CN112538482A - RNA detection and quantification method - Google Patents

RNA detection and quantification method Download PDF

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CN112538482A
CN112538482A CN201910897362.8A CN201910897362A CN112538482A CN 112538482 A CN112538482 A CN 112538482A CN 201910897362 A CN201910897362 A CN 201910897362A CN 112538482 A CN112538482 A CN 112538482A
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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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Abstract

The present invention relates to an aptamer nucleic acid molecule, a complex comprising the aptamer and a small fluorophore molecule, a method for the detection of RNA, DNA or other target molecules inside or outside a cell using the aptamer nucleic acid molecule, and a kit comprising the aptamer. The aptamer can be specifically combined with a small fluorophore molecule, and the fluorescence intensity of the aptamer under the excitation of light with proper wavelength is obviously improved.

Description

RNA detection and quantification method
Technical Field
The present invention relates to an aptamer nucleic acid molecule, a method for detecting RNA, DNA or other target molecules inside or outside cells by using the aptamer nucleic acid molecule, and a kit containing the aptamer. The aptamer of the invention can specifically bind to a small fluorophore molecule and significantly improve the fluorescence intensity of the small fluorophore molecule under excitation of light with appropriate wavelength.
Background
Among all biological macromolecules, RNA exhibits the most diverse range of cellular biological functions. In the central body of biology, RNA, as a transmitter of genetic material (messenger RNA), a template for protein synthesis (ribosomal RNA) and a transport vehicle for amino acids (transfer RNA), constitutes a series of physiological processes, ultimately achieving transcription and expression of genes. Over the past decades, RNA has been increasingly discovered by scientists to perform its vital functions in a variety of vital activities, including many RNA-protein complexes such as telomerase, splicing enzymes, ribozymes, riboswitches, and the like. In addition, some non-coding RNAs in recent years, such as short interfering RNA (sirna), small micro RNA (microrna), and long non-coding RNA (lncrna), play an irreplaceable role in regulation of gene expression at the post-transcriptional level. Real-time monitoring of RNA transport and metabolic processes in cells is crucial for studying the relationship between RNA localization and gene expression and cellular regulatory processes. Scientists have now identified several mechanisms that can lead to different subcellular localization of RNA, such as active transport, passive diffusion, anchoring, etc. In many polar cells, particularly neural cells, the spatially specific expression of mRNA is closely associated with neuronal plasticity, learning, and memory. Thus, these regulatory processes of RNA, once damaged, can lead to neuronal dysfunction and neurological disease.
Biomacromolecule labeling technology is the key to biomolecular imaging. In scientific history, people use fluorescent protein to 'light' intracellular protein, and realize the visualization of protein molecules in the process of life dynamics. Fluorescent protein technology is one of the most important research tools in the modern biological science research; in a short decade or more, its research is awarded the Nobel prize. RNA also has a unique structure, a wide variety of biological functions, and a complex spatiotemporal distribution. RNA is more diverse than proteins, and most species have not been characterized for structural function, and are called "dark materials" in the genome. The identification, function and regulation of different types of RNA and modified forms thereof has now become the international frontier. RNA research also urgently needs a subversive labeling technology similar to fluorescent protein, which is a very useful tool for deeply researching the RNA function mechanism.
The RNA fluorescence in situ hybridization technique is a method widely used for a long time for researching the level and distribution of RNA in cells, and is a technique for carrying out fluorescence labeling on specific RNA molecules through molecular hybridization and further carrying out imaging. However, the operation is complicated and contains an elution step, which can only be used for the research of immobilized cells, i.e. dead cells, and can not be used for monitoring the dynamic change process of RNA in living cells in real time. Molecular beacon technology was the first viable cell RNA imaging technology developed. The stem-loop double-labeled oligonucleotide probe which forms a hairpin structure at the 5 'end and the 3' end is utilized, when the stem-loop double-labeled oligonucleotide probe is combined with target RNA, the quenching effect of a quenching group labeled at one end on a fluorescent group is eliminated, the fluorescent group generates fluorescence, or FRET of the fluorescent groups at two ends disappears. However, molecular beacons have the disadvantages of low fluorescence signal, difficult cell entry, easy degradation, severe non-specific aggregation in cell nucleus, easy influence of RNA secondary structure, and the need of specially customizing oligonucleotide probes for each RNA, which limits the wide application of the technology.
The current method for live cell RNA imaging mainly utilizes the fusion technology of RNA binding protein and fluorescent protein, wherein the most typical is MCP-FPs system, the MCP-FPs can specifically recognize and bind to mRNA molecules fused with multiple copies of MS2 sequence, and the synthesis and distribution of mRNA can be monitored in real time by detecting the signal of fluorescent protein (Ozawa et al, Nature Methods 2007.4: 413-419). However, the signal-to-noise ratio of this method is low because MCP-FPs that do not bind to mRNA molecules generate high background fluorescence. Then, scientists add a nuclear localization signal to the MCP-FPs fusion protein to localize GFP-MS2 not bound to mRNA molecules in the nucleus, reducing non-specific fluorescence in the plasma to some extent and increasing the signal-to-noise ratio of the assay, but still maintaining some non-specific fluorescence.
In addition to the RNA-binding protein-fluorescent protein technology for the detection of cellular RNA, scientists have sought an RNA fluorescent tag similar to GFP for RNA imaging. Scientists have constructed a fluorophore-quencher combination such that when an Aptamer (Aptamer) of the fluorophore binds to the fluorophore, the quencher is unable to quench the fluorescence signal of the fluorophore, and the Aptamer-fluorophore-quencher complex is fluorescent. When the aptamer of the fluorophore is not present, the fluorescence signal of the fluorophore is quenched by the quencher. Based on this principle, scientists have achieved the imaging of mRNA in bacteria (Arora et al. nucleic Acids Research 2015.21: e 144). In addition, a tag called image (intracellular multi aptamer genetic) has been developed, which consists of two different aptamer-small molecule complexes. When the small molecules are combined with the aptamers in the RNA sequence, the Fluorescence Resonance Energy Transfer (FRET) phenomenon occurs between the fluorophores carried by two adjacent small molecules, and the RNA condition in the cell can be detected by detecting the change of the fluorescence signal. However, neither of these methods currently achieves real-time monitoring of RNA in mammalian cells. The S.Jaffrey project group obtained a nucleic acid aptamer called "Spinach" that specifically binds to a fluorophore (3, 5-fluoro-4-hydroxybenzil-dene imine, DFHBI) such that its fluorescence was significantly increased (Paige et al science 2011.333: 642-. The "Spinach" mutant "Spinach 2, which has better stability, provides a good tool for genetically encoding RNA for labeling living cells. This group exchanged one stem-loop structure in "Spinach" for an aptamer that can specifically bind to a cellular metabolite, and developed a means for detecting a cellular metabolite based on the Spinach-DFHBI complex (Paige et al science 2012.335: 1194). To date, this method has been successfully used to monitor and analyze RNA dynamics in bacterial, yeast and mammalian cells separately. Subsequently, the group also developed the Corn-DFHO complex for detecting the activity of RNA polymerase III promoter in mammalian cells (Song et al, Nature Chemical Biology 2017.13: 1187-1194). However, this method also has the following drawbacks that greatly limit its wide application: (1) the aptamer-fluorophore complexes have weak binding capacity with dissociation constants (kd) of several tens to several hundreds nM; (2) the fluorescence signal of the aptamer-fluorophore complex is unstable and quenching occurs very easily, making it difficult to detect the fluorescence signal (Han et al, journal of the American Chemical Society 2013.135: 19033-19038); (3) up to now, the spectra were only green and yellow, and the RNA in vivo in living animals was imaged in the absence of longer wavelength spectra (Song et al journal of the American Chemical Society 2014.136: 1198-1201); (4) corn is a dimer, and may interfere with the function of the target RNA; (5) there are currently no other aptamer-fluorophore complexes that can simultaneously monitor multiple RNAs in a cell.
In view of the above, the currently used RNA labeling techniques all have their own distinct disadvantages. The MCP-FPs labeling technology has non-binding background fluorescence intensity and low signal-to-noise ratio. RNA labeling techniques based on single fluorophore-aptamers appear to be very perfect RNA labeling techniques, however, are limited by the undesirable nature of the complex formed by the current fluorophore (DFHBI, DFHBI-1T, DFHO) and aptamer, and are not widely used. Therefore, there is a continuing need in the scientific community and industry for more efficient fluorophore-aptamer complexes that overcome the shortcomings of previous fluorophore-aptamer complexes for real-time labeling of RNA or DNA in living cells.
Brief description of the invention
The invention provides an aptamer molecule, a DNA molecule encoding the aptamer molecule, a complex of the aptamer molecule and a fluorophore molecule, and uses of the complex.
The technical scheme provided by the invention is as follows:
1. the present invention relates to a nucleic acid aptamer molecule comprising the following nucleotide sequence (a), (b) or (c):
(a) nucleotide sequence N1CACUGGCGCCN12-N13-N14CAAUCGUGGCGUGUCGGN32(referred to as the general formula cpPepper structure) wherein N1、N12、N13、N14And N32Represents a nucleotide fragment of length ≧ 1, and N1And N32At least one pair of bases in the nucleotide sequence form a complementary pair, N12And N14At least one pair of bases in the nucleotide sequence forms a complementary pair;
(b) a nucleotide sequence having at least 70% identity to the nucleotide sequence defined in (a);
(c) not including N in the nucleotide sequence defined in (a)1、N12、N13、N14And N32The aptamer molecule derived from (a) having an aptamer function by substitution, deletion and/or addition of one or several nucleotides.
2. The aptamer molecule according to (1), wherein the sequence has at least 75%, 76%, 78%, 80%, 82%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity to the cppeper structural nucleotide sequence of (a).
3. The aptamer molecule according to (1), wherein the nucleotide sequence (c) is not included in the cpPepper structural nucleotide sequence defined in (a)1、N12、N13、N14And N32The aptamer molecule according to (1) via substitution, deletion and/or addition of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides.
4. The aptamer molecule according to (3), wherein the nucleotide sequence (c) does not include N in the nucleotide sequence defined in (a)1、N12、N13、N14And N32The aptamer molecule obtained by 7, 6, 5, 4, 3, 2 or 1 nucleotide substitution.
5. The aptamer molecule according to (1), wherein N in the nucleotide sequence (a)1And N32Complementary pairing, N1The orientation of the nucleotide sequence is 5 '-3', N32The orientation of the nucleotide sequence is 3 '-5'; n is a radical of12And N14Complementary pairing, N12The orientation of the nucleotide sequence is 5 '-3', N14The orientation of the nucleotide sequence is 3 '-5'.
6. The aptamer molecule according to (5), wherein when N is1And N32When the length of at least one fragment in (a) is more than or equal to 5 nucleotide bases, then N1And N32At least two pairs of bases in the nucleotide sequence form complementary pairs; when N is present12And N14When the length of at least one fragment in (a) is more than or equal to 5 nucleotide bases, then N12And N14At least two pairs of bases in the nucleotide sequence form complementary pairs.
7. The aptamer molecule according to any one of (1) to (6), wherein the substitution of a nucleotide of the general formula cppeper structure is one selected from the group consisting of: c8, G9, C10, C11, C15, A16, A17, U18, C19, G20, U21, G23, C24, G25, U26, C29, G30, C2/G31, C11/G22, C2/G31/C15, C2/G31/A16, C2/G31/A17, C2/G31/G20, C2/G31/C24, C2/G31/U26, C2/G31/C8, C2/G31/C10, C2/G31/C11, C2/G31/C22, C11/G22/G31/C22, C22/G31/C22/G31/C22, C11/G31/C22, C22/G31/C11/C31/C10, C11/C31/C11, C31/C11, C22/, C2A/G31U/C11A/G22U, C2U/G31A/C11U/G22A, C2U/G31A/C11G/G22C, C2U/G31A/C11A/G22U, C2G/G31C/C11G/G22C/C15A, C2G/G31/C11G/G22G/A G/G, C2G/G31/C11G/G22G/A G, C2G/G31/C11G/G22/G/A17G, C2G/G31/C11/G22/G20G, C2G/G31/C11/G72/G22/G/C24/G, C2G/C31/C11/C72/C22/C72/G/C22/G/C72/G/C72/C/G/C24/C72, C2/C/G/C/G/C72/G/C/G, C/C72/G/C32/C72, C72/G/C/G/C/G, C/G G22U/U21A/U26G/C8U.
8. The aptamer molecule according to (7), wherein the substitution of the nucleotide for the structure of the general formula cppeper is selected from one of the following groups: C15A, C15U, A16C, A17C, C19U, G20C, U21A, C24G, C24U, U26G, C8U, C10G, C11U, C2A/G31U, C2U/G31U, C11U/G22U, C11U/G22U, C2U/G31/U/C15U, C2U/G31/U/A16U, C2U/G31/U, C2U/C11/U, C2/U/C3/U, C2/U/C3/U, C2/U, C2/U/C3/U, C3/U, C/U/C/U, C3/U, C2/U/C3/U, C3/U, C2/U, C/U/C/U, C2/U, C2/G31/C11/G22, C2/G31/C11/G22/C15, C2/G31/C11/G22/A16, C2/G31/C11/G22/A17, C2/G31/C11/G22/G20, C2/G31/C11/G22/C24, C2/G31/C11/G22/U26, C2/G31/C11/G22/C8, C2/G31/C11/G22/C10, C2/G31/C11/G22/U18/C8 and C2/G31/C11/G22/U18/C8.
9. The aptamer molecule according to (8), wherein the substitution of the nucleotide for the structure of the general formula cppeper is selected from one of the following groups: C15A, C15U, A16C, A17C, C19U, G20C, U21A, C24G, C24U, U26G, C8U, C10G, C11U, C2A/G31U, C2U/G31A, C2G/G31C, C11U/G22A, C11G/G22C, C11/G22, C2/G31/C15, C2/G31/A16, C2/G31/A17, C2/G31/G20, C2/G31/C24, C2/G31/U26, C2/G31/C8, C2/G31/C10, C2/G31/C11/G22 and C2/G31/C11/G22.
10. The aptamer molecule according to (1) to (9), wherein N in the nucleotide sequence (a)1And N32The nucleotide sequence is F30 or tRNA scaffold RNA sequence.
11. The aptamer molecule according to any of the preceding claims, wherein the aptamer molecule is an RNA molecule or a base-modified RNA molecule.
12. The nucleic acid aptamer molecule according to any preceding claim, wherein the aptamer molecule is a DNA-RNA hybrid molecule or a base-modified DNA-RNA molecule.
13. The aptamer molecule of any preceding claim, wherein N is12-N13-N14Comprising a nucleotide sequence that recognizes the target molecule.
14. The aptamer molecule according to (13), wherein the target molecules include but are not limited to: proteins, nucleic acids, lipid molecules, carbohydrates, hormones, cytokines, chemokines, metabolite metal ions.
15. The aptamer molecule according to (13) or (14), wherein N is12-N13-N14To be at leastTo recognize the nucleotide sequence of GTP and adenosine molecules.
16. The aptamer molecule according to any of the preceding claims, wherein the aptamer function is that the aptamer is capable of increasing 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 100-200 times or at least 500-1000 times.
17. The aptamer molecule according to (1), further comprising a plurality of concatemers capable of binding to the fluorophore moiety, wherein the concatemers are linked together by a spacer sequence of suitable length, and the number of concatemers is 2, 3, 4,5,6, 7, 8 or more. The nucleotides of the concatemer may be selected from, but are not limited to, the sequences of SEQ ID nos: 6. 7, 8, 9, 10, 11, 12, 13, 14 or 15.
18. The aptamer molecule according to any one of the preceding claims, wherein the 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.
19. A complex of an aptamer molecule and a fluorophore molecule, wherein the aptamer molecule is any one of the aptamer molecules of (1) to (17), and the fluorophore molecule has a structure represented by the following formula (I):
Figure BDA0002210709150000061
wherein: d-is X1O-or N (X2) (X3) -; x1, X2 and X3 are respectively and independently selected from hydrogen, C1-10 straight chain or branched chain alkyl and modified alkyl, X2 and X3 are optionally connected with each other to form a saturated or unsaturated ring; r-is selected from hydrogen, cyano, carboxyl, amido, ester group, hydroxyl, straight chain or branched chain alkyl or modified alkyl with 1-10 carbons; ar1 and Ar2 are respectively and independently selected from monocyclic aromatic subunit, monocyclic heteroaromatic subunit, or aromatic subunit with 2-3 ring structures and formed by fusing one or two of monocyclic aryl and monocyclic heteroaromatic;
wherein: the hydrogen atoms in Ar1 and Ar2 can be independently substituted by F, Cl, Br, I, hydroxyl, nitro, aldehyde group, carboxyl group, cyano group, sulfonic group, sulfuric group, phosphoric group, amino group, primary amino group, secondary amino group, linear or branched alkyl group with 1-10 carbons and modified alkyl group;
wherein: a group obtained by replacing any carbon atom of the above-mentioned modified alkyl group, which is an alkyl group, with at least one group selected from the group consisting of F, Cl, Br, I, -O-, -OH, -CO-, -NO2, -CN, -S-, -SO2-, - (S ═ O) -, azido, phenylene, primary amino, secondary amino, tertiary amino, quaternary ammonium group, ethylene oxide, succinate, isocyanate, isothiocyanate, acid chloride, sulfonyl chloride, saturated or unsaturated monocyclic or bicyclic cycloalkylene group, and bridged ester heterocycle, said modified alkyl group having 1to 10 carbon atoms, wherein a carbon-carbon single bond is optionally independently replaced with a carbon-carbon double bond or a carbon-carbon triple bond;
wherein the aptamer molecule and the fluorophore molecule in the complex are present in separate solutions or in the same solution.
20. A complex of fluorophore molecules as described in claim 19, said modified alkyl groups comprising units selected from the group consisting of-OH, -O-, ethylene glycol units, monosaccharide units, disaccharide units, -O-CO-, -NH-CO-, -SO2-O-、-SO-、Me2N-、Et2N-、 -S-S-、-CH＝CH-、F、Cl、Br、I、-NO2And a cyano group;
alternatively, the fluorophore molecule may comprise an aromatic ring selected from the group consisting of structures of formulae (II-1) to (II-15) below:
Figure BDA0002210709150000071
alternatively, the fluorophore molecule is selected from a compound of the formula:
Figure BDA0002210709150000072
Figure BDA0002210709150000081
21. the complex according to (20), wherein the fluorophore molecule is selected from the group consisting of 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.
22. The complex according to any one of (19) - (21), wherein the aptamer molecule in the complex comprises the nucleotide sequence of 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.
23. A complex of any one of (19) to (22) for use in detection or labeling of a target nucleic acid molecule in vitro or in vivo.
24. A complex of any one of (19) to (22) for use in the detection or labeling of an extracellular or intracellular target molecule.
25. A complex of any one of (19) to (22) for use in imaging genomic DNA.
26. A complex of any one of (19) to (22) for detecting the relationship between mRNA and protein content in a cell.
27. A DNA molecule which transcribes the aptamer molecule of any one of (1) to (18).
28. An expression vector comprising the DNA molecule of (27).
29. A host cell comprising the expression system of (28).
30. A kit comprising an aptamer molecule according to any of claims 1to 18 and/or an expression vector according to claim 28 and/or a host cell according to claim 29 and/or a complex according to any of claims 19 to 22.
31. A method of detecting a target molecule comprising the steps of:
a) adding the complex of any one of claims (19) to (22) to a solution comprising the target molecule;
b) exciting the complex with light of a suitable wavelength;
c) detecting the fluorescence of the complex.
32. A method for detecting genomic DNA, which comprises imaging genomic DNA with the composition according to any one of claims (19) to (22).
33. A method for extracting and purifying RNA, comprising extracting and purifying RNA using the complex according to any one of claims (19) to (22).
The invention designs a completely new aptamer molecule, synthesizes a completely new fluorophore molecule, and forms a completely new fluorophore-aptamer complex, wherein the aptamer molecule can obviously improve the fluorescence intensity of the fluorophore molecule under excitation light with a proper wavelength after being combined with the fluorophore molecule, and can be effectively used for real-time labeling of RNA/DNA in living cells. The aptamer of the invention has strong affinity to fluorophore molecules and exhibits different fluorescence spectra and good light and temperature stability. The aptamer-fluorophore molecule complexes can be used for real-time labeling and imaging of RNA/DNA in prokaryotic and eukaryotic cells, or as labels for extraction and purification of RNA.
Drawings
FIG. 1 Secondary Structure prediction of aptamer molecules. The left panel is the predicted generic structure of cpPepper, including N, which can form a stem structure1And N32N, which can form a stem-loop structure12、N13And N14. The right panel shows the predicted structure of cpPepper-1, N1And N32The base sequence of (A) is shown by a dotted frame corresponding to stem 1 in the figure, N12、 N13And N14The base sequence of (2) is shown by a dotted frame corresponding to the stem loop.
FIG. 2 prediction of secondary structure of F30-cpPepper-1.
FIG. 3 prediction of secondary structure of tRNA-cpPepper-2.
FIG. 4 shows fluorescence excitation spectrum and emission spectrum of F30-cpPepper-1-III-3 complex.
FIG. 5. Effect of different base-modified cpPepper on III-3 activation. A "control" is the replacement of the cpPepper-3 or cpPepper-4 aptamer with buffer.
FIG. 6. Effect of different cpPepper concatemers on III-3 activation. (A) Obtaining a cpPepper concatemer according to a 'concatemer 1' mode; (B) the activation effect of the cpPepper concatemer on III-3 is obtained according to a 'tandem 1' mode; (C) obtaining a cpPepper concatemer according to a 'concatemer 2' mode; (D) the activation effect of the cpPepper concatemer on III-3 is obtained according to a 'concatemer 2' mode; (E) obtaining a cpPepper concatemer according to a 'tandem 3' mode; (F) the activation effect of the cpPepper concatemer on III-3 is obtained in a mode of 'concatemer 3'.
FIG. 7F 30-cpPepper-1-III-3 complex for the labeling effect of RNA in bacteria;
FIG. 8 the effect of F30-cpPepper-1-III-3 complex on the labeling of RNA in yeast cells;
FIG. 9 labeling Effect of cpPepper and III-3 and analogs thereof on labeling of RNA in mammalian cells (A) labeling Effect of F30-cpPepper-1-III-3 Complex on RNA in mammalian cells; (B) the effect of F30-8cpPepper-5 and III-3 analogs in labeling RNA in mammalian cells.
FIG. 10. construction of a cpPepper-1 based probe. (A) The detection effect of the adenosine probe; (B) detection effect of GTP probe.
FIG. 11.cpPepper was used to track RNA localization in cells. (A) cpPepper was used to detect the localization of GAPDH mRNA; (B) cpPepper was used to detect the localization of TMED2 mRNA.
FIG. 12. imaging results of cpPepper for detection of genomic DNA.
FIG. 13. detection of the tags that cpPepper can use for RNA extraction and purification.
Detailed Description
The invention is described in detail herein by reference to the following definitions and examples. The contents of all patents and publications, including all sequences disclosed in these patents and publications, referred to herein are expressly incorporated herein by reference. Hereinafter, "nucleotide" and "nucleotide base" are used interchangeably to mean the same.
Aptamer molecules
The "nucleic acid aptamer molecule" according to the invention is also referred to as "aptamer molecule". The aptamer molecule comprises (a) a nucleosideThe sequence is N1CACUGGCGCCN12-N13-N14CAAUCGUGGCGUGUCGGN32(corresponding to the general cppeper structure of fig. 1); or (b) a sequence having at least 70% identity to the nucleotide sequence of (a); wherein N is1And N32At least one pair of bases in the nucleotide sequence form a reverse complementary pair, i.e. N1The orientation of the nucleotide sequence is 5 '-3', N32The orientation of the nucleotide sequence is 3 '-5'. When N is present1And N32At least one nucleotide base is less than or equal to 4 in length, at least one pair of bases is required to form complementary pairing; when N is present1And N32At least two pairs of bases are required to form complementary pairs when at least one nucleotide base is greater than or equal to 5 in length. Wherein N is12And N14At least one pair of bases in the nucleotide sequence form an inverted complementary pair, i.e., N12The orientation of the nucleotide sequence is 5 '-3', N14The orientation of the nucleotide sequence is 3 '-5'. When N is present12And N14At least one nucleotide base is less than or equal to 4 in length, at least one pair of bases is required to form complementary pairing; when N is present12And N14At least two pairs of bases are required to form complementary pairs when at least one nucleotide base is greater than or equal to 5 in length. Wherein N is13Is nucleotide base with any length and any composition; or (c) by 1-7 nucleotide substitution, deletion and/or addition at any position of the nucleotide sequence (a).
The aptamer molecule comprises a substitution of a nucleotide of the general formula cppeper structure selected from one of the following groups: c8, G9, C10, C11, C15, A16, A17, U18, C19, G20, U21, G23, C24, G25, U26, C29, G30, C2/G31, C11/G22, C2/G31/C15, C2/G31/A16, C2/G31/A17, C2/G31/G20, C2/G31/C24, C2/G31/U26, C2/G31/C8, C2/G31/C10, C2/G31/C11, C2/G31/C22, C11/G22/G31/C22, C22/G31/C22/G31/C22, C11/G31/C22, C22/G31/C11/C31/C10, C11/C31/C11, C31/C11, C22/, C2A/G31U/C11A/G22U, C2U/G31A/C11U/G22A, C2U/G31A/C11G/G22C, C2U/G31A/C11A/G22U, C2G/G31C/C11G/G22C/C15A, C2G/G31/C11G/G22G/A G/G, C2G/G31/C11G/G22G/A G, C2G/G31/C11G/G22/G/A17G, C2G/G31/C11/G22/G20G, C2G/G31/C11/G72/G22/G/C24/G, C2G/C31/C11/C72/C22/C72/G/C22/G/C72/G/C72/C/G/C24/C72, C2/C/G/C/G/C72/G/C/G, C/C72/G/C32/C72, C72/G/C/G/C/G, C/G G22U/U21A/U26G/C8U (i.e., aptamer molecular structure in Table 1). These mutants are capable of specifically binding to a fluorophore molecule and, upon binding, can significantly increase the fluorescence intensity of the fluorophore molecule under excitation light of the appropriate wavelength. Wherein the positional sequence of the nucleotides corresponds to the positions in figure 1.
The above mutation indicates that a nucleotide substitution occurs at a corresponding site in the aptamer nucleotide sequence of the structure of the general formula cppeper, e.g., C15A indicates that cytosine nucleotide C at position 3 of cppeper is substituted with adenine nucleotide A, i.e., cppeper in Table 1 (C15A); C2G/G31C indicates that C at position 2 of cpPepper is substituted with G, while G at position 31 is substituted with C, i.e., cpPepper in Table 1 (C2G/G31C).
Table 1: aptamer structure with 7, 6, 5, 4, 3, 2 or 1 nucleotide substitution in CPPepper general structure
Figure BDA0002210709150000121
Figure BDA0002210709150000131
Figure BDA0002210709150000141
Aptamer molecules are single-stranded nucleic acid molecules with a secondary structure of one or more base-pairing regions (stems) and one or more unpaired regions (loops) (FIG. 1). The aptamer molecules of the invention comprise a secondary structure as predicted in FIG. 1. The secondary structure comprises 2 ring structures, 2 stem structures and a stem-loop structure, wherein the stem 1 plays a role in stabilizing the molecular structure of the whole aptamer and can be replaced by other nucleotide base pairs with any length and any composition, and the nucleotide base pairs can form the 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 in vitro or in cells. In a preferred embodiment of the invention, the 5' end of the aptamer molecule is fused to a 5S RNA sequence (Genebank: NR _ 023377.1); in another preferred embodiment of the invention, the 5' end of the aptamer molecule is fused to a GAPDH RNA sequence (Genebank: BC 009081).
The stem-loop structure in FIG. 1 serves to stabilize the overall aptamer molecular structure and can be replaced with other nucleotide base pairs of any length that can form a stem-loop structure. The aptamer molecules of the invention may also comprise an insertion into N12-N13-N14A further nucleotide sequence of positions, the inserted nucleotide sequence replacing the stem-loop structure in figure 1. The nucleotide sequence may specifically recognize/bind to a target molecule. When the target molecule is absent, the aptamer molecule has weak binding capacity to the fluorophore molecule, resulting in the fluorophore molecule exhibiting weak fluorescence; when the target molecule exists, the binding of the target molecule and the aptamer promotes the binding of the aptamer and the fluorophore molecule, and the fluorescence of the fluorophore molecule under excitation light with a proper wavelength is remarkably improved. The target molecule may be a small molecule, a signal molecule on the surface of a cell, etc. These aptamers bind to a specific target molecule non-covalently, which is mainly a binding dependent on intermolecular ionic forces, dipole forces, hydrogen bonding, van der waals forces, positive and negative electron interactions, stacking interactions or the like. The stem-loop structure may be replaced with an RNA sequence that recognizes the target molecule for extracellular or intracellular detection of the target molecule. In a preferred embodiment of the invention, the stem-loop structure of the aptamer molecule can bind to a GTP molecule; in another preferred embodiment of the invention, the stem-loop structure may bind to an adenosine molecule.
In a preferred embodiment of the invention, the 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 a mutated sequence thereof that can bind to a fluorophore molecule to significantly enhance its fluorescence under excitation light of a suitable wavelength.
The aptamer molecules of the invention may further comprise a nucleotide sequence that increases their stability. In a preferred embodiment of the invention, F30 scaffold RNA (sequence 2) is used, which is linked to the aptamer molecule in the manner shown in figure 2; in another preferred embodiment of the invention, tRNA scaffold RNA (SEQ ID NO: 3) is used, which is linked to the aptamer molecule in the manner shown in FIG. 3.
The "aptamer molecule" as used herein is an RNA molecule or a DNA-RNA hybrid molecule in which a part of nucleotides is replaced with deoxyribonucleotides. The nucleotides may be in the form of their D and L enantiomers, as well as derivatives thereof, including, but not limited to, 2 ' -F, 2 ' -amino, 2 ' -methoxy, 5 ' -iodo, 5 ' -bromo-modified polynucleotides. Nucleic acids comprise various modified nucleotides.
Identity of each other
"identity" describes in the present invention the relatedness between two nucleotide sequences. The identity of two aptamer nucleotide sequences of the invention is calculated without including the N in the sequence of (a)1、N12、N13、N14、N32. For The purposes of The present invention, The degree of identity between two nucleotide sequences is determined using The Needle program, such as The EMBOSS Software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al 2000, Trends in Genetics 16: 276-. Optional parameters used are gap penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 instead of the matrix (EMBOSS version of BLOSUM 62). The use of the Needle marker "highest identity (changest identity)y) "(obtained using the-nobrief option) and calculated as percent identity as follows:
(same residue X100)/(alignment length-total number of gaps in alignment).
The sequences of cpPepper (C15A) and cpPepper (C15U) as in Table 1 of the present invention are N1CACUGGCGCCN12-N13-N14 AAAUCGUGGCGUGUCGGN32And N1CACUGGCGCCN12-N13-N14 UAAUCGUGGCGUGUCGGN32The term "identity" when they are compared shall not be construed to include 1 12 13 14 32N, N-N-N and NSo that their sequence identity alignment results in 96.3% (by 1 nucleotide).
Fluorophore molecules
The "fluorophore molecule" described herein is also referred to as a "fluorophore" or a "fluorescent molecule". A "fluorophore molecule" is a class of fluorophore molecules that can be conditionally activated in the present invention. They show lower quantum yields in the absence of aptamers. In particular embodiments, the quantum yield of the fluorophore when not bound to a particular aptamer is less than 0.1, more preferably less than 0.01, and most preferably less than 0.001; when the fluorophore is conjugated with a specific aptamer, the quantum yield of the fluorophore is increased by more than 2 times, preferably by more than 10 times, and most preferably by more than 100 times. The fluorophore molecules are preferably water soluble, non-toxic to cells and membrane-permeable. The fluorophores of the present invention are preferably capable of entering the cytosol or periplasm by active transport or passive diffusion through the cell membrane or cell wall. In embodiments of the invention, the fluorophore may be permeable to the outer and inner membranes of gram-negative bacteria, the cell walls and membranes of plant cells, fungi and cell walls and membranes, the membranes of animal cells, and the membranes of GI and endothelium of living animals.
The aptamer molecule can be specifically combined with a fluorophore, and the fluorescence value of the aptamer molecule under the excitation of a specific wavelength is remarkably increased. The fluorophore molecule is selected from the group consisting of structures (i):
Figure BDA0002210709150000161
(I) wherein: d-is X1O-or N (X)2)(X3)-,X1、X2、X3Each independently selected from hydrogen, C1-C10 linear or branched alkyl and modified alkyl, X2、X3Optionally linked to each other as saturated or unsaturated rings; r-is selected from hydrogen, cyano, carboxyl, amido, ester group, hydroxyl, straight chain or branched chain alkyl or modified alkyl with 1-10 carbons; ar (Ar)1、Ar2Each independently selected from monocyclic aromatic subunit, monocyclic heteroaromatic subunit, or aromatic subunit having 2-3 ring structures and composed of one or two of monocyclic aryl and monocyclic heteroaryl;
wherein: ar (Ar)1、Ar2The hydrogen atoms in (a) may be independently substituted with F, Cl, Br, I, hydroxyl, nitro, aldehyde group, carboxyl group, cyano group, sulfonic group, sulfuric acid group, phosphoric acid group, amino group, primary amino group, secondary amino group, linear or branched alkyl group of 1to 10 carbons and modified alkyl group;
wherein: any carbon atom of the modified alkyl is selected from F, Cl, Br, I, -O-, -OH, -CO-, -NO2、-CN、-S-、-SO2-, - (S ═ O) -, azido, phenylene, primary amino, secondary amino, tertiary amino, quaternary ammonium, oxirane, succinate, isocyanate, isothiocyanate, acid chloride, sulfonyl chloride, saturated or unsaturated monocyclic or bicyclic cycloalkylene, bridged ester heterocycle, said modified alkyl group having from 1to 10 carbon atoms, wherein the carbon-carbon single bond is optionally independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond;
optionally, the fluorophore molecule comprises a modified alkyl group selected from the group consisting of-OH, -O-, ethylene glycol units, monosaccharide units, disaccharide units, -O-CO-, -NH-CO-, -SO2-O-、-SO-、Me2N-、Et2N-、-S-S-、-CH＝CH-、 F、Cl、Br、I、-NO2At least one group selected from a cyano group;
alternatively, the fluorophore molecule may have an aromatic ring selected from the group consisting of the structures of the following formulae (II-1) to (II-15):
Figure BDA0002210709150000171
alternatively, the fluorophore molecule is selected from the group consisting of compounds of the formula:
Figure BDA0002210709150000172
in a preferred embodiment of the invention, 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. "increasing the fluorescence signal", "increasing the fluorescence intensity" in the present invention refer to an increase in the quantum yield of the fluorophore upon irradiation with excitation light of a suitable wavelength, or a shift in the maximum emission peak of the fluorescence signal (relative to the emission peak of the fluorophore itself in ethanol or aqueous solution), or an increase in the molar extinction coefficient, or two or more thereof. In a preferred embodiment of the invention, the increase in quantum yield is at least 2-fold; in another preferred embodiment of the invention, the increase in quantum yield is at least 5-10 fold; in another more preferred embodiment of the invention, the increase in quantum yield is at least 20-50 fold; in another more preferred embodiment of the invention, the increase in quantum yield is at least 100-fold and 200-fold; in another more preferred embodiment of the invention, the increase in quantum yield is at least 500-fold and 1000-fold; in another more preferred embodiment of the invention, the increase in quantum yield is at least 1000-10000 times; in another more preferred embodiment of the invention, the increase in quantum yield is greater than 10000-fold; the light source used to excite the fluorophore to produce a fluorescent signal may be any suitable illumination device, including, for example, LED lamps, incandescent lamps, fluorescent lamps, lasers; excitation light can be either directly emitted from these devices or indirectly acquired through other fluorophores, such as donor fluorophores of FERT, or donor chromophores of BRET.
Target molecules
The target molecules of the present invention may be any biological material or small molecule, including but not limited to: proteins, nucleic acids (RNA or DNA), lipid molecules, carbohydrates, hormones, cytokines, chemokines, metabolite metal ions, and the like. The target molecule may be a molecule associated with a disease or a pathogenic infection.
By substituting the inserted nucleotide sequence for N in the left panel of FIG. 1 in the structure shown in the left panel of FIG. 1 in the aptamer molecule of the invention12、N13、N14The stem-loop structure of (a), the nucleotide sequence being capable of specifically recognizing/binding to a target molecule. When the target molecule does not exist, the aptamer molecule and the fluorophore molecule are not combined or have weak combining capacity, and the fluorescence of the fluorophore molecule under excitation light with proper wavelength cannot be obviously improved; when the target molecule exists, the combination of the target molecule and the nucleotide sequence can promote the combination of the aptamer molecule and the fluorophore molecule, so that the fluorescence of the fluorophore molecule under excitation light with proper wavelength is obviously improved, and the detection, imaging and quantitative analysis of the target molecule are realized.
The target molecule may also be a whole cell or a molecule expressed on the surface of a whole cell. Typical cells include, but are not limited to, cancer cells, bacterial cells, fungal cells, and normal animal cells. The target molecule may also be a viral particle. Many aptamers to the target molecule described above have been identified and can be incorporated into multivalent aptamers of the invention. RNA aptamers that have been reported to bind to target molecules include, but are not limited to: t4RNA polymerase aptamers, HIV reverse transcriptase aptamers, phage R17 capsid protein aptamers.
In a preferred embodiment of the present invention, the target molecule is adenosine (adenosine), and the corresponding probe sequence for recognizing the target molecule is shown in SEQ ID NO: 16; in a preferred embodiment of the present invention, the target molecule is GTP and the corresponding probe sequence for recognizing the target molecule is as shown in SEQ ID NO: 17.
target nucleic acid molecule
"target nucleic acid molecule" also called "target nucleic acid molecule" refers to the nucleic acid molecule to be detected, which may be intracellular or extracellular; including target RNA molecules and target DNA molecules. According to the invention, the target nucleic acid molecule is connected with the nucleic acid aptamer molecule, and the fluorescence value of the fluorophore molecule under excitation light with a proper wavelength is obviously improved by combining the fluorophore molecule with the nucleic acid aptamer molecule, so that the purpose of detecting the content and distribution of the target nucleic acid molecule is realized.
The "target RNA molecule" includes any RNA molecule in the present invention, including but not limited to pre-mRNA, mRNA encoding the cell itself or an exogenous expression product, pre-rRNA, tRNA, hnRNA, snRNA, miRNA, siRNA, shRNA, sgRNA, crRNA, long-chain non-coding RNA, phage capsid protein MCP recognition binding sequence MS2RNA, phage capsid protein PCP recognition binding sequence PP7RNA, lambda phage transcription terminator N recognition binding sequence boxB RNA, and the like. The target RNA may be fused to the 5 'or 3' end of the RNA aptamer molecules of the invention or 12 13 14Position of N-N-N。
The term "sgRNA" as used herein refers to a single guide RNA (sgRNA) formed by modifying tracrRNA and crRNA in CRISPR/Cas9 system, wherein the 5' end of the single guide RNA is 20nt or so, and the single guide RNA is complementary to a DNA site, so as to induce a DNA double strand break in Cas9 protein.
Concatemers of aptamers
The aptamer molecules of the invention may further comprise concatemers that can bind multiple fluorophore molecules. The concatemers are linked together by a spacer of suitable length and the number of cpPepper structures in tandem may be 2, 3, 4,5,6, 7, 8, 9, 10 or more. The form of the concatemer may be varied, and in a preferred embodiment of the invention, the concatemer is in the form of "tandem 1", as shown in FIG. 6A, and the preferred nucleotide sequence is SEQ ID NO: 6. 7, 8 or 9; wherein 2cpPepper-5 represents concatemer 1 having 2cpPepper-5 structures; in another preferred embodiment of the invention, the form of the tandem is "tandem 2", as shown in FIG. 6C, and the preferred nucleotide sequence is SEQ ID NO: 10. 11 or 12; wherein 2 XcpPepper-6 represents a concatemer 2 having a structure of 2 Pepper-6; in another preferred embodiment of the invention, the form of the tandem is "tandem 3", as shown in FIG. 6E, and the preferred nucleotide sequence is SEQ ID NO: 13. 14 or 15; wherein 2X 2cpPepper-5 represents a concatamer 3 having 4cpPepper-5 structures; in either form, the spacer sequence between the concatemers can be replaced.
The aptamer in a monomer form in the invention refers to an aptamer containing only 1 cpPepper structure, namely an aptamer containing 2 stem structures, 2 ring structures and 1 stem-loop structure (left in figure 1).
The aptamer in a multimeric form refers to an aptamer containing more than 1 cpPepper structure, and includes but is not limited to an aptamer composed of several tandem forms shown in fig. 6.
Aptamer-fluorophore complexes
The aptamer-fluorophore complexes of the invention comprise 1 aptamer molecule and 1 or more fluorophore molecules. In one embodiment of the invention, the molecular complex comprising 1 nucleic acid molecule and 1 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.
In another embodiment of the invention, concatemer nucleic acid molecules form complexes with multiple fluorophore molecules, such as complexes formed in a "tandem 1" fashion of F30-8cpPepper-5 containing 8 aptamer units with 8 fluorophore molecules III-3, 8cpPepper-5-8 (III-3), 8cpPepper-5-8 (III-7), 8cpPepper-5-8 (III-6), 8cpPepper-5-8 (III-8), 8cpPepper-5-8 (III-4), 8cpPepper-5-8 (III-15), 8cpPepper-5-8 (III-18), and 8cpPepper-5-8 (III-21). The molecular complexes may be present in vitro in separate solutions, or in the same solution, or may be present in the cell.
Aptamer function
The aptamer function of the invention means that the fluorescence intensity of fluorophore molecules under excitation light with proper wavelength can be obviously improved, and the aptamer can be detected by adopting a common experimental method (V) in a specific embodiment and detecting the function of the aptamer. In a preferred embodiment of the invention, the increase in fluorescence intensity is at least 2-fold (fluorescence intensity measured according to Experimental method (V)); in another preferred embodiment of the invention, the increase in fluorescence intensity is at least 5-10 fold; in another more preferred embodiment of the invention, the increase in fluorescence intensity is at least 20-50 fold; in another more preferred embodiment of the invention, the increase in fluorescence intensity is at least 100-fold and at least 200-fold; in another more preferred embodiment of the invention, the increase in fluorescence intensity is at least 500-fold and 1000-fold; in another more preferred embodiment of the invention, the increase in fluorescence intensity is at least 1000-10000 times; in another more preferred embodiment of the invention, the increase in fluorescence intensity is greater than 10000-fold.
Aptamer secondary structure
The secondary structure of the aptamer in this patent was predicted by simulation using mFold online analysis software (http:// unafold. rna. albany. edu/. The stem structure in the secondary structure refers to that certain regions in the single strand of the aptamer molecule are complementarily paired by hydrogen bonds to form a local double-stranded structure. Generally, the formation of a double-stranded structure does not require that all nucleotides in the region be complementarily paired; in general, N is1And N32And N is12And N14At least 50% of the nucleotides of one of the fragments are complementarily paired with the other fragment to form a stem structure. If N is present1And N32Is a single nucleotide, N is required1And N32Complete complementation will allow the formation of stem structure (as shown on the left of figure 1).
DNA molecules expressing aptamers
The DNA molecule comprises a DNA sequence which may encode a nucleic acid aptamer molecule of the invention. The DNA molecule comprises a nucleotide sequence R1CACUGGCGCCR12-R13-R14CAAUCGUGGCGUGUCGGR32And nucleotide sequences thereof having at least 70% identity. Wherein R is1Encoding N in the structure of the formula cpPepper1，R12Encoding N in the structure of the formula cpPepper12，R13Encoding N in the structure of the formula cpPepper13，R14Encoding N in the structure of the formula cpPepper14，R32Encoding N in the structure of the formula cpPepper32. The DNA molecule may further comprise a promoter for controlling transcription of the DNA, the promoter being operably linked to the DNA sequence encoding the aptamer. In one embodiment of the invention, the DNA molecule comprises the U6 promoter; in another embodiment of the invention, the DNA molecule comprises a CMV promoter. DNA molecules comprising the DNA molecule further may comprise DNA encoding any nucleic acid molecule of interest. In another embodiment of the invention, the DNA molecule encoding the RNA of interest comprises the coding A sequence. In a specific embodiment of the invention, the DNA molecule encoding the target RNA comprises a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a transmembrane emp24 domain containing protein 2(TMED2) (the sequences of the chimeric RNAs are SEQ 19 and 20, respectively, and the sequence of the TagBFP (the sequences of the chimeric RNAs are SEQ ID Nos: 24, respectively).
Promoters
"promoters" in the present invention include eukaryotic and prokaryotic promoters. The promoter sequence of eukaryotic cells is completely different from that of prokaryotic cells. In general, eukaryotic promoters are not recognized by RNA polymerases in prokaryotic cells to mediate transcription of RNA. Similarly, prokaryotic promoters are not recognized by RNA polymerases in eukaryotic cells to mediate transcription of RNA. The strength of different promoters varies widely (strength refers to the ability to mediate transcription). Depending on the actual application, a strong promoter can be used to achieve high levels of transcription. For example, when used for markers, high levels of expression are better, whereas lower levels of transcription may allow the cell to handle the transcription process in a timely manner if the transcription behavior is assessed. Depending on the host cell, one or more suitable promoters may be used. For example, when used in E.coli cells, T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, PR and PL promoter in lambda phage, and other promoters, but are not limited to, lacUV5 promoter, ompF promoter, bla promoter, lpp promoter, etc. In addition, a hybrid trp-lacUV5 promoter (tac promoter) or other E.coli promoters obtained by recombinant or synthetic DNA techniques may be used to transcribe the RNA aptamers of the invention. In bacteria, operator sequences may be combined with promoter sequences to form inducible promoters, and specific inducers may be added to induce transcription of the DNA molecule. For example, the lac operator requires the addition of lactose or lactose analogs (IPTG) to induce expression, and other operators include trp, pro, and the like.
As mentioned above, the control sequence 5' to the coding sequence of the DNA molecule is a promoter. Whether RNA aptamers are obtained by in vitro transcription or aptamers are expressed in cultured cells or tissues, an appropriate promoter needs to be selected depending on the strength of the promoter. Since aptamers can be genetically manipulated for expression in vivo, another type of promoter is an inducible promoter that induces transcription of DNA in response to a particular environment, e.g., expression in a particular tissue, at a particular time, at a particular developmental stage, etc. These different promoters can be recognized by RNA polymerase I, II or III.
Suitable promoters are also required for the initiation of transcription in eukaryotic cells, including but not limited to the β -globin promoter, the CAG promoter, the GAPDH promoter, the β -actin promoter, the Cstf2t promoter, the SV40 promoter, the PGK promoter, the MMTV promoter, the adenovirus Ela promoter, the CMV promoter, and the like. Termination of transcription in eukaryotic cells depends on specific cleavage sites in the RNA sequence. Similarly, the transcription terminator of RNA polymerase is very different from that of RNA polymerase. However, screening for a suitable 3' transcription terminator region is within the routine laboratory skill of a person of skill in the art.
Expression system
The "expression system", also referred to as "expression vector", of the present invention comprises a DNA molecule integrated with an expression aptamer. The expression system of the present invention may be a plasmid or a viral particle.
"expression vector" recombinant viruses can be obtained by transfecting plasmids into cells infected with the virus. Suitable vectors include, but are not limited to, viral vectors such as the lambda vector system gt11, gtWES. tB, Charon 4, plasmid vectors including pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG399, pR290, pKC37, pKC101, pBluescript II SK +/-or KS +/- (see Stratagene cloning systems), the pET28 series, pACYCDuet1, pCDFDuet1, the pRSET series, the pBAD series, pQE, pIH821, pGEX, pIEx 426 RPR, and the like.
A wide variety of host expression systems may be used to express the DNA molecules of the present invention. Mainly, the vector system must be compatible with the host cell used, including but not limited to: transformed phage DNA, or plasmid DNA, or cosmid DNA; yeast comprising a yeast vector; mammalian cells infected with a virus (e.g., adenovirus, adeno-associated virus, retrovirus); insect cells infected with viruses (e.g., baculovirus); infecting bacteria or plant cells transformed by particle bombardment. The strength and properties of the expression elements in the vectors vary widely. Any one or more suitable transcription elements may be selected depending on the host-vector system used.
Once the constructed DNA molecules have been cloned into a vector system, they can be readily transferred into a host cell. Depending on the vector or host cell system, methods include, but are not limited to, transformation, transduction, conjugation, immobilization, electroporation, and the like.
In a specific embodiment of the invention, expression plasmids pET28a-T7-F30-cpPepper-1, pLKO.1-F30-cpPepper-1 and pYES2.1-F30-cpPepper-1 are provided comprising a DNA molecule encoding F30-cpPepper-1 RNA. In another embodiment of the invention, expression plasmid pLKO.1-F30-8cpPepper-5 is provided comprising a DNA molecule encoding F30-8cpPepper-5 RNA. In another embodiment of the present invention, expression plasmids pCDNA3.1hygro (+) -BFP-4cpPepper-8, pCDNA3.1hygro (+) -GAPDH-4cpPepper-8 and pCDNA3.1hygro (+) -TMED2-4cpPepper-8 containing DNA molecules encoding BFP-4cpPepper-8, GAPDH-4cpPepper-8 and TMED2-4cpPepper-8 are provided. In another embodiment of the invention, expression plasmids psgRNA-cpPepper-9(loop1), psgRNA-cpPepper-9 (tetra loop), and psgRNA-cpPepper-9(loop1 and tetra loop) are provided comprising DNA molecules encoding sgRNA-cpPepper-9(loop1), sgRNA-cpPepper-9(tetra loop), sgRNA-cpPepper-9(loop1 and tetra loop).
The present invention also provides an expression vector incorporating a DNA molecule encoding an aptamer, but lacking a DNA sequence encoding a target RNA molecule, wherein the absence of the DNA sequence encoding the target RNA molecule allows the user to select the DNA sequence of the target RNA molecule to be detected, e.g., the DNA sequence corresponding to GAPDH mRNA, insert the DNA sequence into the expression vector of the present invention using standard recombinant DNA techniques, introduce the resulting expression vector into (transfected, transformed, infected, etc.) host cells, and detect the content and distribution of the target RNA.
Host cell
"host cells" in the present invention include, but are not limited to, bacteria, yeast, mammalian cells, insect cells, plant cells, zebrafish cells, drosophila cells, nematode cells. The host cell is more preferably a cultured in vitro cell or whole in vivo living tissue. The host cell of the present invention, which comprises mammalian cells, includes but is not limited to 297T, COS-7, BHK, CHO, HEK293, HeLa, H1299, fertilized egg stem cell, induced totipotent stem cell, primary cell directly isolated from mammalian tissue, etc.; the escherichia coli cells contained by it include, but are not limited to, BL21(DE3), BL21(DE3, Star), TOP10, Mach1, DH5 α; it comprises yeast cells including but not limited to BY4741, BY4742, AH 109.
Detection array
According to the inventionThe detection array comprises one or more aptamer molecules of the invention, wherein the aptamer molecules are anchored at discrete locations on the array surface, which is comprised of a solid support, including but not limited to glass, metal, ceramic, and the like. The anchoring of the aptamer molecules of the invention to the array surface can be achieved by, but is not limited to, the following methods: labeling 5 'or 3' ends of the aptamer molecules by using biotin, coating streptavidin on the surface of an array, and anchoring the aptamer molecules by specific binding of the biotin and the streptavidin; (2) the recognition binding sequence MS2 of the phage capsid protein MCP, the recognition binding sequence PP7 of the phage capsid protein PCP or the recognition binding sequence boxBRNA of the lambda phage transcription termination protein N are fused on the 5 ', 3' or stem-loop structure of the aptamer molecule, and the fusion protein MCP, PP7 or lambda phage transcription termination protein N are recognized and combinedNCoating the protein on the array surface, and coating the protein with MCP protein, PP7 and PCP protein or boxBRNA and lambda by MS2NThe specific action of the protein anchors the aptamer molecule; (3) fusing a section of RNA or DNA sequence at the 5 'or 3' end of the aptamer molecule, anchoring the RNA sequence complementarily paired with the section of RNA sequence or the DNA sequence complementarily paired with the section of DNA sequence on the surface of the array, and anchoring the aptamer molecule on the surface of the array by the principle of molecular hybridization. The detection array can be used for detecting the existence and concentration of target molecules, so that the aptamer molecules can be combined with fluorophore molecules only in the presence of the target molecules, the fluorescence intensity of the aptamer molecules under the appropriate excitation light wavelength is obviously improved, and the higher the concentration of the target molecules is, the higher the fluorescence intensity is within a certain range.
Reagent kit
The kit of the invention comprises the nucleic acid aptamer molecule and/or the fluorophore molecule of the invention, and corresponding instructions; or comprising an expression system for expressing said aptamer molecule and/or a fluorophore molecule, and corresponding instructions; or comprising a host cell expressing an expression system for the nucleic acid aptamer molecule and/or a fluorophore molecule, and corresponding instructions. The aptamer molecules and the fluorophore molecules in the kit are present in separate solutions or in the same solution.
Detailed Description
The invention is further illustrated by the following examples. These examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention in any way. In the examples, the conventional molecular biological cloning methods of genetic engineering are mainly used, and these methods are well known to those skilled in the art, for example: briefly, the handbook of molecular biology laboratory references of ross chems et al, and the translation of j. sambrook, d.w. rasel, huang peitang et al: a relevant section of the molecular cloning guidelines (third edition, 8. 2002, published by scientific Press, Beijing). Those of ordinary skill in the art will readily appreciate that modifications and variations can be made to the present invention as described in the examples below.
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 correctly by Mass Spectrometry by Jerry bioengineering techniques, Inc. The expression plasmids constructed in the examples were subjected to sequencing, which was performed by Jellian sequencer. Taq DNA polymerase used in each example was purchased from assist in Shanghai, Biotech Ltd, PrimeSTAR DNA polymerase was purchased from TaKaRa, and the respective polymerase buffer and dNTP were provided when all of the three polymerases were purchased. Restriction enzymes such as EcoRI, BamHI, BglII, HindIII, NdeI, XhoI, SacI, XbaI, and SpeI, T4 ligase, T4 phosphorylase (T4PNK), and T7RNA polymerase were purchased from Fermentas, and supplied with buffers. Hieff Clone used in the examplesTMOne Step cloning kit was purchased from Shanghai assist saint Biotech, Inc. Unless otherwise stated, the inorganic salt chemicals were purchased from Shanghai chemical company, the national pharmaceutical group. Kanamycin (Kanamycin) was purchased from Ameresco; ampicillin (Amp) was purchased from AmerescoA driver; fluorescent detection blackboards of 384 wells and 96 wells were purchased from Grenier. DFHBI-1T and DFHO are available from Lucerna, Inc. GTP and SAM were purchased from Sigma.
The DNA purification kit used in the examples was purchased from BBI (Canada) and the general plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Ltd. BL21(DE3, Star) strain was purchased from Invitrogen. 293T/17 cells and COS-7 cells were purchased from the cell bank of the culture Collection of the national academy of sciences. BY4741 Yeast strains were purchased from Shanghai Diego Biotechnology, Inc.
The main instruments used in the examples: synergy Neo2 multifunctional microplate reader (Bio-Tek, USA), X-15R high speed refrigerated centrifuge (Beckman, USA), Microfuge22R desk type high speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, Germany), living body imaging system (Kodak, USA), photometer (Japan and light, Inc.), nucleic acid electrophoresis instrument (Shenneng Bo, Inc.).
The abbreviations have the following meanings: "h" refers to hours, "min" refers to minutes, "s" refers to seconds, "d" refers to days, "μ L" refers to microliters, "ml" refers to milliliters, "L" refers to liters, "bp" refers to base pairs, "mM" refers to millimoles, and "μ M" refers to micromoles.
General experimental methods and materials used in the examples
(one) preparation of aptamer molecules:
the cDNA corresponding to the RNA to be detected was amplified using a primer containing the T7 promoter, and the RNA was obtained by transcription using the double-stranded cDNA recovered using T7RNA polymerase (purchased from Fermentas). mu.L of 3M NaAc and 115. mu.L of DEPC water were added to 20. mu.L of the transcription system, and after mixing, 150. mu.L of phenol chloroform-isopropanol mixture (phenol: chloroform: isopropanol: 25: 24: 1) was added, followed by shaking, mixing, and centrifugation at 10000rpm for 5 minutes to obtain a supernatant. Adding equal volume of chloroform solution, shaking, mixing, centrifuging at 10000rpm for 5min, collecting supernatant, and repeating once. Adding 2.5 times volume of anhydrous ethanol into the supernatant, standing in a refrigerator at-20 deg.C for 30min, centrifuging at 4 deg.C at 12000rpm for 5min, discarding the supernatant, and washing the precipitate with 75% precooled anhydrous ethanol for 2 times. After the ethanol is volatilized, adding an appropriate amount of screening buffer solution to resuspend the precipitate, treating at 75 ℃ for 5min, and standing at room temperature for more than 10min for subsequent experiments.
(II) cell culture and transfection:
the cells in this example were all in CO2Culturing in culture box with high-sugar culture medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and streptomycin and penicillin, and subculturing cells when the growth reaches 80-90% confluency. At the time of transfection, use
Figure BDA0002210709150000262
(purchased from Promega) was performed according to the instructions.
(III) fluorescence imaging:
the main imaging experiment in the examples was performed using a Leica SP8 confocal laser microscope, using a HCXPL APO 63.0x1.47 oil lens and a HyD detector. To capture the Pepepr-III-3 complex fluorescence, a 488nm laser was used. For photographing the fluorescence of cpPepper-III-7, cpPepper-III-6, cpPepper-III-8, cpPepper-III-4, cpPepper-III-15, cpPepper-III-18 and cpPepper-III-21, lasers of 458nm, 488nm, 561nm and 561nm were used, respectively.
(IV) recombinant plasmid construction based on homologous recombination method
1. Preparing a linearized vector, namely selecting a proper cloning site, linearizing the vector, and preparing the linearized vector by enzyme digestion or reverse PCR amplification.
And 2, preparing an insert by PCR amplification, namely introducing 15-25bp (not including enzyme cutting sites) of homologous sequences at the tail end of the linearized vector into the 5 ' ends of the forward PCR primer and the reverse PCR primer of the insert, so that the 5 ' ends and the 3 ' ends of the PCR product of the insert are respectively provided with completely consistent sequences corresponding to the two tail ends of the linearized vector.
3. And (3) measuring the concentrations of the linearized vector and the insert, namely performing a plurality of equal-volume dilution gradients on the linearized vector and the insert amplification product, performing agarose gel electrophoresis on 1 mu L of each of the original product and the diluted product, and comparing the brightness of a strip with a DNA molecular weight standard (DNA Marker) to determine the approximate concentration of the product.
4. Recombination reactions
The using amount of the optimal carrier of the recombination reaction system is 0.03 pmol; the molar ratio of the optimal vector to the insert is 1:2 to 1:3, i.e., the optimal insert is used in an amount of 0.06 to 0.09 pmol.
Figure BDA0002210709150000261
Figure BDA0002210709150000271
X and Y are used for obtaining the linearized vector and the insert respectively according to the formula calculation. After the system preparation is finished, the components are mixed uniformly and react for 20min at 50 ℃. When the insert is > 5kb, the incubation temperature can be extended to 25 min. After the reaction was complete, it was recommended to cool the reaction tube on ice for 5 min. The reaction product can be directly transformed, or can be stored at-20 ℃ and can be thawed and transformed when required.
(V) functional assay of nucleic acid aptamers
The cpPepper or cpPepper mutant aptamer molecules were prepared according to the general protocol (one), 5. mu.M aptamer molecules were mixed with 1. mu.M fluorophore molecules in detection buffer (40mM HEPES, pH 7.4,125mM KCl,5mM MgCl)25% DMSO), and detecting and acquiring the maximum excitation peak and the maximum emission peak of the fluorescence of the aptamer-fluorophore molecular complex by using a Synergy Neo2 multifunctional microplate reader. Then, fluorescence intensity of the aptamer-fluorophore molecule complex under the maximum excitation and emission conditions of the aptamer-fluorophore molecule complex is detected by using a Synergy Neo2 multifunctional enzyme labeling instrument, and a control sample (1 mu M fluorophore molecule without the aptamer) is also measured under the same conditions to calculate the ratio of the fluorescence intensity. For example, the complex formed by 5. mu. M F30-cpPepper-1 aptamer and 1. mu.M III-3 fluorophore molecule has a fluorescence maximum excitation peak of 486nm and an emission peak of 531. When the fluorescence intensity of the compound under the excitation of 485 +/-10 nm and the emission of 530nm +/-10 nm is 31000 and the fluorescence intensity of a contrast (1 mu M III-3 fluorophore molecule) under the same detection condition is 10, which is detected by a Synergy Neo2 multifunctional enzyme-linked immunosorbent assay, the F30-cpPepper-1 aptamer pair III-3 the fold activation of the fluorophore molecule is 3100 fold.
Example 1 Secondary Structure of cpPepper aptamer molecules
The secondary structure of the cpPepper aptamer was analyzed using mFold online RNA structure analysis software. cppeper contains 2 stem-loop structures, 2 loop structures and 1 stem-loop structure (fig. 1 left). For one of the stem 1 and stem loop sequences, the predicted secondary structure of cpPepper-1(SEQ ID NO: 1) is shown on the right of FIG. 1.
Example 2 characterization of fluorescence Spectroscopy of the cpPepper-III-3 Complex
To examine the spectroscopic properties of the cpPepper-III-3 complex, F30-cpPepper-1(SEQ ID NO: 2) RNA was prepared according to the general experimental procedure (one). mu.M III-3 was incubated with 5. mu. M F30-cpPepper-1. The detection result shows that the F30-cpPepper-1-III-3 complex has the maximum excitation light of 486nm and the maximum emission light of 531nm (FIG. 4).
Example 3 fluorescent activation Effect of different cpPepper mutants on III-3 fluorophore molecules.
To examine the fluorescence activation effect of different cpPepper mutants on III-3 fluorophore molecules, cpPepper-1 sequences in F30-cpPepper-1 were subjected to point mutation as shown in Table 1, cpPepper mutant RNAs containing different base mutations were prepared according to the general experimental method (one), 1. mu. MIII-3 was incubated with 5. mu.M of different F30-cpPepper-1 mutant RNAs, respectively, and their fluorescence activation fold on III-3 fluorophore molecules according to the general experimental method (five). The detection results show that most F30-cpPepper-1 mutants containing single base mutation retain strong fluorescence activation effect (>10 times) on III-3 (Table 2). Part of the F30-cpPepper-1 mutant with 2-7 base mutations still retained the strong (>100 fold) fluorescence activation effect on III-3 (Table 3). In conclusion, many single-and multi-base mutants of cpPepper still retain the aptamer function of activating the III-3 fluorophore molecule.
TABLE 2 activation Effect of cpPepper mutants with single base mutations on III-3
Mutants Multiple of activation Mutants Multiple of activation Mutants Multiple of activation
F30-cpPepper-1 3100 G22A 365 G20C 875
C15G 256 G22C 652 A3U 65
C15A 2314 G23U 986 A3G 458
C15U 1854 G23A 1125 A3C 96
A16U 1562 G23C 165 C4G 32
A16G 1265 C24G 1758 C4A 462
A4C 1953 C24A 215 G7U 362
A17G 987 C24U 1874 C8G 1574
A17C 1654 G25U 265 C8A 98
U18A 1722 G25A 768 C8U 2953
U18G 1980 G25C 1324 G9U 542
U18C 1236 U26A 1189 G9A 21
C19A 544 U26G 2745 G9C 96
C19U 1265 G27C 5 C10G 1825
G19C 2654 U28A 12 C20U 1236
G20A 154 U28G 47 C10A 6
U21A 1684 C29A 26 C11G 65
U21C 35 C29U 1164 C11U 1786
G22U 651 G30U 963 C11A 1635
Note: F30-cpPepper-1 in Table 2 is a peptide having the sequence of SEQ ID NO: 2; other aptamers are point mutations in the cpPepper-1 sequence of F30-cpPepper-1 at the nucleotide positions corresponding to the cpPepper in FIG. 1 right.
TABLE 3 activation Effect of the cpPepper mutant containing multiple base mutations on III-3
Figure BDA0002210709150000281
Figure BDA0002210709150000291
Example 4 activating Effect of base-modified cpPepper on III-3
To examine the activation effect of base-modified cpPepper on III-3, base-modified cpPepper-3 (SEQ ID NO: 4, SEQ ID NO: GGGCCCACUGGCGCC, SEQ ID NO: 3) was synthesizedGUAGCUUCGGCUACCAAUCGUGGCGUGUCGGGGCCC are deoxyribonucleotide bases) and cppeper-4 (SEQ ID NO: 5, the underlined bases in the sequence GGGCCCACUGGCGCCGUAGCUUCGGCUACCAAUCGUGGCGUGUCGGGGCCC are bases modified with 2 '-F (synthesized by Shanghai Jima pharmaceutical technology, Ltd.), which contain a stem-loop structure base substituted with deoxyribonucleotide and a part of bases modified with 2' -F, respectively. The fluorescence activation effect of these base-modified cppepers on the III-3 fluorophore molecules was examined according to the general experimental procedure (five). The detection results show that the cpPepper-3 and cpPepper-4 modified by the base can still significantly activate the fluorescence of the III-3 fluorophore molecules (FIG. 5).
Example 5 cpPepper concatemer
To examine the activation effect of cppeper concatemers on III-3 fluorescence, cppepers were concatenated in different formats, including three of:
(1) the "head" and "tail" of the cppeper structure are connected in a "head-to-tail" connection manner (fig. 6A), thereby obtaining the ncpper (where n is cppeper which can be any copy). In this example, cDNA encoding F30-2cpPepper-5, F30-4cpPepper-5, F30-8cpPepper-5 and F30-16cpPepper-5 (the sequences encoding RNA aptamers are SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, respectively) were synthesized in their entirety, after PCR amplification, aptamer RNA was prepared according to the general experimental method (one), 0.1. mu.M RNA aptamer was incubated with 10. mu.M MIII-3, and the fluorescence intensity was measured according to the general experimental method (five). The detection result shows that the fluorescence of the ncpPepper-III-3 is increased along with the increase of n (FIG. 6B), which indicates that the fluorescence intensity of the cpPepper-III-3 complex can be improved through the mode of 'tandem 1'.
(2) In the "tandem 2" mode (FIG. 6C), the cpPepper is connected in tandem as a structural unit, thereby obtaining nxcpPepper (where n is cpPepper which can be any copy). In this example, cDNA encoding 2 XcpPepper-6, 4 XcpPepper-6 and 8XcpPepper-6 were synthesized in their respective whole genes (the sequences encoding RNA aptamers were SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, respectively), aptamer RNA was prepared according to the general experimental method (one), and fluorescence intensity was measured according to the general experimental method (five) after 0.1. mu.M RNA aptamer was incubated with 10. mu.MIII-3. the results of the measurement showed that as n increased, the fluorescence of n XcpPepper-III-3 also increased (FIG. 6D), indicating that the fluorescence intensity of the cpPepper-III-3 complex could be increased by "tandem 2".
(3) The "tandem 3" method (FIG. 6E) is a method in which the "tandem 1" and the "tandem 2" are combined, and ncpPepper obtained from the "tandem 1" is connected as a structural unit in the "tandem 2" method, thereby obtaining n1 Xn 2cpPepper (where n1 and n2 are cpPepper which can be any copy). In this example, cDNA encoding 2X 2cpPepper-5, 4X 2cpPepper-5, 8X 2cpPepper-5 (the sequences encoding RNA aptamers are SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, respectively) were synthesized in their entirety, respectively, and nucleic acid aptamer RNA was prepared according to the general experimental method (one), and after incubating 0.1. mu.M RNA aptamer with 20. mu.M MIII-3, the fluorescence intensity was measured according to the general experimental method (five). The detection result shows that the fluorescence intensity of the cpPepper concatemer-III-3 obtained by the 'tandem 3' mode is obviously higher than that of the cpPepper-III-3 (figure 6F), which indicates that the fluorescence intensity of the cpPepper-III-3 complex can be improved by the 'tandem 3' mode.
Example 6 characterization of III-3 analogs
F30-cpPepper-1RNA aptamer molecules are prepared according to the common experimental method I, and basic properties including fluorescence spectrum, molar extinction coefficient, quantum yield, fluorescence activation multiple and binding constant (Kd) of the III-3 analogue and the cpPepper are detected by using the RNA aptamer molecules, and the detection results are shown in Table 4, and the F30-cpPepper-1 can activate the fluorescence intensity of the III-3 analogue to different degrees as can be seen from the data in the table.
Table 4: determination of physicochemical Properties of F30-cpPepper-1RNA aptamer molecule binding to different fluorescent molecules
Figure BDA0002210709150000311
Figure BDA0002210709150000321
Example 7 use of cpPepper-III-3 Complex for labeling RNA in bacteria
To examine the effect of cpPepper-III-3 in bacteria, a bacterial expression plasmid expressing F30-cpPepper-1 was first constructed. F30-cpPepper-1 in example 2 is amplified by using a primer, a promoter and a multiple cloning site region are removed by amplifying pET28a by using the primer, an F30-cpPepper-1DNA fragment obtained by amplification is connected with a pET28a linearized vector according to an experimental method (IV), and an obtained recombinant plasmid is named as pET28 a-T7-F30-cpPepper-1.
The primers used for amplifying the F30-cpPepper-1 fragment are as follows:
upstream primer (P1): 5'-TCGATCCCGCGAAATTAATACGACTCACTATAGGGTTGCCA TGTGTATGTGGG-3'
Downstream primer (P2): 5'-CAAGGGGTTATGCTATTGCCATGAATGATCCCCGACAC-3'
The primers used to amplify the pET28a vector for linearization were:
upstream primer (P3): 5'-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG-3'
Downstream primer (P4): 5'-ATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACG-3'
The recombinant plasmid pET28a-T7-F30-cpPepper-1 was transformed into BL21(DE3, Star) E.coli strain, monoclonal was selected and cultured at 37 ℃ and OD600When the concentration was about 0.2, 1mM IPTG was added to induce the expression of F30-cpPepper-1, and after 4 hours, the cells were harvested and resuspended in a 2. mu.M III-3-containing PBS solution. Coli BL21(DE3, Star) transformed with empty pET28a vector was used as a control. The results show that only if F30-cpPepper-1 is expressed and in the presence of III-3, the bacteria show bright yellow-green fluorescence (FIG. 7), indicating that the cpPepper-III-3 complex can be used for fluorescence labeling of RNA in bacteria.
Example 8 cpPepper-III-3 Complex for labeling RNA in Yeast cells
To examine the effect of cpPepper-III-3 in yeast, a yeast expression plasmid expressing F30-cpPepper-1 was first constructed. The F30-cpPepper-1DNA fragment in example 2 was amplified using primers, and the amplified F30-cpPepper-1 fragment was inserted into pYES2.1TOPO TA vector according to the experimental method (IV), and the resulting recombinant plasmid was named pYES2.1-F30-cpPepper-1.
The primers used for amplifying the F30-cpPepper-1 fragment are as follows:
upstream primer (P5): 5'-GGAATATTAAGCTCGCCCTTTTGCCATGTGTATGTGGG-3'
Downstream primer (P6): 5'-TGACCTCGAAGCTCGCCCTTGTTGCCATGAATGATCCCCGACAC-3' transformation of BY4741 strain with pYES2.1-F30-cpPepper-1 recombinant plasmid, selection of single clone, culture at 30 deg.C, and OD600When the concentration was about 0.1, 1mM galactose was added to induce the expression of F30-cpPepper-1, and the cells were harvested after 10 hours and resuspended in a PBS solution containing 2. mu. MIII-3. Untreated BY4741 strain was used as a control. The results show that only when F30-cpPepper-1 is expressed and in the presence of III-3, the yeast cells show bright yellow-green fluorescence (FIG. 8), indicating that the cpPepper-III-3 complex can be used for fluorescent labeling of RNA in yeast cells.
Example 9 cpPepper and III-3 and analogs thereof for labeling RNA in mammalian cells
In order to detect the marker of cpPepper and III-3 for RNA in mammalian cells, a mammalian cell expression plasmid thereof was constructed. F30-cpPepper-1 from example 2 was amplified with primers P7 and P8, respectively, and the fragment was inserted into pLKO.1puro vector using the experimental method (IV). The resulting expression vector was named pLKO.1-F30-cpPepper-1.
The pLKO.1-F30-cpPepper-1 plasmid is used for transfecting 293T/17 cells, 1 mu M III-3 is added after 24h to mark F30-cpPepper-1, cells which do not express aptamers are used as a control, and the marking effect is detected by an experimental method (III). The results show that the F30-cpPepper-1-III-3 complex exhibited very bright yellow-green fluorescence, and the control exhibited no significant fluorescence (FIG. 9A), indicating that cpPepper-III-3 works well in mammalian cells.
The primers used for amplifying F30-cpPepper-1 were:
upstream primer (P7): 5'-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGG-3'
Downstream primer (P8): 5'-TGTCTCGAGGTCGAGAATTCAAAAAAAGTTGCCATGAATGATCCCCGACAC-3' in order to detect the use of cpPepper and III-3 analogs for labeling RNA in mammalian cells, a mammalian expression plasmid was constructed that expresses F30-8 cpPepper-5. The F30-8cpPepper-5 fragment of example 5 was amplified using primers P7 and P8 of this example, and these fragments were inserted into pLKO.1puro vector using the experimental method (IV). The obtained expression vector was named pLKO.1-F30-8 cpPepper-5.
The pLKO.1-F30-8cpPepper-5 plasmid is transfected into 293T/17 cells, different III-3 analogues are added for marking after 24 hours, and the marking effect is detected by an experimental method (III). The results show that different III-3 analogs can specifically label cells expressing F30-8cpPepper-5, but not control cells not expressing F30-8cpPepper-5 (FIG. 9B), indicating that cpPepper and III-3 and their analogs can be used for labeling RNA in mammalian cells.
Example 10 cpPepper-based Probe construction
In order to construct a cpPepper-based probe for an analyte, nucleotides at a stem-loop structure in a structure of cpPepper-1(SEQ ID No: 2) are replaced by RNA aptamers capable of specifically recognizing and binding adenosine (adenosine) and Guanosine (GTP), the aptamers and the cpPepper-1 are connected by using bases with proper length and composition, probe RNA is prepared according to a common experimental method I, the probe RNA is incubated with III-3, and the fluorescence intensity of the probe RNA in the presence or absence of adenosine or GTP is respectively detected by using a multifunctional microplate reader. The results of the assay showed that for the adenosine probe, the fluorescence of the probe was significantly higher in the presence of adenosine than in the absence of adenosine (fig. 10A), and the corresponding probe RNA sequence was SEQ ID No: 16. likewise, for GTP probes, the fluorescence of the probe in the presence of GTP is significantly higher than in the absence of GTP (fig. 10B), and the corresponding probe RNA sequence is SEQ ID No: 17.
example 11 use of cpPepper for tracing RNA localization in cells
To detect cppeper for RNA localization in tracer cells, expression plasmids of chimeric RNAs fused to different RNAs were first constructed for cppeper. The cDNA of 4cpPepper-8 is synthesized by the whole gene (the sequence of the coding RNA aptamer is SEQ ID No: 18), the 4cpPepper-8 gene segment is amplified by a primer, and the cDNA is inserted into a pCDNA3.1hygro (+) vector which is subjected to double enzyme digestion by HindIII and XhoI by a homologous recombination method to obtain the pCDNA3.1hygro (+) -4cpPepper-8 recombinant plasmid. GAPDH and TMED2 gene fragments (GAPDH and TMED2 coding gene sequences are Genebank: BC009081 and BC025957 respectively) are synthesized by whole genes, the GAPDH and TMED2 gene fragments are amplified by using primers respectively and inserted into a pCDNA3.1hygro (+) -4cpPepper-8 vector which is subjected to double digestion by NheI and HindIII to obtain pCDNA3.1hygro (+) -GAPDH-4cpPepper-8 and pCDNA3.1hygro (+) -TMED2-4cpPepper-8 recombinant plasmids which respectively code GAPDH-4cpPepper-8 and TMED2-4cpPepper-8 chimeric RNAs, and the sequences of the recombinant RNAs are SEQ ID No: 19 and 20
The primers used for amplifying 4cpPepper-8 were:
upstream primer (P9): 5'-TAGCGTTTAAACTTAAGCTTCCATCGGGCCCACTGGCGC-3'
Downstream primer (P10): 5'-ACGGGCCCTCTAGACTCGAGCCATCGGGCCCCGACACGCC-3'
Primers used for amplification of GAPDH were:
upstream primer (P11): 5'-GGAGACCCAAGCTGGCTAGCATGGGGAAGGTGAAGGTCGG-3'
Downstream primer (P12): 5'-CAGTGGGCCCGATGGAAGCTTAACCATGCTCTAGCGAGTGTTACTCCTTGGAGG CCATGT-3'
Primers used for amplification of TMED2 were:
upstream primer (P13): 5'-GGAGACCCAAGCTGGCTAGCATGGTGACGCTTGCTGAACT-3'
Downstream primer (P14): 5'-CAGTGGGCCCGATGGAAGCTTAACCATGCTCTAGCGAGTTAAACAACTCTCCGG ACTTC-3'
After the construction of the plasmids is completed, the inserted sequences are completely correct through sequencing identification, and the plasmids are extracted by using a transfection-grade plasmid extraction reagent box and used for subsequent transfection experiments.
The recombinant plasmids pCDNA3.1hygro (+) -GAPDH-4cpPepper-8 and pCDNA3.1hygro (+) -TMED2-4cpPepper-8 constructed in the example were co-transfected with pCDNA3.1hygro (+) -BFP to obtain COS-7 cells, and the cells were imaged by the fluorescence imaging method described in the specific experimental method (III) after 24 hours of transfection. Imaging results showed that the fluorescence of GAPDH-4cpPepper-8-III-3 was mainly concentrated in the cytoplasm, while the fluorescence of TMED2-4cpPepper-8-III-3 observed endoplasmic reticulum enrichment, which was consistent with previous reports, and also with the results of fluorescence-labeled in situ hybridization (FISH) (FIG. 11), which showed that cpPepper could be used for the localization of trace RNA.
Example 12 use of Pepper for detection of genomic DNA
In order to detect genomic DNA using cpPepper, a recombinant plasmid expressing a chimeric RNA of cpPepper-9 and sgRNA was first constructed. The cDNA of sgRNA-cpPepper-9(loop1), sgRNA-cpPepper-9(tetra loop) and sgRNA-cpPepper-9(loop1 and tetra loop) containing centromere targeting sequences is synthesized by whole genes, and the coded RNA sequences are respectively SEQ ID No: 21. 22 and 23. The cDNA of the chimeric RNA was amplified using primers P15 and P16, the psgRNA plasmid was amplified using primers P17 and P18 (Shao et al. nucleic acids research 2016.44: e86), the amplified cDNA was ligated to the linearized psgRNA vector using the experimental method (IV), and the resulting plasmids were named psgRNA-cpPepper-9(loop1), psgRNA-cpPepper-9(loop2), and psgRNA-cpPepper-9(loop1 and tetraop loop), respectively. The dCas9-GFP gene fragment was amplified using pSLQ1645(dCas9-GFP) (Shao et al nucleic acids research 2016.44: e86) as a template using primers P19 and P20, and was inserted into a HindIII and XhoI double digested pCDNA3.1hygro (+) vector using the experimental method (IV), and the resulting plasmid was named pCDNA3.1hygro (+) -dCas 9-GFP.
Primers used for amplifying cDNA corresponding to the cpPepper and sgRNA chimeric RNA are as follows:
upstream primer (P15): 5'-AAAGGACGAAACACCGAATCTGCAAGTGGATATTGTTTGAG-3'
Downstream primer (P16): 5'-TGATCTAGAAAAAAAGCACCGACTCGGTGCCAC-3'
Primers for amplifying the psgRNA plasmid to linearize it were:
upstream primer (P17): 5'-TTTTTTTCTAGATCATAATCAGCCATACC-3'
Downstream primer (P18): 5'-GGTGTTTCGTCCTTTCCACAAG-3'
Primers used for amplifying SpdCas9-GFP were:
upstream primer (P19): 5'-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCACCATGGCCCC-3'
Downstream primer (P20): 5'-ACGGGCCCTCTAGACTCGAGTTACTTGTACAGCTCGTCCATGC-3'
pCDNA3.1hygro (+) -dCas9-GFP was co-transfected with psgRNA-cpPepper-9(loop1), psgRNA-cpPepper-9(loop2) and psgRNA-cpPepper-9(loop1 and tetra loop) recombinant plasmids, respectively, into COS-7 cells, after 24h transfection, the cells were labeled with 1. mu. MIII-21 and Hoechst, and fluorescence of cpPepper-9-III-21, GFP and Hoechst was observed with a fluorescence microscope. The imaging results showed that the fluorescence of cpPepper-9-III-21 was concentrated mainly in the nucleus and clustered in spots (centromere), almost completely coinciding with the fluorescence of dCas9-GFP (FIG. 12), indicating that cpPepper can be used for imaging gene DNA.
Example 13 tag for extraction and purification of RNA by cpPepper
To examine the use of cpPepper for RNA extraction and purification, a TagBFP gene fragment was amplified using Easyfusion T2A-H2B-TagBFP (Addge: 113086) as a template using primers P21 and P22, respectively, and inserted into a pCDNA3.1hygro (+) -GAPDH-4cpPepper-8 vector digested with NheI and HindIII to obtain a recombinant plasmid pCDNA3.1hygro (+) -TagBFP-4cpPepper-8 encoding TagBFP-4cpPepper-8 having an RNA sequence of SEQ ID No: 24.
primers used for amplifying TagBFP were:
upstream primer (P21): 5'-GGAGACCCAAGCTGGCTAGCATGAGCGAGCTGA TTAAGGA-3'
Downstream primer (P22): 5'-CAGTGGGCCCGATGGAAGCTTCTCCCAAACCATGCTCTAGCG AGTGTTAATTGAGCTTGTGCCCCA-3'
Transfecting the pCDNA3.1hygro (+) -TagBFP-4cpPepper-8 recombinant plasmid into COS-7 cells, collecting the cells after 24h, and utilizing 40mM HEPES, pH 7.4,125mM KCl and 5mM MgCl2Is resuspended in the buffer of (1). After washing Activated thio Sepharose 4B (GE healthcare) twice with 500. mu.L PBS, 10mM TCEP (Sigma) in PBS was added and incubated for 1h at room temperature. After washing twice with 500. mu.L of PBS, a maleimide-containing III-3 fluorophore molecule (Mal-III-3) was added thereto, and the mixture was reacted at room temperature for 30min and washed three times with 500. mu.L of PBS. After disruption of the resuspended cells, incubation with the above treated beads at room temperature, 30min later, centrifugation at 4000rpm for 2min, discarding the supernatant, and application of 40mM HEPES, pH 7.4,125mM KCl,5mM MgCl2The agarose beads were washed 6 times with the buffer solution and the supernatant was centrifuged off each time. Reselecting the microbeads by DEPC water, treating for 10min at 70 ℃, centrifuging for 2min at 4000rpm, and collecting the supernatant. Adding 1/10 volume NaAc and 2.5 volume times of anhydrous ethanol into the collected supernatant, standing in a refrigerator at-80 deg.C for 20min, centrifuging at 14000rpm at 4 deg.C for 10min, leaving precipitate, discarding the supernatant, washing the precipitate with precooled 70% ethanol solution, centrifuging at 14000rpm at 4 deg.C for 10min, leaving precipitate, discarding the supernatant, and repeating the steps. And (3) placing the precipitate at room temperature for 5min, and adding a small volume of DEPC water to resuspend the precipitate after the alcohol is volatilized.
The supernatants after cell disruption and the eluates after final high temperature elution (with 40mM HEPES, pH 7.4,125mM KCl,5mM MgCl) were assayed separately2Buffer 1: 1) fluorescence after incubation with fluorophore III-3, control broken supernatants from blank cells. The detection result shows that the fluorescence of the eluate after incubation with III-3 is significantly higher than that of the supernatant after crushing before sample loading (FIG. 13), which indicates that TagBFP-4cpPepper-8RNA is well enriched, and indicates that cpPepper can be used as oneThe label is used for separating and purifying RNA.
Example 14 Synthesis of III-3 and analogs thereof
Compound III-1:
Figure BDA0002210709150000371
4-N, N-dimethyl-benzaldehyde (0.35g, 2.3mmol) and 4-cyano-benzyl cyanide (0.4g, 2.8mmol) are placed in a 100ml round bottom flask, 40ml of absolute ethyl alcohol is added for dissolution, two drops of piperidine are added, oil bath heating and refluxing are carried out under the protection of Ar for 2h, after the reaction is finished, the mixture is cooled to room temperature, a large amount of solid is separated out, filtration is carried out, the filter cake is washed three times by cold ethyl alcohol, and orange solid (0.60g, 95%) is dried in vacuum.1H NMR(400MHz,DMSO-d6):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 C18H16O3[M+H]+:274.1344.Found:274.1345.
Compound III-2:
Figure BDA0002210709150000381
reference was made to the procedure for the synthesis of compound III-1 (0.34g, 89%).1H NMR(400MHz,DMSO-d6):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),8.09 ppm(s,1H).HRMS(ESI-TOF):Calcd.For C20H20O3[M+H]+:302.1657.Found:302.1658.
Compound III-2:
Figure BDA0002210709150000382
reference was made to the procedure for the synthesis of compound III-1 (0.34g, 89%).1H NMR(400MHz,DMSO-d6):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 C20H20O3[M+H]+:302.1657.Found: 302.1658.
Compound III-3:
Figure BDA0002210709150000383
reference was made to the procedure for the synthesis of compound III-1 (0.33g, 95%).1H NMR(400MHz,DMSO-d6):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＝28.0 Hz,4H),3.04(s,1H).LR-HRMS(ESI-TOF):Calcd.For C19H18N3O[M+H]+:304.1450. Found:304.1451.
Compound III-4:
Figure BDA0002210709150000384
adding TEA (0.25g, 2.2mmol) into 40mL of dry DCM compound III-3 (0.61g, 2.0mmol), slowly adding a solution of p-toluenesulfonyl chloride (0.38g, 2.0mmol) in 10mL of DCM at 0 ℃, slowly raising the temperature to room temperature under the protection of Ar, adding 2mL of water to quench the reaction after the reaction is finished, separating an organic phase, and Na2SO4The organic solvent was removed under reduced pressure and the residue was used in the next step without further treatment.
Dissolving the residue in 20ml acetonitrile, adding 1ml methylamine in methanol, heating and refluxing the system in oil bath under Ar protection overnight, removing solvent under pressure after reaction, dissolving the system in 50ml DCM, washing with water and saturated brine respectively (2X 100ml), and washing the organic phase with Na2SO4Drying, removing the solvent under pressure, and separating the residue by column chromatography to obtain orange-red solid (0.54g, 82%).1H NMR(400MHz,CDCl3):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 C21H23N4[M+H]+:331.1923. Found:331.1925.
Compound iii-5:
Figure BDA0002210709150000391
4-hydroxy-3, 5-difluoro-benzaldehyde (0.32g,2.0mmol) and 4-cyano-benzyl cyanide (0.35g, 2.4mmol) are placed in a 100ml round bottom flask, 40ml of absolute ethyl alcohol is added for dissolving, two drops of piperidine are added, oil bath heating and refluxing are carried out for 2h under the protection of Ar, after the reaction is finished, the mixture is cooled to room temperature, a large amount of solid is separated out, filtration is carried out, cold ethyl alcohol is used for washing a filter cake for three times, and orange solid is dried in vacuum.1H NMR(400MHz,CDCl3):δ＝7.80(d,J＝9.0Hz,2H),7.74–7.66 (m,4H),7.48(s,1H).LR-MS(ESI-TOF):Calcd.For C16H9F2N2O[M+H]+:283.0683.Found: 283.0684.
Compound 5- (N-methyl-N-hydroxyethyl) amino-pyrazine-2-carbaldehyde:
Figure BDA0002210709150000392
4-N-methyl-N-hydroxyethylamine (2.6g, 35mmol), 5-chloro-pyrazine-2-carbaldehyde (0.50g, 3.5mmol) was dissolved in a 100ml round-bottomed flask by adding 20ml of anhydrous acetonitrile, and K was added2CO3(0.71g, 5.3mmol), oil bath heating under Ar protection and refluxing for 24h, after the reaction is finished, cooling to room temperature, filtering, removing the solvent in vacuum, dissolving the residue in 100ml DCM, washing with water and saturated brine respectively (2X 100ml), drying the organic phase with Na2SO4, removing the organic solvent, and separating the residue by column chromatography to obtain 5- (N-methyl-N-hydroxyethyl) -pyrazine-2-aldehyde (0.48g, 76%).1H NMR(400MHz,CDCl3):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 C8H12N3O2 [M+H]+:182.1.Found:182.1.
Compound III-6:
Figure BDA0002210709150000401
reference was made to the procedure for the synthesis of compound III-1 (0.36g, 96%).1H NMR(400MHz,CDCl3):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 C17H16N5O [M+H]+:306.1355.Found:306.1357.
Compound III-7:
Figure BDA0002210709150000402
reference was made to the procedure for the synthesis of compound III-4 (0.21g, 67%).1H NMR(400MHz,DMSO-d6):δ 8.37(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 C19H21N6[M+H]+: 333.1828.Found:333.1829.
Compound 6- (N-methyl-N-hydroxyethyl) amino-pyrazine-3-aldehyde:
Figure BDA0002210709150000403
according to the synthesis of compound 5- (N-methyl-N-hydroxyethyl) amino-pyrazine-2-aldehyde, (0.45g, 68%).1H NMR(400MHz,CDCl3):δ＝9.69(s,1H),8.43(d,J＝2.1Hz,1H),7.86(dd,J＝9.0,2.3 Hz,1H),6.56(d,J＝9.1Hz,1H),3.86–3.79(m,4H),3.15(s,3H).LR-MS(ESI-TOF): Calcd.For C9H13O2N2[M+H]+:181.1.Found:181.1.
Compound III-8:
Figure BDA0002210709150000404
referencingSynthesis of Compound III-1, (0.39g, 89%).1H NMR(400MHz,DMSO-d6):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 C18H27N4O[M+H]+: 305.1402.Found:305.1401.
Compound III-9:
Figure BDA0002210709150000411
reference was made to the procedure for the synthesis of compound III-4 (0.31g, 92%).1H NMR(400MHz,DMSO-d6):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 C22H26N5[M+H]+:360.2188.Found:360.2187.
4-N, N-dimethyl-6 aldehyde-pyridine:
Figure BDA0002210709150000412
reference was made to the procedure for the synthesis of the compound 4-N-methyl-N- (2-N ', N' -dimethyl-ethyl) -benzaldehyde, (0.31g, 49%).1H NMR(400MHz,DMSO-d6):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 C8H11N2O[M+H]+:151.1.Found:151.1.
Compound iii-10:
Figure BDA0002210709150000413
reference was made to the procedure for the synthesis of compound III-1 (0.36g, 96%).1H NMR(400MHz,DMSO-d6):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 C17H15N4[M+H]+:275.1297.Found:275.1298.
2- (N-methyl-N-hydroxyethyl) amino-5-formyl-pyrimidine:
Figure BDA0002210709150000414
reference was made to the procedure for the synthesis of the compound 4-N-methyl-N- (2-N ', N' -dimethyl-ethyl) -benzaldehyde, (0.42g, 72%).1H NMR(400MHz,DMSO-d6):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 C8H12N3O[M+H]+:182.1. Found:182.1.
Compound III-11:
Figure BDA0002210709150000421
reference was made to the procedure for the synthesis of compound III-1 (0.36g, 96%).1H NMR(400MHz,DMSO-d6):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 C17H16N5O[M+H]+:306.1355.Found:306.1356.
5- (N-methyl-N-hydroxyethyl) amino-2-formyl-pyrimidine:
Figure BDA0002210709150000422
reference was made to the procedure for the synthesis of the compound 4-N-methyl-N- (2-N ', N' -dimethyl-ethyl) -benzaldehyde, (0.42g, 72%).1H NMR(400MHz,DMSO-d6):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 C8H12N3O2[M+H]+:182.1. Found:182.1.
1-cyano-1- (4-phenylacetonitrile) -2-2- (5- (N-methyl-N-hydroxyethyl) amino-) pyrimidine-ethylene:
Figure BDA0002210709150000423
reference was made to the procedure for the synthesis of compound III-1 (0.56g, 89%).1H NMR(400MHz,DMSO-d6):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 C17H16N5O[M+H]+:306.1.Found:306.1.
Compound III-12:
Figure BDA0002210709150000424
reference was made to the procedure for the synthesis of compound III-4 (0.36g, 96%).1H NMR(400MHz,DMSO-d6):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 C19H21N6[M+H]+:333.1828.Found: 333.1829.
2-acetonitrile-5-cyano-pyridine:
Figure BDA0002210709150000431
dissolving 2-bromomethyl-5-cyanopyridine (0.50g, 2.5mmol) in 50ml THF in 100ml round bottom flask, adding 10ml NaCN 2M aqueous solution under Ar protection, heating in oil bath under reflux for 12h, cooling the system to room temperature after reaction, DCM extracting (3X 100ml), combining organic phases, washing with water and saturated brine respectively (2X 100ml), drying the organic phase with NaSO4, removing under reduced pressureThe solvent and residue were purified by column chromatography to give 2-acetonitrile-5-cyanopyridine (0.19g, 56%).1H NMR(400MHz,DMSO-d6):δ＝8.78(s,1H),7.95(m,1H),7.56(m, 1H),4.01(s,2H).LR-MS(ESI-TOF):Calcd.For C8H6N3[M+H]+:144.1.Found:144.1.
Compound III-13:
Figure BDA0002210709150000432
reference was made to the procedure for the synthesis of compound III-1 (0.45g, 95%).1H NMR(400MHz,DMSO-d6):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 C18H17N4O[M+H]+:305.1402.Found:305.1403.
2-cyano-5-acetonitrile-pyrazine:
Figure BDA0002210709150000433
2-chloro-pyrazine-5-acetonitrile (0.32g,2.0mmol) and CuCN (0.93g, 10.0mmol) are put into a 100ml round bottom flask, 30ml of dry DMSO is added for dissolution, the mixture is heated in an oil bath at 80 ℃ under the protection of Ar for 12h, after the reaction is finished, the system is poured into 100ml of water, DCM is used for extraction (4X 50ml), organic phases are combined, the mixture is washed with water and saturated saline water (2X 100ml), the organic phase is dried by Na2SO4, the organic solvent is removed under reduced pressure, and the residue is separated by column chromatography to obtain 2-cyano-pyrazine-5-acetonitrile (0.20g, 69%).1H NMR(400MHz,DMSO-d6):δ＝8.60(s,1H),8.48(s, 1H),3.92(s,2H).LR-MS(ESI-TOF):Calcd.For C7H5N4[M+H]+:145.1.Found:145.1.
Compound III-14:
Figure BDA0002210709150000441
reference was made to the procedure for the synthesis of compound III-1 (0.25g, 91%).1H NMR(400MHz,DMSO-d6):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 C17H16 N5O[M+H]+:306.1355.Found:306.1354.
Compound III-15:
Figure BDA0002210709150000442
reference was made to the procedure for the synthesis of compound III-1 (0.25g, 91%).1H NMR(400MHz,DMSO-d6):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 C19H18N3O2[M+H]+:320.1399.Found:320.1397.
Compound III-16:
Figure BDA0002210709150000443
reference was made to the procedure for the synthesis of compound III-1 (0.29g, 94%).1H NMR(400MHz,DMSO-d6):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 C19H18N3OS[M+H]+: 336.1171.Found:336.1170.
6-methylamine-benzo [ b ] thiophene-2-carbaldehyde:
Figure BDA0002210709150000451
6-bromobenzo [ b ]]Thiophene-2-Formaldehyde (0.42g, 1.7mmol), dimethylethylamine (40% in water, 1g, 8.9mmol), CuI (13.9mg, 0.073mmol), K3PO4·H2O (155.4mg, 0.73mmol), methylamine (33% aqueous solution, 1g) in 100ml pressure bottle, heating in oil bath at 60 deg.C under sealed condition for 12h, cooling the system to room temperature, adding 50ml water, extracting with DCM (3X 100ml), combining organic phases, Na2SO4The organic solvent was removed under reduced pressure, and the residue was purified by column chromatography (0.23g, 68%).1H NMR(400MHz,DMSO-d6):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 C10H10NOS[M+H]+:192.0.Found:192.0.
Compound III-17:
Figure BDA0002210709150000452
reference was made to the procedure for the synthesis of compound III-1 (0.29g, 94%).1H NMR(400MHz,DMSO-d6):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 C19H14N3S[M+H]+:360.1171.Found:360.1173.
6-N-methyl-N-hydroxyethyl-benzo [ b ] thiophene-2-carbaldehyde:
Figure BDA0002210709150000453
reference compound 6-methylamine-benzo [ b]Method for the synthesis of thiophene-2-carbaldehyde, (0.54g, 79%).1H NMR(400 MHz,DMSO-d6):δ＝9.91(s,1H),8.14(s,1H),7.81(d,J＝5.2Hz,1H),7.17(d,J＝2.0Hz,1 H),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 C12H14NO2S,[M+H]+:235.1.Found 236.1.
Compound III-18:
Figure BDA0002210709150000461
reference was made to the procedure for the synthesis of compound III-1 (0.21g, 95%).1H NMR(400MHz,DMSO-d6):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 C21H19N3OS [M+H]+:360.1171.Found:360.1173.
5-N, N-dimethylamine-2-carbondithiophene:
Figure BDA0002210709150000462
reference compound 6-N-methyl-N-hydroxyethyl-benzo [ b]Method for the synthesis of thiophene-2-carbaldehyde, (0.54g, 79%).1H NMR(400MHz,DMSO-d6):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 C9H12NOS2[M+H]+:214.0；found 214.0.
Compound III-19:
Figure BDA0002210709150000463
reference was made to the procedure for the synthesis of compound III-1 (0.31g, 90%).1H NMR(400MHz,DMSO-d6):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 C18H14N3S2[M+H]+: 336.0629.Found:336.0630.
5-N, N-diethylamine-2-carbondithiophene:
Figure BDA0002210709150000464
reference compound 6-N-methyl-N-hydroxyethyl-benzo [ b]Method for the synthesis of thiophene-2-carbaldehyde, (0.44g, 75%).1H NMR(400MHz,DMSO-d6):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 C9H12NOS2[M+H]+: 214.0；found 214.0.
Compound iii-20:
Figure BDA0002210709150000471
reference was made to the procedure for the synthesis of compound III-1 (0.31g, 90%).1H NMR(400MHz,DMSO-d6):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 C20H18N3S2[M+H]+:364.0942.Found:364.0943.
Example 21:
5- (N-methyl-N-hydroxyethyl) amino-2-formylbithiophene:
Figure BDA0002210709150000472
reference compound 6-N-methyl-N-hydroxyethyl-benzo [ b]Method for the synthesis of thiophene-2-carbaldehyde, (0.44g, 75%).1H NMR(400MHz,DMSO-d6):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 C10H12NO2S2[M+H]+:241.0；found 242.0.
Compound III-21:
Figure BDA0002210709150000473
reference was made to the procedure for the synthesis of compound III-1 (0.31g, 90%).1H NMR(400MHz,DMSO-d6):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 C19H16N3OS2[M+H]+:366.0735.Found:366.0736.
It should be understood that the amounts used, reaction conditions, etc. in the various examples of the present specification are approximate unless otherwise indicated, and similar results can be obtained with slight variations in practice. Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All documents mentioned herein are incorporated by reference into this application. While the present invention has been described with reference to preferred embodiments for illustrative purposes, those skilled in the art will appreciate that the present invention may be practiced with methods and materials similar to those described herein, with various modifications and changes within the scope of the present invention as defined by the appended claims.
SEQUENCE LISTING
<110> university of east China's college of science
<120> RNA detection and quantification method
<130> 2019-09-23
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 47
<212> RNA
<213> Synthetic Sequence
<400> 1
ucccacuggc gccguagcuu cggcuaccaa ucguggcgug ucgggga 47
<210> 2
<211> 99
<212> RNA
<213> Synthetic Sequence
<400> 2
uugccaugug uauguggguu cgcccacaua cucugaugau cccacuggcg ccguagcuuc 60
ggcuaccaau cguggcgugu cggggaucau ucauggcaa 99
<210> 3
<211> 112
<212> RNA
<213> Synthetic Sequence
<400> 3
gcccggauag cucagucggu agagcagcgc ugcacuggcg ccguagcuuc ggcuaccaau 60
cguggcgugu cggcagcgcg gguccagggu ucaagucccu guucgggcgc ca 112
<210> 4
<211> 51
<212> RNA
<213> Synthetic Sequence
<400> 4
gggcccacug gcgccguagc uucggcuacc aaucguggcg ugucggggcc c 51
<210> 5
<211> 51
<212> RNA
<213> Synthetic Sequence
<400> 5
gggcccacug gcgccguagc uucggcuacc aaucguggcg ugucggggcc c 51
<210> 6
<211> 136
<212> RNA
<213> Synthetic Sequence
<400> 6
uugccaugug uauguggguu cgcccacaua cucugaugau cccacuggcg ccguagccac 60
uggcgccgua gcuucggcua ccaaucgugg cgugucgggc uaccaaucgu ggcgugucgg 120
ggaucauuca uggcaa 136
<210> 7
<211> 210
<212> RNA
<213> Synthetic Sequence
<400> 7
uugccaugug uauguggguu cgcccacaua cucugaugau cccacuggcg ccguagccac 60
uggcgccgua gccacuggcg ccguagccac uggcgccgua gcuucggcua ccaaucgugg 120
cgugucgggc uaccaaucgu ggcgugucgg gcuaccaauc guggcguguc gggcuaccaa 180
ucguggcgug ucggggauca uucauggcaa 210
<210> 8
<211> 358
<212> RNA
<213> Synthetic Sequence
<400> 8
uugccaugug uauguggguu cgcccacaua cucugaugau cccacuggcg ccguagccac 60
uggcgccgua gccacuggcg ccguagccac uggcgccgua gccacuggcg ccguagccac 120
uggcgccgua gccacuggcg ccguagccac uggcgccgua gcuucggcua ccaaucgugg 180
cgugucgggc uaccaaucgu ggcgugucgg gcuaccaauc guggcguguc gggcuaccaa 240
ucguggcgug ucgggcuacc aaucguggcg ugucgggcua ccaaucgugg cgugucgggc 300
uaccaaucgu ggcgugucgg gcuaccaauc guggcguguc ggggaucauu cauggcaa 358
<210> 9
<211> 654
<212> RNA
<213> Synthetic Sequence
<400> 9
uugccaugug uauguggguu cgcccacaua cucugaugau cccacuggcg ccguagccac 60
uggcgccgua gccacuggcg ccguagccac uggcgccgua gccacuggcg ccguagccac 120
uggcgccgua gccacuggcg ccguagccac uggcgccgua gccacuggcg ccguagccac 180
uggcgccgua gccacuggcg ccguagccac uggcgccgua gccacuggcg ccguagccac 240
uggcgccgua gccacuggcg ccguagccac uggcgccgua gcuucggcua ccaaucgugg 300
cgugucgggc uaccaaucgu ggcgugucgg gcuaccaauc guggcguguc gggcuaccaa 360
ucguggcgug ucgggcuacc aaucguggcg ugucgggcua ccaaucgugg cgugucgggc 420
uaccaaucgu ggcgugucgg gcuaccaauc guggcguguc gggcuaccaa ucguggcgug 480
ucgggcuacc aaucguggcg ugucgggcua ccaaucgugg cgugucgggc uaccaaucgu 540
ggcgugucgg gcuaccaauc guggcguguc gggcuaccaa ucguggcgug ucgggcuacc 600
aaucguggcg ugucgggcua ccaaucgugg cgugucgggg aucauucaug gcaa 654
<210> 10
<211> 130
<212> RNA
<213> Synthetic Sequence
<400> 10
ggggccccac uggcgccgua gcuucggcua ccaaucgugg cgugucgggg gcccccuagc 60
uacuagcuag caucgggggc cccacuggcg ccguagcuuc ggcuaccaau cguggcgugu 120
cgggggcccc 130
<210> 11
<211> 280
<212> RNA
<213> Synthetic Sequence
<400> 11
ggggccccac uggcgccgua gcuucggcua ccaaucgugg cgugucgggg gcccccuagc 60
uacuagcuag caucgggggc cccacuggcg ccguagcuuc ggcuaccaau cguggcgugu 120
cgggggcccc cuagcuacua gcuagcaucg ggggccccac uggcgccgua gcuucggcua 180
ccaaucgugg cgugucgggg gcccccuagc uacuagcuag caucgggggc cccacuggcg 240
ccguagcuuc ggcuaccaau cguggcgugu cgggggcccc 280
<210> 12
<211> 580
<212> RNA
<213> Synthetic Sequence
<400> 12
ggggccccac uggcgccgua gcuucggcua ccaaucgugg cgugucgggg gcccccuagc 60
uacuagcuag caucgggggc cccacuggcg ccguagcuuc ggcuaccaau cguggcgugu 120
cgggggcccc cuagcuacua gcuagcaucg ggggccccac uggcgccgua gcuucggcua 180
ccaaucgugg cgugucgggg gcccccuagc uacuagcuag caucgggggc cccacuggcg 240
ccguagcuuc ggcuaccaau cguggcgugu cgggggcccc cuagcuacua gcuagcaucg 300
ggggccccac uggcgccgua gcuucggcua ccaaucgugg cgugucgggg gcccccuagc 360
uacuagcuag caucgggggc cccacuggcg ccguagcuuc ggcuaccaau cguggcgugu 420
cgggggcccc cuagcuacua gcuagcaucg ggggccccac uggcgccgua gcuucggcua 480
ccaaucgugg cgugucgggg gcccccuagc uacuagcuag caucgggggc cccacuggcg 540
ccguagcuuc ggcuaccaau cguggcgugu cgggggcccc 580
<210> 13
<211> 200
<212> RNA
<213> Synthetic Sequence
<400> 13
ggggcccacu ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc 60
gggcuaccaa ucguggcgug ucggggcccc cuagcuacua gcuagcaucg ggggcccacu 120
ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc gggcuaccaa 180
ucguggcgug ucggggcccc 200
<210> 14
<211> 420
<212> RNA
<213> Synthetic Sequence
<400> 14
ggggcccacu ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc 60
gggcuaccaa ucguggcgug ucggggcccc cuagcuacua gcuagcaucg ggggcccacu 120
ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc gggcuaccaa 180
ucguggcgug ucggggcccc cuagcuacua gcuagcaucg ggggcccacu ggcgccguag 240
ccacuggcgc cguagcuucg gcuaccaauc guggcguguc gggcuaccaa ucguggcgug 300
ucggggcccc cuagcuacua gcuagcaucg ggggcccacu ggcgccguag ccacuggcgc 360
cguagcuucg gcuaccaauc guggcguguc gggcuaccaa ucguggcgug ucggggcccc 420
<210> 15
<211> 860
<212> RNA
<213> Synthetic Sequence
<400> 15
ggggcccacu ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc 60
gggcuaccaa ucguggcgug ucggggcccc cuagcuacua gcuagcaucg ggggcccacu 120
ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc gggcuaccaa 180
ucguggcgug ucggggcccc cuagcuacua gcuagcaucg ggggcccacu ggcgccguag 240
ccacuggcgc cguagcuucg gcuaccaauc guggcguguc gggcuaccaa ucguggcgug 300
ucggggcccc cuagcuacua gcuagcaucg ggggcccacu ggcgccguag ccacuggcgc 360
cguagcuucg gcuaccaauc guggcguguc gggcuaccaa ucguggcgug ucggggcccc 420
cuagcuacua gcuagcaucg ggggcccacu ggcgccguag ccacuggcgc cguagcuucg 480
gcuaccaauc guggcguguc gggcuaccaa ucguggcgug ucggggcccc cuagcuacua 540
gcuagcaucg ggggcccacu ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc 600
guggcguguc gggcuaccaa ucguggcgug ucggggcccc cuagcuacua gcuagcaucg 660
ggggcccacu ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc 720
gggcuaccaa ucguggcgug ucggggcccc cuagcuacua gcuagcaucg ggggcccacu 780
ggcgccguag ccacuggcgc cguagcuucg gcuaccaauc guggcguguc gggcuaccaa 840
ucguggcgug ucggggcccc 860
<210> 16
<211> 75
<212> RNA
<213> Synthetic Sequence
<400> 16
ggggcccacu ggcgccucag ggaagaaacu guggcacuuc ggugccagcu gacaaucgug 60
gcgugucggg gcccc 75
<210> 17
<211> 67
<212> RNA
<213> Synthetic Sequence
<400> 17
ggggcccacu ggcgccuaau agaagagcac guauacgcaa auuacaaucg uggcgugucg 60
gggcccc 67
<210> 18
<211> 172
<212> RNA
<213> Synthetic Sequence
<400> 18
ccaucgggcc cacuggcgcc guagccacug gcgccguagc cacuggcgcc guagccacug 60
gcgccguagc uucggcuacc aaucguggcg ugucgggcua ccaaucgugg cgugucgggc 120
uaccaaucgu ggcgugucgg gcuaccaauc guggcguguc ggggcccgau gg 172
<210> 19
<211> 1203
<212> RNA
<213> Synthetic Sequence
<400> 19
auggggaagg ugaaggucgg agucaacgga uuuggucgua uugggcgccu ggucaccagg 60
gcugcuuuua acucugguaa aguggauauu guugccauca augaccccuu cauugaccuc 120
aacuacaugg uuuacauguu ccaauaugau uccacccaug gcaaauucca uggcaccguc 180
aaggcugaga acgggaagcu ugucaucaau ggaaauccca ucaccaucuu ccaggagcga 240
gaucccucca aaaucaagug gggcgaugcu ggcgcugagu acgucgugga guccacuggc 300
gucuucacca ccauggagaa ggcuggggcu cauuugcagg ggggagccaa aagggucauc 360
aucucugccc ccucugcuga ugcccccaug uucgucaugg gugugaacca ugagaaguau 420
gacaacagcc ucaagaucau cagcaaugcc uccugcacca ccaacugcuu agcaccccug 480
gccaagguca uccaugacaa cuuugguauc guggaaggac ucaugaccac aguccaugcc 540
aucacugcca cccagaagac uguggauggc cccuccggga aacuguggcg ugauggccgc 600
ggggcucucc agaacaucau cccugccucu acuggcgcug ccaaggcugu gggcaagguc 660
aucccugagc ugaacgggaa gcucacuggc auggccuucc guguccccac ugccaacgug 720
ucaguggugg accugaccug ccgucuagaa aaaccugcca aauaugauga caucaagaag 780
guggugaagc aggcgucgga gggcccccuc aagggcaucc ugggcuacac ugagcaccag 840
guggucuccu cugacuucaa cagcgacacc cacuccucca ccuuugacgc uggggcuggc 900
auugcccuca acgaccacuu ugucaagcuc auuuccuggu augacaacga auuuggcuac 960
agcaacaggg ugguggaccu cauggcccac auggccucca aggaguaacu cgcuagagca 1020
ugguuaagcu uccaucgggc ccacuggcgc cguagccacu ggcgccguag ccacuggcgc 1080
cguagccacu ggcgccguag cuucggcuac caaucguggc gugucgggcu accaaucgug 1140
gcgugucggg cuaccaaucg uggcgugucg ggcuaccaau cguggcgugu cggggcccga 1200
ugg 1203
<210> 20
<211> 571
<212> RNA
<213> Synthetic Sequence
<400> 20
ugggaaauac acauuugcug cucacaugga uggaacauac aaauuuuguu uuaguaaccg 60
gauguccacc augacuccaa aaauagugau guucaccauu gauauugggg aggcuccaaa 120
aggacaagau auggaaacag aagcucacca gaacaagcua gaagaaauga ucaaugagcu 180
agcaguggcg augacagcug uaaagcacga acaggaauac auggaagucc gggagagaau 240
acacagagcc aucaacgaca acacaaacag cagagugguc cuuugguccu ucuuugaagc 300
ucuuguucua guugccauga cauugggaca gaucuacuac cugaagagau uuuuugaagu 360
ccggagaguu guuuaacucg cuagagcaug guuaagcuuc caucgggccc acuggcgccg 420
uagccacugg cgccguagcc acuggcgccg uagccacugg cgccguagcu ucggcuacca 480
aucguggcgu gucgggcuac caaucguggc gugucgggcu accaaucgug gcgugucggg 540
cuaccaaucg uggcgugucg gggcccgaug g 571
<210> 21
<211> 142
<212> RNA
<213> Synthetic Sequence
<400> 21
gaaucugcaa guggauauug uuugagagcu aggccccacu ggcgccguag cuucggcuac 60
caaucguggc gugucggggg ccuagcaagu ucaaauaagg cuaguccguu aucaacuuga 120
aaaaguggca ccgagucggu gc 142
<210> 22
<211> 142
<212> RNA
<213> Synthetic Sequence
<400> 22
gaaucugcaa guggauauug uuugagagcu agaaauagca aguucaaaua aggcuagucc 60
guucucaacu uggccccacu ggcgccguag cuucggcuac caaucguggc gugucggggg 120
ccaaguggca ccgagucggu gc 142
<210> 23
<211> 189
<212> RNA
<213> Synthetic Sequence
<400> 23
gaaucugcaa guggauauug uuugagagcu aggccccacu ggcgccguag cuucggcuac 60
caaucguggc gugucggggg ccuagcaagu ucaaauaagg cuaguccguu aucaacuugg 120
ccccacuggc gccguagcuu cggcuaccaa ucguggcgug ucgggggcca aguggcaccg 180
agucggugc 189
<210> 24
<211> 911
<212> RNA
<213> Synthetic Sequence
<400> 24
augagcgagc ugauuaagga gaacaugcac augaagcugu acauggaggg caccguggac 60
aaccaucacu ucaagugcac auccgagggc gaaggcaagc ccuacgaggg cacccagacc 120
augagaauca agguggucga gggcggcccu cuccccuucg ccuucgacau ccuggcuacu 180
agcuuccucu acggcagcaa gaccuucauc aaccacaccc agggcauccc cgacuucuuc 240
aagcaguccu ucccugaggg cuucacaugg gagagaguca ccacauacga agacgggggc 300
gugcugaccg cuacccagga caccagccuc caggacggcu gccucaucua caacgucaag 360
aucagagggg ugaacuucac auccaacggc ccugugaugc agaagaaaac acucggcugg 420
gaggccuuca ccgagacgcu guaccccgcu gacggcggcc uggaaggcag aaacgacaug 480
gcccugaagc ucgugggcgg gagccaucug aucgcaaaca ucaagaccac auauagaucc 540
aagaaacccg cuaagaaccu caagaugccu ggcgucuacu auguggacua cagacuggaa 600
agaaucaagg aggccaacaa cgagaccuac gucgagcagc acgagguggc aguggccaga 660
uacugcgacc ucccuagcaa acuggggcac aagcucaauu aacacucgcu agagcauggu 720
ugguaccgua gucaagcuuc caucgggccc acuggcgccg uagccacugg cgccguagcc 780
acuggcgccg uagccacugg cgccguagcu ucggcuacca aucguggcgu gucgggcuac 840
caaucguggc gugucgggcu accaaucgug gcgugucggg cuaccaaucg uggcgugucg 900
gggcccgaug g 911

Claims (33)

1. A nucleic acid aptamer molecule comprising the following nucleotide sequence (a), (b) or (c):
(a) nucleotide sequence N1CACUGGCGCCN12-N13-N14CAAUCGUGGCGUGUCGGN32In which N is1、N12、N13、N14And N32Represents a fragment of ≧ 1 nucleotide in length, and N1And N32At least one pair of bases in the nucleotide sequence form a complementary pair, N12And N14At least one pair of bases in the nucleotide sequence forms complementary pairing;
(b) a nucleotide sequence having at least 70% identity to the nucleotide sequence defined in (a);
(c) not including N in the nucleotide sequence defined in (a)1、N12、N13、N14And N32The aptamer molecule derived from (a) having an aptamer function by substitution, deletion and/or addition of one or several nucleotides.
2. The aptamer molecule of claim 1, wherein the sequence has at least 75%, 76%, 78%, 80%, 82%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity to the cppeper structural nucleotide sequence of (a).
3. The aptamer molecule according to claim 1, wherein the nucleotide sequence (c) does not include N in the cpPepper structural nucleotide sequence defined in (a)1、N12、N13、N14And N32The positions of (2) are 10, 9, 8 and 76, 5, 4, 3, 2 or 1 nucleotide substitution, deletion and/or addition.
4. The aptamer molecule of claim 3, wherein nucleotide sequence (c) does not include N in the nucleotide sequence defined in (a)1、N12、N13、N14And N32The aptamer molecule obtained by 7, 6, 5, 4, 3, 2 or 1 nucleotide substitution.
5. The aptamer molecule of any one of claims 1to 4, wherein N in nucleotide sequence (a)1And N32Complementary pairing, N1The orientation of the nucleotide sequence is 5 '-3', N32The orientation of the nucleotide sequence is 3 '-5'; n is a radical of12And N14Complementary pairing, N12The orientation of the nucleotide sequence is 5 '-3', N14The orientation of the nucleotide sequence is 3 '-5'.
6. The aptamer molecule of claim 5 wherein when N is1And N32When the length of at least one fragment in (a) is more than or equal to 5 nucleotide bases, then N1And N32At least two pairs of nucleotide bases in the nucleotide sequence form complementary pairing; when N is present12And N14When the length of at least one fragment in (a) is more than or equal to 5 nucleotide bases, then N12And N14At least two pairs of bases in the nucleotide sequence form complementary pairs.
7. The aptamer molecule of any one of claims 1to 6 wherein the substitution of a nucleotide to the structure of the general formula cpPepper is selected from one of the group consisting of: c8, G9, C10, C11, C15, A16, A17, U18, C19, G20, U21, G23, C24, G25, U26, C29, G30, C2/G31, C11/G22, C2/G31/C15, C2/G31/A16, C2/G31/A17, C2/G31/G20, C2/G31/C24, C2/G31/U26, C2/G31/C8, C2/G31/C10, C2/G31/C11, C2/G31/C22, C11/G22/G31/C22, C22/G31/C22/G31/C22, C11/G31/C22, C22/G31/C11/C31/C10, C11/C31/C11, C31/C11, C22/, C2/G31/C11/G22, C2/G31/C11/G22/C15, C2/G31/C11/G22/A16, C2/G31/C11/G22/A17, C2/G31/C11/G22/G20, C2/G31/C11/G22/C24, C2/G31/C11/G22/U26, C2/G31/C11/G22/C8, C2/G31/C11/G22/C10, C2/G31/C11/G22/U18/C8, C2/G31/C11/G22/U21/C11/G22/U21/C11/C22/U21/C21/U21/C21 U26G/C8U.
8. The aptamer molecule of claim 7, wherein the substitution of a nucleotide for the structure of the general formula cppeper is selected from one of the group consisting of: C15A, C15U, A16C, A17C, C19U, G20C, U21A, C24G, C24U, U26G, C8U, C10G, C11U, C2A/G31U, C2U/G31U, C11U/G22U, C11U/G22U, C2U/G31/U/C15U, C2U/G31/U/A16U, C2U/G31/U, C2U/C11/U, C2/U/C3/U, C2/U/C3/U, C2/U, C2/U/C3/U, C3/U, C/U/C/U, C3/U, C2/U/C3/U, C3/U, C2/U, C/U/C/U, C2/U, C2/G31/C11/G22, C2/G31/C11/G22/C15, C2/G31/C11/G22/A16, C2/G31/C11/G22/A17, C2/G31/C11/G22/G20, C2/G31/C11/G22/C24, C2/G31/C11/G22/U26, C2/G31/C11/G22/C8, C2/G31/C11/G22/C10, C2/G31/C11/G22/U18/C8 and C2/G31/C11/G22/U18/C8.
9. The aptamer molecule of claim 8, wherein the substitution of a nucleotide for the structure of the general formula cppeper is selected from one of the following groups: C15A, C15U, A16C, A17C, C19U, G20C, U21A, C24G, C24U, U26G, C8U, C10G, C11U, C2A/G31U, C2U/G31A, C2G/G31C, C11U/G22A, C11G/G22C, C11/G22, C2/G31/C15, C2/G31/A16, C2/G31/A17, C2/G31/G20, C2/G31/C24, C2/G31/U26, C2/G31/C8, C2/G31/C10, C2/G31/C11/G22 and C2/G31/C11/G22.
10. The aptamer molecule of claims 1-9 wherein N in nucleotide sequence (a)1And N32The nucleotide sequence is F30 or tRNA scaffold RNA sequence.
11. The aptamer molecule according to any preceding claim, wherein the aptamer molecule is an RNA molecule or a base-modified RNA molecule.
12. The nucleic acid aptamer molecule according to any preceding claim, wherein the aptamer molecule is a DNA-RNA hybrid molecule or a base-modified DNA-RNA molecule.
13. The aptamer molecule of any preceding claim, wherein N is12-N13-N14Comprising a nucleotide sequence that recognizes the target molecule.
14. The aptamer molecule of claim 13 wherein the target molecules include but are not limited to: proteins, nucleic acids, lipid molecules, carbohydrates, hormones, cytokines, chemokines, metabolite metal ions.
15. The aptamer molecule of claim 13 or 14 wherein N is12-N13-N14Is the nucleotide sequence capable of recognizing GTP and adenosine molecules.
16. The aptamer molecule according to any of the preceding claims, wherein the aptamer function is that the aptamer is capable of increasing 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 100-200 times or at least 500-1000 times.
17. The aptamer molecule according to claim 1, further comprising a plurality of concatemers of fluorophore moiety that can bind to the concatemers, wherein the concatemers are linked together by a spacer sequence of suitable length, and wherein the number of concatemers is 2, 3, 4,5,6, 7, 8 or more. The nucleotides of the concatemer may be selected from, but are not limited to, the sequences of SEQ ID nos: 6. 7, 8, 9, 10, 11, 12, 13, 14 or 15.
18. The aptamer molecule according to any preceding claim, wherein the 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.
19. A complex of an aptamer molecule and a fluorophore molecule, wherein the aptamer molecule is according to any one of claims 1to 17 and the fluorophore molecule has the structure of formula (I):
Figure FDA0002210709140000041
wherein: d-is X1O-or N (X2) (X3) -; x1, X2 and X3 are independently selected from hydrogen, linear or branched alkyl groups of 1-10 carbons and modified alkyl groups, X2 and X3 are optionally connected with each other to form a saturated or unsaturated ring; r-is selected from hydrogen, cyano, carboxyl, amido, ester group, hydroxyl, straight chain or branched chain alkyl or modified alkyl with 1-10 carbons; ar1 and Ar2 are respectively and independently selected from monocyclic aromatic subunit, monocyclic heteroaromatic subunit, or aromatic subunit with 2-3 ring structures and formed by fusing one or two of monocyclic aryl and monocyclic heteroaromatic;
wherein: the hydrogen atoms in Ar1 and Ar2 can be independently substituted by F, Cl, Br, I, hydroxyl, nitro, aldehyde group, carboxyl group, cyano group, sulfonic group, sulfuric group, phosphoric group, amino group, primary amino group, secondary amino group, linear or branched alkyl group with 1-10 carbons and modified alkyl group;
wherein: any carbon atom of the above-mentioned modified alkyl group is a group obtained by replacing any carbon atom of an alkyl group with at least one group selected from the group consisting of F, Cl, Br, I, -O-, -OH, -CO-, -NO2, -CN, -S-, -SO2-, - (S ═ O) -, azido, phenylene, primary amino, secondary amino, tertiary amino, quaternary ammonium group, ethylene oxide, succinate, isocyanate, isothiocyanate, acid chloride, sulfonyl chloride, saturated or unsaturated monocyclic or bicyclic cycloalkylene group, and bridged ester heterocycle, the modified alkyl group having 1to 10 carbon atoms, wherein a carbon-carbon single bond is optionally independently replaced with a carbon-carbon double bond or a carbon-carbon triple bond;
wherein the aptamer molecule and the fluorophore molecule in the complex are present in separate solutions or in the same solution.
20. The complex of fluorophore molecules according to claim 19, said modified alkyl groups containing groups selected from-OH, -O-, ethylene glycol units, monosaccharide units, disaccharide units, -O-CO-, -NH-CO-, -SO2-O-、-SO-、Me2N-、Et2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、-NO2And a cyano group;
alternatively, the fluorophore molecule may comprise an aromatic ring selected from the group consisting of structures of formulae (II-1) to (II-15) below:
Figure FDA0002210709140000042
Figure FDA0002210709140000051
alternatively, the fluorophore molecule is selected from a compound of the formula:
Figure FDA0002210709140000052
21. the complex of claim 20, wherein the fluorophore molecule is selected from the group consisting of 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.
22. The complex of any one of claims 19-21, wherein the aptamer molecule in the complex comprises the nucleotide sequence of 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.
23. A complex according to any one of claims 19 to 22 for use in the detection or labelling of a target nucleic acid molecule in vitro or in vivo.
24. A complex according to any one of claims 19 to 22 for use in the detection or labelling of an extracellular or intracellular target molecule.
25. A complex according to any one of claims 19 to 22 for use in imaging genomic DNA.
26. A complex according to any one of claims 19 to 22 for use in detecting mRNA versus protein content in a cell.
27. A DNA molecule that transcribes the nucleic acid aptamer molecule of any one of claims 1to 18.
28. An expression vector comprising the DNA molecule of claim 27.
29. A host cell comprising the expression system of claim 28.
30. A kit comprising the aptamer molecule of any one of claims 1to 18 and/or the expression vector of claim 28 and/or the host cell of claim 29 and/or the complex of any one of claims 19 to 22.
31. A method of detecting a target molecule comprising the steps of:
a) adding the complex of any one of claims 19-22 to a solution comprising the target molecule;
b) exciting the complex with light of a suitable wavelength;
c) detecting the fluorescence of the complex.
32. A method for detecting genomic DNA comprising imaging genomic DNA with the complex of any one of claims 19-22.
33. A method for extracting and purifying RNA comprising extracting and purifying RNA using the complex of any one of claims 19-22.
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