluorophore is less than 0.1, more preferably less than 0.01, and most preferably less than 0.001; when the fluorophore is bound by a specific aptamer After that, the quantum yield of the fluorophore is increased by more than 2 times, more preferably by more than 10 times, and most preferably by more than 100 times.
  • the fluorophore molecule is preferably water-soluble, non-toxic to cells and easily penetrates the membrane.
  • the fluorophore of the present 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.
  • the fluorophore molecule is selected from structure (I):
  • D- is X 1 O- or N(X 2 )(X 3 )-, X 1 , X 2 , and X 3 are each independently selected from hydrogen, linear or branched chains of 1-10 carbons Alkyl and modified alkyl, X 2 and X 3 are optionally connected to each other to form a saturated or unsaturated ring;
  • R- is selected from hydrogen, cyano, carboxy, amide, ester, hydroxyl, 1-10 carbon Straight or branched chain alkyl or modified alkyl;
  • Ar 1 and Ar 2 are each independently selected from monocyclic aryl, monocyclic heteroaryl, or from monocyclic aryl and monocyclic heteroaryl.
  • the hydrogen atoms in Ar 1 and Ar 2 can be independently replaced by F, Cl, Br, I, hydroxyl, nitro, aldehyde, carboxy, cyano, sulfonic, sulfate, phosphoric, amino, and primary amino groups. , Secondary amino, 1-10 carbon linear or branched alkyl and modified alkyl substitution;
  • the aromatic ring contained in the above-mentioned fluorophore molecule is selected from the structures in the following formulas (II-1) to (II-15):
  • the aforementioned fluorophore molecule is selected from compounds of the following formula:
  • the fluorophore molecule comprises 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-21.
  • “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 quantum yield is increased by at least 2 times; in another preferred embodiment of the present application, the quantum yield is increased by at least 5-10 times; in another more preferred embodiment of the present application In another more preferred embodiment of the present application, the increase in quantum yield is at least 100-200 times; in another more preferred embodiment of the present application, the increase in quantum yield is 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 may be a molecule related to a disease or pathogen infection.
  • the inserted nucleotide sequence replaces the stem-loops of N 12 , N 13 , and N 14 in the left image of FIG. 1 in the structure shown in the left image of FIG. 1 Structure, 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 sequences will promote the combination of aptamer molecules and fluorophore molecules, and significantly increase the fluorescence of fluorophore molecules under excitation light at a suitable wavelength, so as to realize the detection, imaging and quantitative analysis of target molecules.
  • the target molecule can also be a whole cell or a molecule expressed on the surface of the whole cell. Typical cells include but are not limited to cancer cells, bacterial cells, fungal cells and normal animal cells.
  • the target molecule can also be a virus particle.
  • many aptamers of the above-mentioned target molecules have been identified, and they can be integrated into the multivalent nucleic acid aptamers in this application.
  • RNA aptamers that can bind to 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: 16; in a preferred embodiment of the present application, the target molecule is GTP, its corresponding probe sequence for identifying the target molecule is as SEQ ID NO: 17.
  • Target nucleic acid molecule
  • Target nucleic acid molecule also known as “target nucleic acid molecule” refers to the nucleic acid molecule to be detected, which can be intracellular or extracellular; including target RNA molecules and target DNA molecules.
  • the target nucleic acid molecule is connected to the nucleic acid aptamer molecule, and the fluorophore molecule is combined with the nucleic acid aptamer molecule to significantly increase the fluorescence value of the fluorophore molecule under excitation light of a suitable wavelength, thereby realizing the detection of the target nucleic acid molecule.
  • the content and the purpose of distribution is described in this application.
  • 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 and binding sequence MS2 RNA, phage capsid protein PCP recognition and binding sequence PP7 RNA, lambda phage transcription termination protein N recognition and binding sequence boxB RNA, etc.
  • the target RNA can be fused to the 5'end or 3'end or the N 12 -N 13 -N 14 position of the RNA aptamer molecule of the present application.
  • sgRNA refers to a 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 concatemers are connected together by a spacer sequence of appropriate length, and the number of cpPepper structures connected in series can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. There can be many forms of the concatenation.
  • the concatenation form is "series 1", as shown in Figure 6A, and the preferred nucleotide sequence is SEQ ID NO: 6, 7, 8 Or 9; where 2cpPepper-5 represents a concatenation 1 with two cpPepper-5 structures; in another preferred embodiment of the present application, the concatenation form is "concatenation 2", as shown in Figure 6C, the preferred core
  • the nucleotide sequence is SEQ ID NO: 10, 11 or 12; where 2 ⁇ cpPepper-6 represents the concatemer 2 with two Pepper-6 structures; in another preferred embodiment of the present application, the concatenated form is " Concatenation 3", as shown in Figure 6E, the preferred nucleotide sequence is SEQ ID NO: 13, 14 or 15; where 2 ⁇ 2cpPepper-5 represents concatenation 3 with 4 cpPepper-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 1 cpPepper structure, that is, an aptamer containing 2 stem structures, 2 loop structures and 1 stem loop structure (Figure 1 left). Ligand.
  • An aptamer in a multimeric form refers to an aptamer containing more than one cpPepper 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-cpPepper-1-III-3, F30-cpPepper-1-III-7, F30-cpPepper- 1-III-6, F30-cpPepper-1-III-8, F30-cpPepper-1-III-4, F30-cpPepper-1-III-15, F30-cpPepper-1-III-18 and F30-cpPepper- 1-III-21.
  • the nucleic acid molecule of the concatemer and multiple fluorophore molecules form a complex, for example, the F30-8cpPepper-5 and the F30-8cpPepper-5 containing 8 aptamer units formed in a "tandem 1" manner
  • 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 the aptamer Detection.
  • the increase in fluorescence intensity is at least 2 times (fluorescence intensity is detected according to experimental method (5)); in another preferred embodiment of the present application, the increase in fluorescence intensity is at least 5-10 In another more preferred embodiment of the present application, the increase in fluorescence intensity is at least 20-50 times; in another more preferred embodiment of the present application, the increase in fluorescence intensity is at least 100-200 times; in the present application 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 12 and N 14 Complementary pairing between the nucleotides and 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 on the left 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 a nucleotide sequence R 1 CACUGGCGCCR 12 -R 13 -R 14 CAAUCGUGGCGUGUCGGR 32 and a nucleotide sequence with at least 70% identity.
  • R 1 encodes N 1 in the cpPepper structure
  • R 12 encodes N 12 in the cpPepper structure
  • R 13 encodes N 13 in the cpPepper structure
  • R 14 encodes N 14 , R in the cpPepper structure.
  • N 32 in the 32- encoding general formula cpPepper structure.
  • the DNA molecule may also include a promoter that controls DNA transcription, and the promoter is operably linked to the DNA sequence encoding the nucleic acid aptamer.
  • the DNA molecule includes a U6 promoter; in another specific embodiment of the present application, the DNA molecule includes a CMV promoter.
  • the DNA molecule includes the DNA molecule and may further include DNA encoding any target nucleic acid molecule. In another specific embodiment of the present application, the DNA molecule encoding the target RNA includes a coding A sequence.
  • the DNA molecule encoding the target RNA includes a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a transmembrane emp24 domain containing protein 2 (TMED2) (sequence of chimeric RNA) They are SEQ 19 and 20, respectively, and the DNA sequence of TagBFP (the sequence of the chimeric RNA is SEQ ID No: 24, respectively).
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • TMED2 transmembrane emp24 domain containing protein 2
  • the "promoter” in this application includes eukaryotic and prokaryotic promoters.
  • the promoter sequence of eukaryotic cells is completely different from the promoter sequence of prokaryotic cells.
  • eukaryotic promoters cannot be recognized by RNA polymerase in prokaryotic cells to mediate RNA transcription.
  • prokaryotic promoters cannot be recognized by RNA polymerase in eukaryotic cells and mediate RNA transcription.
  • the strength of different promoters varies greatly (strength refers to the ability to mediate transcription). Depending on the actual application, strong promoters can be used to achieve high-level transcription.
  • a high level of expression is better, and if the transcription behavior is evaluated, a lower level of transcription can allow cells to process the transcription process in a timely manner.
  • 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 the transcription of DNA molecules. For example, the lac operator needs to add lactose or lactose analog (IPTG) to induce its expression, and other operators include trp, pro, etc.
  • IPTG lactose or lactose analog
  • the regulatory sequence at the 5'end of the coding sequence of the DNA molecule is a promoter. Whether it is to obtain RNA aptamers by in vitro transcription or express aptamers in cultured cells or tissues, it is necessary to select a suitable promoter according to the strength of the promoter. Since the expression of aptamers in vivo can be genetically manipulated, another type of promoter is an inducible promoter that induces DNA transcription in response to a specific environment, such as expression in a specific tissue, a specific time, and a specific developmental stage. These different promoters can be recognized by RNA polymerase I, II or III.
  • RNA polymerase transcribes genes differently, and its transcription terminator is also very different. However, screening suitable 3'transcription terminator regions is a daily experimental skill of human sources in this field.
  • the "expression system” of the present application also referred to as "expression vector”, includes a DNA molecule integrated with an expression nucleic acid aptamer.
  • the expression system of this application can be a plasmid or a virus particle.
  • the "expression vector" recombinant virus can be obtained by transfecting a plasmid into a virus-infected cell.
  • Suitable vectors include but are not limited to viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, plasmid vectors include pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG399, pR290, pKC37, pKC101, pBluescript II SK+/- or KS+/- (see Stratagene cloning system), pET28 series, pACYCDuet1, pCDFDuet1, pRSET series, pBAD series, pQE, pIH821, pGEX, pIIIEx426RPR, etc.
  • the host vector system includes, but is not limited to: transformed phage DNA, or plasmid DNA, or coliform plasmid DNA; yeast containing yeast vectors; virus-infected mammals Animal cells (such as adenovirus, adeno-associated virus, retrovirus); 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.
  • methods include, but are not limited to, transformation, transduction, conjugation, fixation, electrotransduction, etc.
  • expression plasmids pET28a-T7-F30-cpPepper-1, pLKO.1-F30-cpPepper-1 and pYES2.1-F30 containing DNA molecules encoding F30-cpPepper-1 RNA are provided -cpPepper-1.
  • an expression plasmid pLKO.1-F30-8cpPepper-5 containing a DNA molecule encoding F30-8cpPepper-5 RNA is provided.
  • an expression plasmid pCDNA3.1 hygro(+)-BFP-4cpPepper-8 containing DNA molecules encoding BFP-4cpPepper-8, GAPDH-4cpPepper-8 and TMED2-4cpPepper-8 is provided , PCDNA3.1 hygro(+)-GAPDH-4cpPepper-8 and pCDNA3.1 hygro(+)-TMED2-4cpPepper-8.
  • an expression plasmid psgRNA-containing DNA molecules encoding sgRNA-cpPepper-9 (loop1), sgRNA-cpPepper-9 (tetraloop), sgRNA-cpPepper-9 (loop1 and tetraloop) 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 vacant.
  • the coding DNA sequence vacancy of the target RNA molecule allows the user to select the DNA sequence of the target RNA molecule to be detected.
  • the coding DNA sequence corresponding to GAPDH mRNA 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, Drosophila cells, and nematode cells.
  • the host cells are more preferably cultured in vitro cells or whole in vivo living tissues.
  • the host cell in this application includes mammalian cells including but not limited to 297T, COS-7, BHK, CHO, HEK293, HeLa, H1299, fertilized egg stem cells, induced pluripotent stem cells, and the original directly isolated from mammalian tissues. Generation cells, etc.; it contains E. coli cells including but not limited to BL21 (DE3), BL21 (DE3, Star), TOP10, Mach1, DH5 ⁇ ; it contains yeast cells including but not limited to BY4741, BY4742, AH109.
  • the detection array 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 to bind streptavidin to The aptamer molecules are anchored by the specific binding of biotin and streptavidin; (2) the phage capsid protein MCP recognizes the binding sequence MS2, the phage capsid protein PCP recognition binding sequence PP7 or lambda phage transcription terminator protein N recognition binding sequence boxB RNA sequence is fused to the 5', 3'or stem-loop structure of the nucleic acid aptamer molecule to recognize the bound protein MCP, PP7 or lambda N The protein is coated on the surface of the array, and the nucleic acid aptamer molecule is anchored by the specific action of MS2 and MCP protein, PP7 and PCP protein or boxB RNA and ⁇ N protein; (3) A piece of RNA or DNA
  • the detection array can be used to detect the presence or absence and concentration of target molecules. Therefore, only in the presence of the target molecules, the nucleic acid aptamer molecules can bind to the fluorophore molecules, which significantly improves their ability to excite light at a suitable wavelength. Within a certain range, the higher the concentration of target molecules, the higher the fluorescence intensity.
  • the kit of the present 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 a host cell and/or fluorophore molecule containing an expression system for expressing nucleic acid aptamer molecules, and corresponding instructions.
  • the nucleic acid aptamer molecule and the fluorophore molecule in the kit are in separate solutions, or the nucleic acid aptamer molecule and the fluorophore molecule are in the same solution.
  • the pCDNA3.1 hygro(+) plasmid vector used in the examples was purchased from Invitrogen, the pLKO.1-puro plasmid vector was purchased from Sigma, the pET28a plasmid vector was purchased from Novagen, and the pYES2.1 TOPO TA plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Jereh Bioengineering Technology Co., Ltd. The expression plasmids constructed in the examples have all undergone sequence determination, which was completed by Jie Li Sequencing Company.
  • the Taq DNA polymerase used in each example was purchased from Shanghai Yisheng Biotechnology Co., Ltd., and the PrimeSTAR DNA polymerase was purchased from TaKaRa Company.
  • the three polymerases were purchased with corresponding polymerase buffer and dNTP.
  • Restriction enzymes such as EcoRI, BamHI, BglII, HindIII, NdeI, XhoI, SacI, XbaI, SpeI, T4 ligase, T4 phosphorylase (T4 PNK), and T7 RNA polymerase were purchased from Fermentas, with the purchase attached The corresponding buffer, etc.
  • the Hieff CloneTM One Step cloning kit used in the examples was purchased from Shanghai Yisheng Biotechnology Co., Ltd.
  • inorganic salt chemical reagents were purchased from Sinopharm Shanghai Chemical Reagent Company.
  • Kanamycin was purchased from Ameresco Company;
  • Ampicillin was purchased from Ameresco Company;
  • 384-well and 96-well fluorescence detection blackboards were purchased from Grenier Company.
  • DFHBI-1T and DFHO were purchased from Lucerna Company.
  • GTP and SAM were purchased from Sigma Company.
  • the DNA purification kit used in the examples was purchased from BBI Company (Canada), and the common plasmid mini-pumping 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 (Sheneng Group).
  • 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.
  • cpPepper-III-7, cpPepper-III-6, cpPepper-III-8, cpPepper-III-4, cpPepper-III-15, cpPepper-III-18 and cpPepper-III-21 458nm, 458nm, 488nm, 488nm, 488nm, 561nm, 561nm laser.
  • Preparation of linearized vector select a suitable cloning site and linearize the vector.
  • the linearized vector can be prepared by restriction enzyme digestion or reverse PCR amplification.
  • the 'and 3'ends respectively have identical sequences corresponding to the two ends of the linearized vector.
  • the optimal amount of vector used in the recombination reaction system is 0.03pmol; the optimal molar ratio of vector to insert is 1:2-1:3, that is, the optimal amount of insert used is 0.06-0.09pmol.
  • X and Y are calculated according to the formula to obtain the linearized vector and insert fragment. After the preparation of the system is complete, mix the components and place 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.
  • cpPepper or cpPepper mutant nucleic acid aptamer molecules according to the commonly used experimental method (1). Combine 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 and obtain 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 fluorescence maximum excitation peak of the complex formed by 5 ⁇ M F30-cpPepper-1 nucleic acid aptamer and 1 ⁇ M III-3 fluorophore molecule is 486 nm, and the maximum emission peak is 531.
  • Synergy Neo2 multifunctional microplate reader to detect the fluorescence intensity of the complex under 485 ⁇ 10nm excitation and 530nm ⁇ 10nm emission conditions is 31000, while the fluorescence intensity of the control (1 ⁇ M III-3 fluorophore molecule) under the same detection conditions is 10. Then the F30-cpPepper-1 nucleic acid aptamer activates the III-3 fluorophore molecule by 3100 times.
  • cpPepper contains 2 stem structures, 2 loop structures and 1 stem loop structure ( Figure 1 left).
  • stem 1 and stem loop sequences the secondary structure predicted by cpPepper-1 (SEQ ID NO: 1) is shown on the right of Figure 1.
  • F30-cpPepper-1 (SEQ ID NO: 2) RNA was prepared according to the common experimental method (1). Incubate 1 ⁇ M III-3 with 5 ⁇ M F30-cpPepper-1. The test results show that the maximum excitation light of the F30-cpPepper-1-III-3 complex is 486nm and the maximum emission light is 531nm ( Figure 4).
  • the cpPepper-1 sequence in F30-cpPepper-1 was subjected to point mutations as shown in Table 1, and prepared according to common experimental methods (1) containing different bases Incubate the cpPepper mutant RNA with base mutation at 1 ⁇ M III-3 and 5 ⁇ M different F30-cpPepper-1 mutant RNA respectively, and according to the common experimental method (5) their fluorescence activation times for III-3 fluorophore molecules.
  • F30-cpPepper-1 in Table 2 is a nucleic acid aptamer with the sequence SEQ ID NO: 2; other aptamers are in the cpPepper-1 sequence of F30-cpPepper-1 and correspond to the cpPepper on the right of Figure 1 Point mutations at nucleotide positions.
  • synthetic base-modified cpPepper-3 (SEQ ID NO: 4, the sequence GGGCCCACUGGCGCC GUAGCUUCGGCUAC CAAUCGUGGCGUGUGUCGGGGCCC underlined base is deoxyribonucleotide base Base) and cpPepper-4 (SEQ ID NO: 5, the sequence GGGCCCACUGGCGCCGUA G CUUCGGC U ACCAAUCGUGGCGUGUCGGGGCCC underlined bases are bases modified by 2'-F) (synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.), they Respectively, the bases containing stem-loop structure were replaced with deoxyribonucleotides and some of the bases were modified by 2'-F.
  • cpPepper In order to detect the activation effect of cpPepper concatemers on III-3 fluorescence, cpPepper is connected in series according to different forms, including the following three types:
  • nucleic acid aptamer RNA After PCR amplification, prepare nucleic acid aptamer RNA according to the common experimental method (1), and combine 0.1 ⁇ M RNA aptamer with 10 ⁇ M III -3 After incubation, detect the fluorescence intensity according to the commonly used experimental method (5).
  • the test results show that with the increase of n, the fluorescence of ncpPepper-III-3 also increases ( Figure 6B), indicating that the fluorescence intensity of the cpPepper-III-3 complex can be increased through the "tandem 1" method.
  • SEQ ID NO: 12 prepare nucleic acid aptamer RNA according to common experimental method (1), incubate 0.1 ⁇ M RNA aptamer with 10 ⁇ M III-3, and detect fluorescence intensity according to common experimental method (5).
  • the test results show that, With the increase of n, the fluorescence of n ⁇ cpPepper-III-3 also increases ( Figure 6D), indicating that the fluorescence intensity of the cpPepper-III-3 complex can be increased through the "tandem 2" method.
  • the coding cDNAs of 2 ⁇ 2cpPepper-5, 4 ⁇ 2cpPepper-5, and 8 ⁇ 2cpPepper-5 were respectively synthesized for the whole gene (the sequences of the coding RNA aptamers are SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15), prepare nucleic acid aptamer RNA according to common experimental method (1), after incubating 0.1 ⁇ M RNA aptamer with 20 ⁇ M III-3, detect fluorescence intensity according to common experimental method (5).
  • Example 7 Use of cpPepper-III-3 complex for the labeling of RNA in bacteria
  • a bacterial expression plasmid expressing F30-cpPepper-1 was first constructed.
  • the primers were used to amplify F30-cpPepper-1 in Example 2, and the primers were used to amplify pET28a.
  • the promoter and multiple cloning site regions were removed, and the amplified DNA fragment of F30-cpPepper-1 was linear with pET28a.
  • the chemical vector was connected according to the experimental method (4), and the obtained recombinant plasmid was named pET28a-T7-F30-cpPepper-1.
  • the primers used to amplify the F30-cpPepper-1 fragment are:
  • Upstream primer (P1) 5’-TCGATCCCGCGAAATTAATACGACTCACTATAGGGTTGCCATGTGTATGTGGG-3’
  • the primers used to amplify the pET28a vector to linearize it are:
  • Upstream primer 5’-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG-3’
  • a yeast expression plasmid expressing F30-cpPepper-1 was first constructed.
  • the F30-cpPepper-1 DNA fragment in Example 2 was amplified using primers, and the amplified F30-cpPepper-1 fragment was inserted into the pYES2.1 TOPO TA vector according to the experimental method (4) to obtain the recombinant plasmid Named pYES2.1-F30-cpPepper-1.
  • the primers used to amplify the F30-cpPepper-1 fragment are:
  • Upstream primer (P5): 5’-GGAATATTAAGCTCGCCCTTTTGCCATGTGTATGTGGG-3’
  • the pYES2.1-F30-cpPepper-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-cpPepper-1, and the bacteria were harvested after 10 hours. Resuspend in PBS containing 2 ⁇ M III-3.
  • the untreated BY4741 strain was used as a control.
  • Example 9 cpPepper and III-3 and their analogs are used for the labeling of RNA in mammalian cells
  • a mammalian cell expression plasmid was constructed.
  • the primers P7 and P8 were used to amplify the F30-cpPepper-1 in Example 2, and the fragment was inserted into the pLKO.1 puro vector using experimental method (4).
  • the resulting expression vector was named pLKO.1-F30-cpPepper-1.
  • the pLKO.1-F30-cpPepper-1 plasmid was transfected into 293T/17 cells. After 24 hours, 1 ⁇ M III-3 was added to label F30-cpPepper-1. The cells that did not express the aptamer were used as controls. ) Detect the marking effect. The results show that the F30-cpPepper-1-III-3 complex exhibits very bright yellow-green fluorescence, while the control has no obvious fluorescence ( Figure 9A), indicating that cpPepper-III-3 can work well in mammalian cells.
  • the primers used to amplify F30-cpPepper-1 are:
  • Upstream primer (P7) 5’-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGG-3’
  • a mammalian expression plasmid expressing F30-8cpPepper-5 was constructed.
  • the primers P7 and P8 in this example were used to amplify the F30-8cpPepper-5 fragment in Example 5, and these fragments were inserted into the pLKO.1 puro vector using experimental method (4).
  • the resulting expression vector was named pLKO.1-F30-8cpPepper-5.
  • the pLKO.1-F30-8cpPepper-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 F30-8cpPepper-5, but not control cells that do not express F30-8cpPepper-5 ( Figure 9B), indicating that cpPepper and III-3 and their Analogs can be used to label RNA in mammalian cells.
  • RNA aptamers In order to construct a cpPepper-based analyte probe, the nucleotides at the stem-loop structure in the cpPepper-1 (SEQ ID No: 2) structure were replaced with those that can specifically recognize and bind adenosine (adenosine) and guanosine (GTP). ) RNA aptamers, these aptamers and cpPepper-1 are connected with bases of appropriate length and composition.
  • the probe RNA is prepared according to the common experimental method (1) and incubated with III-3.
  • the functional microplate reader detects their fluorescence intensity in the presence or absence of adenosine or GTP.
  • test results show that for the adenosine probe, the fluorescence of the probe when adenosine is present is significantly higher than when adenosine is not present (FIG. 10A ), and the corresponding probe RNA sequence is SEQ ID No: 16.
  • the fluorescence of the probe is significantly higher than when GTP is not present ( Figure 10B), and the corresponding probe RNA sequence is SEQ ID No: 17.
  • Example 11 cpPepper is used to track RNA localization in cells
  • RNA expression plasmid in which cpPepper fused with different RNAs was first constructed.
  • the cDNA of 4cpPepper-8 was synthesized by the whole gene (the sequence encoding the RNA aptamer is SEQ ID No: 18), and the 4cpPepper-8 gene fragment was amplified by primers, and inserted into the HindIII and XhoI pairs by homologous recombination.
  • the pCDNA3.1 hygro(+) vector was digested to obtain the pCDNA3.1 hygro(+)-4cpPepper-8 recombinant plasmid.
  • GAPDH and TMED2 coding gene sequences are Genebank: BC009081, BC025957, respectively
  • use primers to amplify GAPDH and TMED2 gene fragments and insert them into pCDNA3 digested with NheI and HindIII .1
  • pCDNA3.1 hygro(+)-GAPDH-4cpPepper-8 and pCDNA3.1 hygro(+)-TMED2-4cpPepper-8 recombinant plasmids are obtained, which respectively code for GAPDH-4cpPepper -8 and TMED2-4cpPepper-8 chimeric RNA, their sequence is SEQ ID No: 19 and 20
  • the primers used to amplify 4cpPepper-8 are:
  • Upstream primer (P9): 5’-TAGCGTTTAAACTTAAGCTTCCATCGGGCCCACTGGCGC-3’
  • the primers used to amplify GAPDH are:
  • the primers used to amplify TMED2 are:
  • Upstream primer (P13): 5’-GGAGACCCAAGCTGGCTAGCATGGTGACGCTTGCTGAACT-3’
  • Downstream primer (P14): 5’-CAGTGGGCCCGATGGAAGCTTAACCATGCTCTAGCGAGTTAAACAACTCTCCGGACTTC-3’
  • the inserted sequence was identified by sequencing to be completely correct, and the plasmid was extracted with a transfection-grade plasmid extraction kit for subsequent transfection experiments.
  • the pCDNA3.1 hygro(+)-GAPDH-4cpPepper-8 and pCDNA3.1 hygro(+)-TMED2-4cpPepper-8 recombinant plasmids constructed in this example were co-transfected with pCDNA3.1 hygro(+)-BFP, respectively. -7 cells, 24 hours 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-4cpPepper-8-III-3 is mainly concentrated in the cytoplasm, while the fluorescence of TMED2-4cpPepper-8-III-3 can observe the phenomenon of endoplasmic reticulum enrichment, which is different from the previous
  • the reports are 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 genomic DNA
  • a recombinant plasmid expressing the chimeric RNA of cpPepper-9 and sgRNA was first constructed.
  • Full-gene synthesis of sgRNA-cpPepper-9 (loop1), sgRNA-cpPepper-9 (tetraloop) and sgRNA-cpPepper-9 (loop1 and tetraloop) cDNAs containing centromere targeting sequences, and the encoded RNA sequences are SEQ respectively ID No: 21, 22, and 23.
  • the primers used to amplify the cDNA corresponding to the chimeric RNA of cpPepper and sgRNA are:
  • Upstream primer 5’-AAAGGACGAAACACCGAATCTGCAAGTGGATATTGTTTGAG-3’
  • the primers for amplifying psgRNA plasmid to linearize it are:
  • the primers used to amplify SpdCas9-GFP are:
  • Upstream primer (P19): 5’-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCACCATGGCCCC-3’
  • the imaging results show that the fluorescence of cpPepper-9-III-21 is mainly concentrated in the nucleus and aggregates in dots (centromeres), which is almost completely consistent with the fluorescence of dCas9-GFP ( Figure 12), indicating that cpPepper can be used for Genomic DNA imaging.
  • Example 13 Tags used by cpPepper for RNA extraction and purification
  • primers P21 and P22 were used to amplify the TagBFP gene fragment with EasyFusion T2A-H2B-TagBFP (Addgene: 113086) as the template, and insert it into the NheI and HindIII double enzyme digestion
  • the pCDNA3.1 hygro(+)-GAPDH-4cpPepper-8 vector the pCDNA3.1 hygro(+)-TagBFP-4cpPepper-8 recombinant plasmid was obtained, which encodes TagBFP-4cpPepper-8, and its RNA sequence is SEQ ID No: 24 .
  • the primers used to amplify TagBFP are:
  • Downstream primer (P22): 5’-CAGTGGGCCCGATGGAAGCTTCTCCCAAACCATGCTCTAGCGAGTGTTAATTGAGCTTGTGCCCCA-3’
  • the recombinant plasmid pCDNA3.1 hygro(+)-TagBFP-4cpPepper-8 was transfected into COS-7 cells. After 24 hours, the cells were collected and resuspended in a buffer of 40 mM HEPES, pH 7.4, 125 mM KCl, and 5 mM MgCl 2. After Activated Thiol Sepharose 4B (GE Healthcare) was washed twice with 500 ⁇ L PBS, it was added with PBS containing 10 mM TCEP (Sigma) and incubated for 1 h at room temperature.
  • a maleamide-containing III-3 fluorophore molecule (Mal-III-3) was added to react at room temperature for 30 minutes, and then washed three times with 500 ⁇ L PBS.
  • the resuspended cells were disrupted and incubated with the above-treated beads at room temperature. After 30 minutes, they were centrifuged at 4000 rpm for 2 minutes. The supernatant was discarded.
  • the agarose beads were washed 6 times with a buffer of 40 mM HEPES, pH 7.4, 125 mM KCl, and 5 mM MgCl 2 , Centrifuge to remove the supernatant each time.
  • the beads were re-selected with DEPC water, treated at 70°C for 10 minutes, centrifuged at 4000 rpm for 2 minutes, and collected the supernatant.
  • the solution washes the precipitate, centrifuges at 14000 rpm for 10 min at 4°C, saves the precipitate, discards the supernatant, and repeats the procedure once. Place the precipitate at room temperature for 5 minutes. After the alcohol has evaporated, add a small volume of DEPC water to resuspend the precipitate.
  • 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) and methylamine (33% aqueous solution, 1g) were placed in a 100ml pressure-resistant bottle, heated in an oil bath at 60°C for 12h under sealed conditions, the system was cooled to room temperature, 50ml of water was added, and DCM was extracted ( 3 ⁇ 100ml), 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

L'invention concerne une molécule d'acide nucléique aptamère comprenant un complexe de l'aptamère et d'une petite molécule fluorophore. L'invention concerne également un procédé de détection d'ARN, d'ADN ou d'autres molécules cibles à l'intérieur ou à l'extérieur d'une cellule au moyen de la molécule d'acide nucléique aptamère, et un kit contenant l'aptamère. L'aptamère peut se lier spécifiquement à une petite molécule fluorophore et peut améliorer significativement l'intensité de fluorescence de celle-ci sous l'excitation d'une lumière d'une longueur d'onde appropriée.
PCT/CN2020/117252 2019-09-23 2020-09-23 Molécule d'acide nucléique aptamère Ceased WO2021057816A1 (fr)

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CN115479923B (zh) * 2021-05-31 2024-06-18 北京大学 一种基于荧光共振能量转移的活细胞单分子rna标记方法
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CN115704026A (zh) * 2021-08-06 2023-02-17 华东理工大学 一种新型rna核酸分子及其复合物和应用
CN115704025A (zh) * 2021-08-06 2023-02-17 华东理工大学 一种新型核酸分子检测与定量技术
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