CN115704029A - RNA detection and quantification method - Google Patents
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- CN115704029A CN115704029A CN202110903889.4A CN202110903889A CN115704029A CN 115704029 A CN115704029 A CN 115704029A CN 202110903889 A CN202110903889 A CN 202110903889A CN 115704029 A CN115704029 A CN 115704029A
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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
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 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 remarkably improved.
Background
In living cells, RNA has a unique structure, a wide variety of biological functions, and a complex spatiotemporal distribution. The identification, function and regulation research of different kinds of RNA and modified forms thereof has become the international frontier. Development of techniques to trace and analyze them in real time is critical to understanding various cellular life processes.
At present, the more mature technical means for RNA imaging mainly include an RNA fluorescence in situ hybridization technique, a molecular beacon technique, an RNA binding protein-fluorescent protein MCP-FPs system and a fluorescent RNA technique. Among them, the RNA fluorescence in situ hybridization technique is a method widely used for a long time to study the level and distribution of RNA in cells, and is a technique of performing fluorescence labeling of specific RNA molecules by molecular hybridization and then performing imaging. However, the operation is complicated and comprises an elution step, which can only be used for the research of immobilized cells, namely 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 probe is a stem-loop double-labeled oligonucleotide probe which forms a hairpin structure at the 5 'end and the 3' end, when the 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, serious non-specific aggregation in nuclei, 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 MCP-FPs system, wherein MCP-FPs can specifically recognize and combine mRNA molecules fused with multiple copies of MS2 sequences, and the synthesis and distribution of mRNA can be monitored in real time by detecting signals 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 the GFP-MS2 not bound to the mRNA molecules in the cell nucleus, thereby reducing the non-specific fluorescence in the cytoplasm of the cell to a certain extent and improving the signal-to-noise ratio of the detection.
Professor qianmen (Roger Tsien), one of the nobel chemical prize winners for fluorescent proteins, suggested that RNA aptamers could be used to specifically recognize, bind to and activate dye molecules, and thereby enable RNA visualization. Based on the principle, the screening of a subject group obtains molecules capable of combining and activating triphenylmethane dye Malachite Green (MG) dye, and the fluorescence enhancement multiple can reach 2360 times. Unfortunately, MG itself is a biological stain and therefore cannot be used for RNA labeling of living cells. Inspired by this discovery, over the past decade scientists have also developed other RNA aptamer-fluorophore complexes, including fluorescent RNAs based on Hoechst derivatives, cyanine-like dyes, fluorophore-quenching groups (FQs), and fluorescent protein chromophore derivatives. Compared with other RNA imaging technologies, people only need to fuse and express the RNA aptamer coding sequence and the target RNA coding sequence, and can image the RNA marker by adding the dye molecule without introducing other nucleic acid live protein molecules, so that the method is the most direct RNA imaging method and is the most promising living cell RNA marking and imaging technology.
Jaffrey subject group obtained aptamer called "Spinach" using SELEX method and DFHBI (3, 5-difluoro-4-hvdroxybenzyl-dene iminazolinone), a derivative of HBI, a chromophore of green fluorescent protein, as a screening target. After the Spinach is combined with the DFHBI, the fluorescence signal of the DFHBI can be obviously enhanced. Using the Spinach-DFHBI complex, they first achieved the tracking of target RNA in mammalian cells (Paige et al science 2011.333: 642-646). The group developed a tool for detecting cellular metabolites based on the Spinach-DFHBI complex by replacing one stem-loop structure in "Spinach" with an aptamer that can specifically bind to cellular metabolites (Paige et al science 2012.335: 1194). To date, this method has been successfully used for monitoring and analysis of RNA dynamics in bacterial, yeast and mammalian cells. Then, the group optimizes aptamer molecules and fluorophores respectively to obtain Spinach2-DFHBI-1T, and the properties of the novel compound are further improved. In 2014, the subject group combined SELEX with flow cytometry and screened to obtain Broccoli (Filonov et al. Journal of American Chemical society.2014.136: 16299-16308), which has significantly improved fluorescence intensity, stability and the like compared to previous Spinach-DFHBI and Spinach 2-DFHBI-1T. However, broccoli-DFHBI-1T still has the problems of serious dependence on magnesium ions and poor photostability. In 2020, the subject group further optimized DFHBI-1T, resulting in a new fluorophore BI and finally a new complex Broccoli-BI (Li et al. Angewandte chemical International Edition in English.2020.59: 4511-4518). In 2017, the group also developed a 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.
In 2019, a combined offensive team consisting of Yangying education and education in China made a breakthrough in the field of fluorescent RNA. Based on a brand-new molecular design concept, the method designs and synthesizes a brand-new fluorophore molecule HBC, and screens and obtains the aptamer Pepper with high affinity with the HBC. Meanwhile, chemical modification is carried out on the basis of HBC to obtain a series of derivatives, and the spectrum of the compound combined with the Pepper covers cyan, green, orange and red. The Pepper-HBC620 compound can emit bright red fluorescence, has better light stability, and can realize SIM super-resolution imaging.
In view of the above, the currently used RNA labeling techniques all have their own disadvantages. Background fluorescence intensity caused by nonspecific binding exists in the MCP-FPs labeling technology, and the signal-to-noise ratio is low. RNA labeling technologies based on aptamer-fluorophore-quencher complexes have only achieved RNA labeling in bacteria, and have not achieved RNA labeling in mammalian cells. The RNA labeling technology based on single fluorophore-aptamer seems to be a very perfect RNA labeling technology, however, it is limited by the few kinds of currently available bio-orthogonal fluorescent RNAs, and most of the complexes are located in green band, there is strong dependence of magnesium ion, poor photostability, etc., and thus the technology is 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. a nucleic acid aptamer molecule comprising the following nucleotide sequence (a), (b) or (c):
(a) The nucleotide sequence is as follows: n is a radical of 1 UGUAGUAN 9 -N 10 -N 11 GGAAGAAUUGAUCUCGGN 29 In which N is 1 、N 9 、N 10 、N 11 And N 29 Represents a nucleotide fragment of length ≥ 1, and N 1 And N 29 At least one pair of bases in the nucleotide sequence forms a complementary pair, N 9 And N u At least one pair of bases in the nucleotide sequence forms complementary pairing;
(b) A nucleotide sequence having at least 58% identity to the nucleotide sequence defined in (a);
(c) Not including N in the nucleotide sequence defined in (a) 1 、N 9 、N 10 、N 11 And N 29 The 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 58%,63%,67%,71%,75%,79%,83%,94%,96%,98% or 100% identity to the Paprika structural nucleotide sequence of (a).
3. The aptamer molecule of claim 1 wherein nucleotide sequence (c) does not include N in the Paprika structural nucleotide sequence defined in (a) 1 、N 9 、N 10 、N 11 And N 29 The aptamer molecule of (4) through substitution, deletion and/or addition of 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 nucleotides.
4. The aptamer molecule of claim 3, wherein nucleotide sequence (c) does not include N in the nucleotide sequence defined in (a) 1 、N 9 、N 10 、N 11 And N 29 The 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) 1 And N 29 Complementary pairing, N 1 The orientation of the nucleotide sequence is 5'-3', N 29 The orientation of the nucleotide sequence is 3'-5'; n is a radical of hydrogen 9 And N 11 Complementary pairing, N 9 The orientation of the nucleotide sequence is 5'-3', N 11 The orientation of the nucleotide sequence is 3'-5'.
6. The aptamer molecule of claim 5 wherein when N is 1 And N 29 When the length of at least one fragment in (1) is more than or equal to 5 nucleotide bases, then N 1 And N 29 At least two pairs of nucleotide bases in the nucleotide sequence form complementary pairing; when N is present 9 And N 11 When the length of at least one fragment in (1) is more than or equal to 5 nucleotide bases, then N 9 And N 11 At least two pairs of bases in the nucleotide sequence form complementary pairs.
7. The nucleic acid aptamer molecule of any one of claims 1to 6, wherein the substitution of a nucleotide for the structure of formula Paprika is selected from one of the following: U2A, U2C, U2G, G3A, G3U, G3C, U4A, U4C, U4G, A5U, A5C, A5G, G6A, G6U, G6C, U7A, U7C, U7G, A8U, A8C, A8G, G12A, G12U, G12C, G13A, G13U, G13C, A14U, A14C, A14G, A15U, A15C, A15G, G16A, G16U, G16C, A17U, A17C, A17G, A18U, A18C, A18G, U19A, U19C U19G, U20A, U20C, U20G, G21A, G21U, G21C, A22U, A22C, A22G, U23A, U23C, U23G, C24A, C24U, C24G, U25A, U25C, U25G, C26A, C26U, C26G, G27A, G27U, G27C, G28A, G28U, G28C, A5U/U25A, A5C/U25G, A5G/U25C, G6A/C24U, G6U/C24A, G6C/C24G, U4A/G12A, U4A/G13U, U U4A/G16A, U4A/U20C, U4A/A22C, G12A/G13U, G12A/G16A, G12A/U20C, G12A/A22C, G13U/G16A, G13U/U20C, G13U/A22C, G16A/U20C, G16A/A22C, U20C/A22C, U4A/G12A/G13U, U4A/G12A/G16A, U4A/G12A/A22C, G12A/G13U/G16A, G12A/G13U/U20C, G12A/G13U/A22C G13U/G16A/U20C, G13U/G16A/A22C, G16A/U20C/A22C, U4A/G12A/G13U/G16A/U20C, U4A/G12A/G13U/G16A/A22C, U4A/G13U/G16A/U20C/A22C, U4A/G12A/G13U/U20C/A22C, A5U/U25A/U4A/G12A/G13U, A5U/U25A/U4A/G12A/G16A, G6A/C24U/U4A/G12A/A22C, G6A/C24U/G12A/G13U/G16A, G6A/C24U/G12A/G13U/U20C, G6A/C24U/G12A/G13U/A22C.
8. The aptamer molecule of claim 7, wherein the substitution of a nucleotide for the structure of formula Paprika is selected from one of the following: U2A, U2C, U2G, U4A, U4C, U4G, A5C, A5G, G6U, U7A, U7C, U7G, A8U, A8C, A8G, G12A, G12U, G12C, G13A, G13U, G13C, A14U, A14C, A14G, A15U, A15C, A15G, G16A, G16U, G16C, A17U, A17C, A17G, A18U, A18C, U19A, U19C, U19G, U20A, U20C, U20G, G21A G21U, A22C, A22G, U23A, U23C, U23G, C24A, C24U, C24G, U25A, U25C, C26A, C26U, G27A, G27U, G27C, G28A, G28U, G28C, A5U/U25A, A5C/U25G, A5G/U25C, G6A/C24U, G6U/C24A, G6C/C24G, U4A/G12A, U4A/G13U, U4A/G16A, U4A/U20C, U4A/A22C, G12A/G13U G12A/G16A, G12A/U20C, G12A/A22C, G13U/G16A, G13U/U20C, G13U/A22C, G16A/U20C, G16A/A22C, U20C/A22C, U4A/G12A/G13U, U4A/G12A/G16A, U4A/G12A/A22C, G12A/G13U/G16A, G12A/G13U/U20C, G12A/G13U/A22C, G13U/G16A/U20C, G13U/G16A/A22C G16A/U20C/A22C, U4A/G12A/G13U/G16A/U20C, U4A/G12A/G13U/G16A/A22C, U4A/G13U/G16A/U20C/A22C, U4A/G12A/G13U/U20C/A22C, A5U/U25A/U4A/G12A/G13U, A5U/U25A/U4A/G12A/G16A, G6A/C24U/U4A/G12A/A22C, G6A/C24U/G12A/G13U/G16A, G6A/C24U/G12A/G13U/U20C, G6A/C24U/G12A/G13U/A22C.
9. The aptamer molecule of claim 8, wherein the substitution of a nucleotide for the structure of formula Paprika is selected from one of the following: U2A, U2C, U2G, U4A, U4C, U4G, A5C, A5G, G6U, U7C, U7G, A8U, A8C, A8G, G12A, G12U, G12C, G13A, G13U, G13C, A14U, A14C, A14G, A15U, A15C, A15G, G16A, G16U, G16C, A17U, A17C, A17G, U19A, U19C, U20A, U20C, U20G, G21U, A22C, A22G U23A, U23C, U23G, C24A, C24G, U25C, C26U, G27A, G27U, G27C, G28A, G28U, G28C, A5U/U25A, A5C/U25G, A5G/U25C, G6A/C24U, G6U/C24A, G6C/C24G, U4A/G12A, U4A/G13U, U4A/G16A, U4A/U20C, U4A/A22C, G12A/G13U, G12A/G16A, G12A/U20C, G12A/A22C G13U/G16A, G13U/U20C, G13U/A22C, G16A/U20C, G16A/A22C, U20C/A22C, U4A/G12A/G13U, U4A/G12A/G16A, U4A/G12A/A22C, G12A/G13U/G16A, G12A/G13U/U20C, G12A/G13U/A22C, G13U/G16A/U20C, G13U/G16A/A22C, G16A/U20C/A22C, U4A/G12A/G13U/G16A/U20C U4A/G12A/G13U/G16A/A22C, U4A/G13U/G16A/U20C/A22C, U4A/G12A/G13U/U20C/A22C, A5U/U25A/U4A/G12A/G13U, A5U/U25A/U4A/G12A/G16A, G6A/C24U/U4A/G12A/A22C, G6A/C24U/G12A/G13U/G16A, G6A/C24U/G12A/G13U/U20C, G6A/C24U/G12A/G13U/A22C.
10. The aptamer molecule of claims 1-9 wherein N in nucleotide sequence (a) 1 And N 29 The nucleotide sequence of (A) is F30 or tRNA scaffold RNA sequence.
11. The nucleic acid aptamer molecule of 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 according to any preceding claim, wherein the aptamer function is that the aptamer increases the fluorescence intensity of the fluorophore molecule under excitation light of a suitable wavelength by at least 2 fold, at least 5-10 fold, at least 20-50 fold, at least 100-200 fold, at least 200-1000 fold, or at least 1000-5000 fold.
14. The aptamer molecule of claim 1 further comprising concatemers that bind multiple fluorophore molecules, said concatemers being linked together by a spacer sequence of appropriate length, and the number of concatemers being 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: 1.2, 3,4,5,6,7,8,9, 10, 11, 12, 13 and 14.
15. A complex of an aptamer molecule and a fluorophore molecule, wherein the aptamer molecule is according to any one of claims 1to 14 and the fluorophore molecule has the structure of formula (I):
electron donor moiety-D is-NX 1-X2, X1 is selected from hydrogen, alkyl, or modified alkyl, X2 is selected from hydrogen, alkyl, or modified alkyl, X1, X2 are optionally linked to each other, forming together with the N atom a lipo-heterocycle;
the conjugated system E is formed by at least one conjugated connection selected from double bonds, triple bonds, aromatic rings and aromatic heterocycles, wherein each contained hydrogen atom is optionally and independently substituted by a substituent selected from halogen atoms, hydroxyl groups, amino groups, primary amino groups, secondary amino groups, hydrophilic groups, alkyl groups and modified alkyl groups, and the substituents are optionally connected with each other to form an alicyclic ring or an alicyclic heterocyclic ring;
x1, X2, independently, are joined to a conjugated structure E to form a lipoheterocycle;
the electron acceptor A moiety has a cyclic structure represented by the following formula (I-1-a):
ra is independently selected from hydrogen, halogen atoms, nitro groups, alkyl groups, aryl groups, heteroaryl groups, hydrophilic groups or modified alkyl groups; rb is independently selected from hydrogen, halogen atom, hydroxyl, carboxyl, amino, nitro, alkyl, aryl, heteroaryl, hydrophilic group or modified alkyl or a group formed by conjugated connection of double bond and at least one of aromatic ring and aromatic heterocyclic ring;
each Y is 1 Independently selected from-O-, -S-, - (S = O) -, and- (NR) i ) -, wherein R i Selected from hydrogen, amino, alkyl orA modified alkyl group;
each Y is 2 Independently selected from = O, = S = O and = NR i Wherein R is i Selected from hydrogen, alkyl or modified alkyl;
each Y is 3 Independently selected from = O, = S = O and = NR i Wherein R is i Selected from hydrogen, alkyl or modified alkyl;
or, each Y 3 Independently is = C (R) e ) (CN); wherein R is e Selected from hydrogen, ester group, amide group, sulfonic group, sulfonamide group, sulfonic ester group;
wherein,
said "alkyl" is C 1 -C 30 Linear or branched alkyl of (a); preferably, is C 1 -C 10 A linear or branched alkyl group; preferably, is C 1 -C 7 A linear or branched alkyl group; preferably, is C 1 -C 5 A linear or branched alkyl group; <xnotran> , , , , , , , , , ,1- ,2- ,3- , ,1- , , ,1- ,2- ,3- , ,1,1- ,2,2- ,3,3- ,1,2- ,1,3- ,2,3- ,2- , ,2- ,3- ,2,2- ,3,3- ,2,3- ,2,4- ,3- 2,2,3- ; </xnotran>
The "modified alkyl" is an alkyl in which any carbon atom is substituted with a halogen atom, -O-, -OH, -CO-, -CS-, -NO 2 、 -CN、-S-、-SO 2 -、-(S=O)-、At least one of phenyl, phenylene, primary amino, secondary amino, tertiary amino, quaternary ammonium group, saturated or unsaturated monocyclic or bicyclic cyclic hydrocarbon group, biaryl heterocycle and bridged alicyclic heterocycleA group resulting from group replacement, said modified alkyl group having from 1to 30 carbon atoms, the carbon-carbon single bond of which is optionally independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond;
the carbon atom is replaced, and the carbon atom or the carbon atom and the hydrogen atom on the carbon atom are replaced by the corresponding group;
the alicyclic ring is a saturated or unsaturated 4-to 10-membered monocyclic or polycyclic alicyclic ring;
the "aliphatic heterocyclic ring" is a saturated or unsaturated 4-to 10-membered monocyclic or polycyclic aliphatic heterocyclic ring containing at least one hetero atom selected from N, O, S or Si in the ring, and when the aliphatic heterocyclic ring contains an S atom, it is-S-, -SO-or-SO 2 -; the aliphatic heterocyclic ring is optionally substituted with a halogen atom, a nitro group, an alkyl group, an aryl group, a hydrophilic group and a modified alkyl group;
the "aryl or aromatic ring" is a 5-to 10-membered monocyclic or fused bicyclic aromatic group;
the heteroaryl or the aromatic heterocycle is a 5-to 10-membered monocyclic or fused bicyclic heteroaromatic group containing at least one heteroatom selected from N, O, S or Si on the ring;
the halogen atoms are respectively and independently selected from F, cl, br and I;
the "hydrophilic group" is a hydroxyl group, a sulfonic group, a carboxyl group, a phosphite group, a primary amino group, a secondary amino group or a tertiary amino group;
the bridged lipoheterocycle is a 5-20-membered bridged lipoheterocycle containing at least one heteroatom selected from N, O or S on the ring;
the "ester group" is an R (C = O) OR' group;
said "phosphite" RP (= O) (OH) 2 group;
the "sulfonic acid group" is RSO 3 A H group;
the "sulfonate group" is RSO 3 An R' group;
the "sulfonic acid amino group" is RSO 2 A NR' R "group;
the "primary amino group" is RNH 2 A group;
said "secondary amino" is an RNHR' group;
the "tertiary amino" group is an RNR' R "group;
said "quaternary ammonium salt group" R 'R "R'" N + group;
each R, R ', R ", R'" is independently a single bond, an alkyl group, an alkylene group, a modified alkyl group, or a modified alkylene group, the modified alkyl or modified alkylene is Cl-C10 (preferably C1-C6) alkyl or alkylene, any carbon atom of which is substituted by one or more groups selected from-O-, (IV) -one of-OH, -CO-, -CS-, - (S = O) -, replacing the resulting group;
optionally, the modified alkyl or modified alkylene is each independently a polymer containing a unit selected from the group consisting of-OH, -O-, and ethylene glycol (- (CH) 2 CH 2 <xnotran> O) n-), C1 ~ C8 , C1 ~ C8 , C1 ~ C8 , C1 ~ C8 , , , , -O-CO-, -NH-CO-, - (-NH-CHR "" -CO-) n-, -SO </xnotran> 2 -O-、 -SO-、-SO 2 -NH-, -S-S-, -CH = CH-, a halogen atom, a cyano group, a nitro group, an o-nitrophenyl group, a phenacyl group, a phosphate group, wherein n is 1to 100, preferably 1to 50, more preferably 1to 30, more preferably 1to 10; r "" is H or the residue of an alpha amino acid; said "phosphate group" has the definition as described above;
the "monosaccharide units" are saccharide units that can no longer be simply hydrolyzed to smaller sugar molecules;
the disaccharide unit is a saccharide unit formed by dehydration of two monosaccharides;
the polysaccharide unit is a saccharide unit formed by dehydration of more than ten monosaccharides;
optionally, the C1-C8 alkyl is methyl, ethyl, propyl, isopropyl, the C1-C8 alkoxy is methoxy, ethoxy, propoxy, isopropoxy, the C1-C8 acyloxy is acetoxy, ethyl, propyl, isopropyl, the C1-C8 haloalkyl is trifluoromethyl, chloromethyl, bromomethyl;
optionally, the lipoheterocycle is selected from azetidine, pyrrolidine, piperidine, tetrahydrofuran, tetrahydropyran, morpholine, thiomorpholine.
Alternatively, the conjugated system E is selected from the structures of the following formulas (I-2-1) to (I-2-39):
alternatively, the conjugated system E is conjugated with-NX 1 -X 2 Forming a lipoheterocycle as shown in (I-3-1) (I-3-5) below:
alternatively, the conjugated system E is with-NX 1 -X 2 The structure shown in the following (I-3-6) is formed:
optionally, the electron acceptor moiety is one selected from the following formulae (I-4-1) to (I-4-42):
16. optionally, the fluorescent probe as described above, wherein said fluorescent probe of formula (I) is selected from the group consisting of compounds of the following formulae:
17. the composite of claim 16 wherein the at least one of the group consisting of, wherein the fluorophore molecule is selected from the group consisting of I-5-1, I-5-2, I-5-3, I-5-4, I-5-5, I-5-6, I-5-7, I-5-8, I-5-9, I-5-10, I-5-11, I-5-12, I-5-13, I-5-14, I-5-15, I-5-16, I-5-17, I-5-18, I-5-19, I-5-20, I-5-21, I-5-22, I-5-23, I-5-24, I-5-4, I-5-5, I-5-6, I-5-9, I-5-14, I-5-9, I-5-24, I-5-9, I-5-5, I I-5-25, I-5-26, I-5-27, I-5-28, I-5-29, I-5-30, I-5-31, I-5-32, I-5-33, I-5-34, I-5-35, I-5-36, I-5-37, I-5-38, I-5-39, I-5-40, I-5-41, I-5-42, I-5-43, I-5-44, I-5-45, I-5-46, I-5-47, I-5-48, I-5-49, I-5-50, I-5-51, I-5-52, I-5-53, I-5-54, I-5-55, I-5-56, I-5-57, I-5-58, I-5-59, I-5-60, I-5-61, I-5-62, I-5-63, I-5-64, I-5-65, I-5-66, I-5-67, I-5-68, I-5-69, I-5-70, I-5-71, I-5-72, I-5-73, I-5-74, I-5-75, I-5-76, I-5-77, I-5-78, I-5-79, I-5-80, I-5-81, I-5-82, I-5-83, I-5-84.
18. The complex of any one of claims 15-17, 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 and 14.
19. A complex according to any one of claims 15 to 18 for use in the detection or labelling of a target nucleic acid molecule in vitro or in vivo.
20. A DNA molecule that transcribes the nucleic acid aptamer molecule of any one of claims 1-14.
21. An expression vector comprising the DNA molecule of claim 20.
22. A host cell comprising the expression system of claim 21.
23. A kit comprising the aptamer molecule of any one of claims 1to 14 and/or the expression vector of claim 21 and/or the host cell of claim 22 and/or the complex of any one of claims 15 to 18.
24. A method of detecting a target molecule comprising the steps of:
a) Adding the complex of any one of claims 15-18 to a solution comprising a target molecule;
b) Exciting the complex with light of a suitable wavelength;
c) Detecting the fluorescence of the complex.
25. A method for extracting and purifying RNA comprising extracting and purifying RNA using the complex of any one of claims 15-18.
The invention designs novel aptamer molecules to form a novel fluorophore-aptamer complex, wherein the fluorescence intensity of the fluorophore molecules under excitation light with proper wavelength can be obviously improved after the aptamer molecules are combined with the fluorophore molecules, the defects of the fluorophore-aptamer complex in the prior art are overcome, and the novel aptamer molecules can be effectively used for real-time RNA/DNA labeling in living cells. The aptamer of the invention has strong affinity to fluorophore molecules and shows good light stability and temperature stability. The aptamer-fluorophore molecule complexes can be used for real-time marking and imaging of RNA/DNA in prokaryotic and eukaryotic cells, detecting protein-RNA interaction, researching the relation between mRNA content and protein in cells, or labeling for RNA extraction and purification, and the like.
Drawings
FIG. 1 Secondary Structure prediction of aptamer molecules. (A) Predicted generic structure of Paprika, comprising N that can form stem structure 1 And N 29 N, which can form a stem-loop structure 9 、N 10 And N 11 . (B) Predicted Structure of Paprika-1, N 1 And N 29 The base sequence of (A) is shown by a dotted frame corresponding to stem 1 in the figure, N 9 、N 10 And N 11 The base sequence of (2) is shown by a dotted frame corresponding to the "stem loop".
FIG. 2 prediction of secondary structure of Paprika-1. (a) secondary structure prediction of F30-Paprika-1; (B) Secondary Structure prediction of tRNA-Paprika-1.
And 3. The spectral property identification of Paprika-1-I-5-57 complex. (A) Imaging of the Paprika-1-I-5-57 complex in nuclear magnetic tubes; (B) Fluorescence excitation spectrum and emission spectrum of Paprika-1-I-5-57 complex; (C) The absorption spectra of the Paprika-1-I-5-57 complex and fluorophore molecule I-5-57; (D) Dissociation constants for Paprika-1 binding to I-5-57 were determined.
FIG. 4.Paprika-1-I-5-57 complex stability characterization. (A) Determination of the pH stability of the Paprika-1-I-5-57 complex; (B) Paprika-1-I-5-57 complex pair Mg 2+ (ii) a dependency determination of; (C) Paprika-1-I-5-57 Complex temperature stability assay (supplemented with 5mM Mg additional) 2+ ) (ii) a (D) Paprika-1-I-5-57 complex pair K + (iii) determining the dependence of (a).
FIG. 5. Effect of Paprika-2 and Paprika-3 on I-5-57 activation with various modifications in the Paprika-1 sequence. (A) Schematic secondary structure of Paprika-2 aptamer containing deoxyribonucleotides (marked with light color in the figure); (B) Schematic representation of the secondary structure of a 2' F-modified (light colored in the figure) containing Paprika-3 aptamer; (C) The activating effects of Paprika-2 and Paprika-3 on I-5-57 were quantitatively analyzed. A "control" is the replacement of the Paprika-2 or Paprika-3 aptamer with buffer.
FIG. 6. Effect of different Paprika-1 concatemers on I-5-57 activation. (A) Paprika-1 to obtain Paprika-4 concatemer in "concatemer 1" manner; (B) Paprika-1 obtains a Paprika-5 concatemer according to a 'concatemer 2' mode; (C) Obtaining a Paprika-4 concatemer according to a 'concatemer 3' mode; (D) The activation effect of the Paprika-4 concatemer on I-5-57 is obtained according to a 'tandem 1' mode; (E) The activation effect of the Paprika-5 concatemer on I-5-57 is obtained according to a 'tandem 2' mode; (F) The activation effect of the Paprika-4 concatemer on I-5-57 is obtained in a 'tandem 3' manner.
FIG. 7.F30-Paprika-1-I-5-57 complex is used for the labeling effect of RNA in bacteria (scale bar: 10 μm).
FIG. 8 shows that the complex of circular-Paprika-1 and I-5-57 is expressed in mammalian cells (scale bar: 50 μm).
FIG. 9.F30-Paprika-1 and I-5-57 complex are expressed in mammalian cells (scale bar: 50 μm).
FIG. 10 Paprika-1-I-5-57 complex was used to label U6 spliceosome RNA localization (scale bar: 10 μm).
FIG. 11.Paprika-1-I-5-57 complex was used to mark the mRNA localization of fibrillar actin ACTB (scale bar: 10 μm).
FIG. 12 quantitative results of Paprika-1 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 by reference. Hereinafter, "nucleotide" and "nucleotide base" are used interchangeably to mean the same.
Hereinafter, some terms related to the present application are explained in detail.
Aptamer molecules
The "nucleic acid aptamer molecule" according to the invention is also referred to as "aptamer molecule". The aptamer molecule comprises (a) a nucleotide sequence of:
N 1 UGUAGUAN 9 -N 10 -N 11 GGAAGAAUUGAUCUCGGN 29 (corresponding to the general structure of Paprika of FIG. 1A); or (b) a sequence having at least 58% identity to the nucleotide sequence of (a); wherein N is 1 And N 29 At least one pair of bases in the nucleotide sequence form a reverse complementary pair, i.e. N 1 The orientation of the nucleotide sequence is 5'-3', N 29 The orientation of the nucleotide sequence is 3'-5'. When N is present 1 And N 29 At 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 present 1 And N 29 When the length of at least one nucleotide base is more than or equal to 5, at least two pairs of bases are required to form complementary matchesAnd (4) pairing. Wherein N is 9 And N 11 At least one pair of bases in the nucleotide sequence form a reverse complementary pair, i.e. N 9 The orientation of the nucleotide sequence is 5'-3', N 11 The orientation of the nucleotide sequence is 3'-5'. When N is present 9 And N 11 At 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 present 9 And N 11 At 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 is 10 Is nucleotide base with any length and any composition; or (c) by 1-10 nucleotide substitution, deletion and/or addition at any position of the nucleotide sequence (a).
The aptamer molecule comprises a substitution of a nucleotide having the structure of Paprika selected from one of the following groups: U2A, U2C, U2G, G3A, G3U, G3C, U4A, U4C, U4G, A5U, A5C, A5G, G6A, G6U, G6C, U7A, U7C, U7G, A8U, A8C, A8G, G12A, G12U, G12C, G13A, G13U, G13C, A14U, A14C, A14G, A15U, A15C, A15G, G16A, G16U, G16C, A17U, A17C, A17G, A18U, A18C, A18G, U19A, U19C U19G, U20A, U20C, U20G, G21A, G21U, G21C, A22U, A22C, A22G, U23A, U23C, U23G, C24A, C24U, C24G, U25A, U25C, U25G, C26A, C26U, C26G, G27A, G27U, G27C, G28A, G28U, G28C, A5U/U25A, A5C/U25G, A5G/U25C, G6A/C24U, G6U/C24A, G6C/C24G, U4A/G12A, U4A/G13U, U U4A/G16A, U4A/U20C, U4A/A22C, G12A/G13U, G12A/G16A, G12A/U20C, G12A/A22C, G13U/G16A, G13U/U20C, G13U/A22C, G16A/U20C, G16A/A22C, U20C/A22C, U4A/G12A/G13U, U4A/G12A/G16A, U4A/G12A/A22C, G12A/G13U/G16A, G12A/G13U/U20C, G12A/G13U/A22C G13U/G16A/U20C, G13U/G16A/A22C, G16A/U20C/A22C, U4A/G12A/G13U/G16A/U20C, U4A/G12A/G13U/G16A/A22C, U4A/G13U/G16A/U20C/A22C, U4A/G12A/G13U/U20C/A22C, A5U/U25A/U4A/G12A/G13U, A5U/U25A/U4A/G12A/G16A, G6A/C24U/U4A/G12A/A22C, G6A/C24U/G12A/G13U/G16A, G6A/C24U/G12A/G13U/U20C, G6A/C24U/G12A/G13U/A22C. (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 sequence of positions of the nucleotides corresponds to the positions in figure 1A.
The above mutation indicates a nucleotide substitution at the corresponding site in the aptamer nucleotide sequence of the structure of the general formula Paprika, e.g., U2A indicates that the uracil nucleotide U at position 2 of Paprika is substituted with adenine nucleotide a, i.e., paprika-U2A in table 1; paprika-A5U/U25A indicates that the adenine nucleotide A at position 5 of Paprika is substituted with the uracil nucleotide U, while the uracil nucleotide U at position 25 of Paprika is substituted with the adenine nucleotide A, namely Paprika-A5U/U25A in Table 1.
Table 1: aptamer structure with the general structure of Paprika with 5, 4, 3,2 or 1 nucleotide substitutions
Aptamer molecules are single-stranded nucleic acid molecules that have a secondary structure of one or more base-paired regions (stems) and one or more unpaired regions (loops) (FIG. 1A). The aptamer molecules of the invention comprise a secondary structure as predicted in FIG. 1A. The secondary structure comprises 2 stem structures, 2 ring structures and 1 stem-ring 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, which 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 nucleic acid aptamer molecule is fused to an ACTB RNA sequence (ACCESSION NM-001101).
The stem-loop structure in FIG. 1A serves to stabilize the overall aptamer molecular structure and can be replaced with other nucleotide base pairs of any length and arbitrary composition that can form a stem-loop structure. The aptamer molecules of the invention may further comprise an insertion into N 9 -N 10 -N 11 The other nucleotide sequence of position, this inserted nucleotide sequence replacing the stem-loop structure in FIG. 1A, gives the structure in FIG. 1B (SEQ ID NO: 1). The nucleotide sequence may specifically recognize/bind to a target molecule. When the target molecule is absent, the aptamer molecule has a weak binding capacity to the fluorophore molecule, resulting in the fluorophore molecule exhibiting weak fluorescence; when the target molecule is present, the binding of the target molecule to the aptamer facilitates the binding of the aptamer to the fluorophore molecule, significantly increasing the fluorescence of the fluorophore molecule under excitation light of the appropriate wavelength. The target molecule may be a small molecule, a cell surface signaling molecule, 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 aptamer molecule is preferably SEQ ID NO: 1.2, 3,4,5,6,7,8,9, 10, 11, 12, 13, or 14, can be combined with fluorophore molecules to significantly enhance their mutated sequences that fluoresce under excitation light of the appropriate wavelength.
The aptamer molecules of the invention may further comprise a nucleotide sequence that increases their stability. In a preferred embodiment of the present invention, F30 scaffold RNA (SEQ ID NO: 2) is used, which is attached to the aptamer molecule in the manner shown in FIG. 2A; 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. 2B.
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 their D and L enantiomeric forms, and also include 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 the two aptamer nucleotide sequences of the invention is calculated without including the N in the sequence of (a) 1 、N 9 、N 10 、N 11 、N 29 . 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. Optional parameters used are gap penalty (gap penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM 62). The output result of Needle labeled "highest identity" (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:
(same residues x 100)/(alignment length-total number of gaps in alignment).
The sequences of Paprika (U2A) and Paprika (U2C) as in table 1 of the invention are Paprika (U2A): n is a radical of 1 AGUAGUAN 9 -N 10 -N 11 GGAAGAAUUGAUCUCGGN 29 And Paprika (U2C): n is a radical of 1 CGUAGUAN 9 -N 10 -N 11 GGAAGAAUUGAUCUCGGN 29 When comparing their identities, the definition according to the invention shall not include 1 9 N、N- 10 H 29 N-N and NAnd thus their sequence identity alignment results in 96% (1 nucleotide difference).
Fluorophore molecules
The "fluorophore molecule" described herein is also referred to as a "fluorophore" or a "fluorescent molecule". "fluorophore molecules" are 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 bound by a specific aptamer, the quantum yield of the fluorophore is increased by more than 2 times, more preferably by more than 10 times, and most preferably by more than 100 times. The fluorophore 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 of a cell by active transport or passive diffusion through the cell membrane or cell wall. In embodiments of the invention, the fluorophore is 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 the 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 structure (I):
electron donor moiety-D is-NX 1-X2, X1 is selected from hydrogen, alkyl, or modified alkyl, X2 is selected from hydrogen, alkyl, or modified alkyl, X1, X2 are optionally linked to each other, forming together with the N atom a lipo-heterocycle;
the conjugated system E is formed by at least one conjugated connection selected from double bonds, triple bonds, aromatic rings and aromatic heterocycles, wherein each contained hydrogen atom is optionally and independently substituted by a substituent selected from halogen atoms, hydroxyl groups, amino groups, primary amino groups, secondary amino groups, hydrophilic groups, alkyl groups and modified alkyl groups, and the substituents are optionally connected with each other to form an alicyclic ring or an alicyclic heterocyclic ring;
x1, X2, independently, are joined to the conjugated structure E to form a lipoheterocycle;
the electron acceptor A moiety has a cyclic structure of the following formula (I-1-a):
ra is independently selected from hydrogen, halogen, nitro, alkyl, aryl, heteroaryl, hydrophilic groups or modified alkyl; r is b Independently selected from hydrogen, halogen atom, hydroxyl, carboxyl, amino, nitro, alkyl, aryl, heteroaryl, hydrophilic group or modified alkyl or a group formed by conjugated connection of double bond and at least one of aromatic ring and aromatic heterocycle;
each Y is 1 Independently selected from the group consisting of-O-, -S-, - (S = O) -, and- (NR) i ) -, wherein R i Selected from hydrogen, amino, alkyl or modified alkyl;
each Y 2 Independently selected from = O, = S = O and = NR i Wherein R is i Selected from hydrogen, alkyl or modified alkyl;
each Y 3 Independently selected from = O, = S = O and = NR i Wherein R is i Selected from hydrogen, alkyl or modified alkyl;
or, each Y 3 Independently = C (R) e ) (CN); wherein R is e Selected from hydrogen, ester group, amide group, sulfonic group, sulfonamide group, sulfonic ester group;
wherein,
said "alkyl" is C 1 -C 30 Straight or branched chain ofA chain alkyl group; preferably, is C 1 -C 10 A linear or branched alkyl group; preferably, is C 1 -C 7 A linear or branched alkyl group; preferably, is C 1 -C 5 A linear or branched alkyl group; <xnotran> , , , , , , , , , ,1- ,2- ,3- , ,1- , , ,1- ,2- ,3- , ,1,1- ,2,2- ,3,3- ,1,2- ,1,3- ,2,3- ,2- , ,2- ,3- ,2,2- ,3,3- ,2,3- ,2,4- ,3- 2,2,3- ; </xnotran>
The "modified alkyl" is an alkyl in which any carbon atom is substituted with a halogen atom, -O-, -OH, -CO-, -CS-, -NO 2 、 -CN、-S-、-SO 2 -、-(S=O)-、A group obtained by replacing at least one group of phenyl, phenylene, primary amino, secondary amino, tertiary amino, quaternary ammonium base, saturated or unsaturated monocyclic or bicyclic cyclic hydrocarbon, biaryl heterocyclic ring and bridged alicyclic ring, wherein the modified alkyl has 1to 30 carbon atoms, and a carbon-carbon single bond of the modified alkyl is optionally and independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond;
the carbon atom is replaced, and the carbon atom or the carbon atom and the hydrogen atom on the carbon atom are replaced by the corresponding group;
the alicyclic ring is a saturated or unsaturated 4-to 10-membered monocyclic or polycyclic alicyclic ring;
the "aliphatic heterocyclic ring" is a saturated or unsaturated 4-to 10-membered monocyclic or polycyclic aliphatic heterocyclic ring containing at least one hetero atom selected from N, O, S or Si in the ring, and when the aliphatic heterocyclic ring contains an S atom, it is-S-, -SO-or-SO 2 -; the aliphatic heterocyclic ring being optionally substituted by halogen atoms, nitro groupsAlkyl, aryl, hydrophilic groups and modified alkyl substitutions;
the "aryl or aromatic ring" is a 5-to 10-membered monocyclic or fused bicyclic aromatic group;
the heteroaryl or the aromatic heterocycle is a 5-to 10-membered monocyclic or fused bicyclic heteroaromatic group containing at least one heteroatom selected from N, O, S or Si on the ring;
the halogen atoms are respectively and independently selected from F, cl, br and I;
the hydrophilic group is hydroxyl, sulfonic group, carboxyl, phosphite group, primary amino, secondary amino or tertiary amino;
the bridged lipoheterocycle is a 5-to 20-membered bridged lipoheterocycle containing at least one heteroatom selected from N, O or S on the ring;
the "ester group" is an R (C = O) OR' group;
the "phosphite" RP (= O) (OH) 2 group;
the "sulfonic acid group" is RSO 3 A H group;
the "sulfonate group" is RSO 3 An R' group;
the "sulfonic acid amino group" is RSO 2 A NR' R "group;
the "primary amino group" is RNH 2 A group;
said "secondary amino" is an RNHR' group;
the "tertiary amino" group is an RNR' R "group;
said "quaternary ammonium salt group" R 'R "R'" N + group;
each R, R' is independently a single bond, an alkyl group, an alkylene group, a modified alkyl group, or a modified alkylene group, the modified alkyl or modified alkylene is C1-C10 (preferably C1-C6) alkyl or alkylene, any carbon atom of which is selected from-O-, (II) -one of-OH, -CO-, -CS-, - (S = O) -, replacing the resulting group;
optionally, the modified alkyl or modified alkylene is each independently a polymer containing a unit selected from the group consisting of-OH, -O-, and ethylene glycol (- (CH) 2 CH 2 O) n-), C1-C8 alkyl,C1-C8 alkoxy, C1-C8 acyloxy, C1-C8 haloalkyl, a monosaccharide radical disaccharide groups, polysaccharide groups, -O-CO-, -NH-CO-, - (-NH-CHR "" CO-) n-, -SO 2 -O-、 -SO-、-SO 2 -NH-, -S-S-, -CH = CH-, a halogen atom, a cyano group, a nitro group, an o-nitrophenyl group, a phenacyl group, a phosphate group, wherein n is 1to 100, preferably 1to 50, more preferably 1to 30, more preferably 1to 10; r "" is H or the residue of an alpha amino acid; said "phosphate group" has the definition as described above;
the "monosaccharide units" are saccharide units that can no longer be simply hydrolyzed to smaller sugar molecules;
the disaccharide unit is a saccharide unit formed by dehydration of two monosaccharides;
the polysaccharide unit is a saccharide unit formed by dehydration of more than ten monosaccharides;
optionally, the C1-C8 alkyl is methyl, ethyl, propyl, isopropyl, the C1-C8 alkoxy is methoxy, ethoxy, propoxy, isopropoxy, the C1-C8 acyloxy is acetoxy, ethyl, propyl, isopropyl, the C1-C8 haloalkyl is trifluoromethyl, chloromethyl, bromomethyl;
optionally, the lipoheterocycle is selected from azetidine, pyrrolidine, piperidine, tetrahydrofuran, tetrahydropyran, morpholine, thiomorpholine.
Alternatively, the conjugated system E is selected from the structures of the following formulas (I-2-1) to (I-2-39):
alternatively, the conjugated system E is with-NX 1 -X 2 Form a lipoheterocycle represented by the following (I-3-1) (I-3-5):
alternatively, the conjugated system E is with-NX 1 -X 2 The structure shown in the following (I-3-6) is formed:
optionally, the electron acceptor moiety is one selected from the group consisting of the following formulas (I-4-1) to (I-4-42):
optionally, the fluorescent probe is characterized in that the formula (I) of the fluorescent probe is selected from the following compounds:
in a preferred embodiment of the present invention, the fluorophore molecule comprises I-5-1, I-5-2, I-5-3, I-5-4, I-5-5, I-5-6, I-5-7, I-5-8, I-5-9, I-5-10, I-5-11, I-5-12, I-5-13, I-5-14, I-5-15, I-5-16, I-5-17, I-5-18, I-5-19, I-5-20, I-5-21, I-5-22, I-5-23, I-5-24I-5-25, I-5-26, I-5-27, I-5-28, I-5-29, I-5-30, I-5-31, I-5-32, I-5-33, I-5-34, I-5-35, I-5-36, I-5-37, I-5-38, I-5-39, I-5-40, I-5-41, I-5-42, I-5-43, I-5-44, I-5-45, I-5-46, I-5-47, I-5-48, I-5-49, I-5-50, I-5-51, I-5-52, I-5-53, I-5-54, I-5-55, I-5-56, I-5-57, I-5-58, I-5-59, I-5-60, I-5-61, I-5-62, I-5-63, I-5-64, I-5-65, I-5-66, I-5-67, I-5-68, I-5-69, I-5-70, I-5-71, I-5-72, I-5-73, I-5-74, I-5-75, I-5-76, I-5-77, I-5-78, I-5-79, I-5-80, I-5-81, I-5-82, I-5-83, I-5-84. "increasing the fluorescence signal", "increase in fluorescence", "increasing the fluorescence intensity" in the present invention refer to an increase in the quantum yield of a fluorophore under excitation light of a suitable wavelength, or a shift in the maximum emission peak of a fluorescence signal (relative to the emission peak of the fluorophore itself in ethanol or aqueous solutions), 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 100-200 fold; in another more preferred embodiment of the invention, the increase in quantum yield is at least 500-1000 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; the excitation light may be emitted directly from these devices or may be indirectly obtained through other fluorophores, such as a donor fluorophore for FRET or a donor chromophore for 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 infection by a pathogenic bacterium.
By having the aptamer molecule of the invention, such as the structure shown in FIG. 1B, the inserted nucleotide sequence replaces N in FIG. 1A 9 、N 10 、N 11 The 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 ability, so that the fluorescence of the fluorophore molecule under excitation light with proper wavelength cannot be obviously improved; when the target molecules exist, the combination of the target molecules and the nucleotide sequences can promote the combination of the aptamer molecules and the fluorophore molecules, so that the fluorescence of the fluorophore molecules under excitation light with proper wavelength is obviously improved, and the detection, imaging and quantitative analysis of the target molecules 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 above target molecules 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: t4 RNA polymerase aptamers, HIV reverse transcriptase aptamers, phage R17 capsid protein aptamers.
Target nucleic acid molecule
The "target nucleic acid molecule" also called "target nucleic acid molecule" refers to a nucleic acid molecule to be detected, which can 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 aim of detecting the content and distribution of the target nucleic acid molecule is fulfilled.
The "target RNA molecule" in the present invention includes any RNA molecule, 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 MS2 RNA, phage capsid protein PCP recognition binding sequence PP7 RNA, 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 25 26 27 Position 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, and its sequence 20nt or so from the 5' end targets a DNA site through base pair complementation, which causes the Cas9 protein to induce DNA double strand break at the site.
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 sequence of appropriate length, and the number of tandem Paprika-1 structures may be 2,3, 4,5,6,7,8,9, 10 or more. The concatemer can be in a variety of forms, 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. and 7; wherein 2Paprika-4 represents concatemer 1 having 2 structures of Paprika-4; in another preferred embodiment of the invention, the form of the tandem is "tandem 2", as shown in FIG. 6B, and the preferred nucleotide sequence is SEQ ID NO: 8 and 9; wherein 2X Paprika-5 represents a concatamer 2 having 2 structures of Paprika-5; in another preferred embodiment of the invention, the tandem form is "tandem 3", as shown in fig. 6C, and the preferred nucleotide sequence is SEQ ID NO:10 and 11; 2x2Paprika-4 represents concatemer 3 having 2 structures of 2 Paprika-4; in either form, the spacer sequence between the concatemers can be replaced.
The aptamer in a monomer form according to the present invention refers to an aptamer having only 1 Paprika-1 structure, that is, an aptamer having 2 stem structures, 2 loop structures and 1 stem-loop structure (FIG. 1B).
The aptamer in a multimeric form refers to the aptamer containing more than 1 Paprika-1 structure, and includes but is not limited to the 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-Paprika-1-I-5-57.
In another embodiment of the invention, the nucleic acid molecules of the concatemer form complexes with multiple fluorophore molecules, such as F30-4Paprika-4, formed in a "concatemer 1" fashion, comprising 4 aptamer units, and 4 fluorophore molecules, forming complexes F30-4Paprika-4-4 × (I-5-57). The molecular complex may be present in vitro in separate two 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, the nucleic acid aptamer can be prepared by adopting the common experimental method (I) in the specific embodiment, and the fluorescence value can be detected according to the common experimental method (II) in the embodiment. In a preferred embodiment of the invention, the increase in fluorescence intensity is at least 2-fold; 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 100-200 fold; in another more preferred embodiment of the present invention, the increase in fluorescence intensity is at least 200-2000 fold.
Aptamer secondary structure
The secondary structure of the aptamer in this patent was predicted by simulation using mFold online analysis software (http:// www.unaford. Org/RNA _ form. Php). 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. In general, formation of a double-stranded structure does not require that all nucleotides in the region be complementarily paired; in general, N is 1 And N 29 And N is 9 And N 11 At least 50% of the nucleotides in one of the fragments are complementarily paired with the other fragment to form a stem structure. If N is present 1 And N 29 Is a single nucleotide, then N is required 1 And N 29 Complete complementation will result in the formation of the stem structure (as shown in figure 1A).
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 R 1 TGTAGTAR 9 -R 10 -R 11 GGAAGAATTGATCTCGGN 29 And nucleotide sequences thereof having at least 58% identity. Wherein R is 1 Encoding N in the structure of the general formula Paprika 1 ,R 9 Encoding N in the structural structure of the general formula Paprika 9 ,R 10 Encoding N in the structural structure of the general formula Paprika 10 ,R 11 Encoding N in the structural structure of the general formula Paprika 11 ,R 29 Encoding N in the structural structure of the general formula Paprika 29 . 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 a U6 promoter; in another embodiment of the invention, the DNA molecule comprises a CMV promoter. DNThe A molecule comprises said DNA molecule and may further comprise a DNA sequence encoding any nucleic acid molecule of interest. In one embodiment of the present invention, the DNA molecule encoding the target RNA comprises a DNA sequence encoding the fibrillar actin ACTB (SEQ ID No:14 for chimeric RNA, 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 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, the T7 phage promoter, the lac promoter, the trp promoter, the recA promoter, the ribosomal RNA promoter, the PR and PL promoters 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-1acUV5 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 described above, the control sequence 5' to the coding sequence of the DNA molecule is a promoter. Whether the RNA aptamers are obtained by in vitro transcription or the 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.
Initiation of transcription in eukaryotic cells also requires a suitable promoter, including but not limited to β -globin promoter, CAG promoter, GAPDH promoter, β -actin promoter, cstf2t promoter, SV40 promoter, PGK promoter, MMTV promoter, adenovirus Ela promoter, CMV promoter, etc. Termination of transcription in eukaryotic cells depends on specific cleavage sites in the RNA sequence. Similarly, RNA polymerase transcribes genes whose transcription terminators vary widely. 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 nucleic acid 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, gt WES. 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, pACYCDuetl, pCDFDuet1, pRSET series, pBAD series, pQE, pIH821, pGEX, pIII 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 expression elements in the vectors vary widely in strength and properties. 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 present invention, there is provided an expression plasmid pET28a-T7-F30-Paprika-1, pLKO.1-F30-Paprika-1, containing a DNA molecule encoding F30-Paprika-1 RNA. In another embodiment of the present invention, an expression plasmid pLKO.1-F30-5x (F30-2 xPaprika-1) comprising a DNA molecule encoding F30-2xPaprika-1 RNA is provided. In another embodiment of the present invention, expression plasmids pCDNA3.1/hygro (+) -EGFP-F30-5x (F30-2 xPaprika-1) and pCDNA3.1/hygro (+) -ACTB-F30-5x (F30-2 xPaprika-1) containing DNA molecules encoding EGFP-F30-5x (F30-2 xPaprika-1) and ACTB-F30-5x (F30-2 xPaprika-1) are provided. In another embodiment of the present invention, there is provided an expression plasmid pLKO.1-Paprika-1-MS2 containing a DNA molecule encoding Paprika-1-MS2.
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. Host cells in the present invention, which comprise mammalian cells, include, but are not limited to, 297T, COS-7, BHK, CHO, HEK293, heLa, H1299, fertilized egg stem cells, induced totipotent stem cells, primary cells isolated directly from mammalian tissue, and the like; coli cells it contains include but are not limited to BL21 (DE 3), BL21 (DE 3, star), TOP10, mach1, DH5 α; it comprises yeast cells including but not limited to BY4741, BY4742, AH109.
Detection array
The detection arrays of the invention comprise one or more aptamer molecules of the invention 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: (1) Labeling the 5 'or 3' end of the aptamer molecules by using biotin, coating streptavidin on the surface of the array, and anchoring the aptamer molecules by specific binding of the biotin and the streptavidin; (2) The RNA sequence of 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 boxB of the lambda phage transcription termination protein N is fused on the 5', 3' or stem-loop structure of the nucleic acid aptamer molecule, and the RNA sequence recognizes the combined protein MCP, PP7 or lambda phage transcription termination protein N Coating the protein on the surface of the array by MS2 and MCP protein, PP7 and PCP protein or boxB RNA and lambda N The specific action of the protein anchors the aptamer molecule; (3) Fusing a section of RNA or DNA sequence on the 5 'or 3' end of the nucleic acid aptamer molecule, anchoring the RNA sequence which is complementarily paired with the section of RNA sequence or the DNA sequence which is complementarily paired with the section of DNA sequence on the surface of the array, and anchoring the nucleic acid aptamer molecule on the surface of the array by the principle of molecular hybridization. The detection array can be used to detect the presence and concentration of target molecules, and therefore, only the target moleculesIn the presence of the molecule, the aptamer molecule can be combined with a fluorophore molecule, so that the fluorescence intensity of the aptamer molecule under a proper excitation light wavelength is remarkably improved, and in a certain range, the higher the concentration of the target molecule is, the higher the fluorescence intensity is.
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 for illustrative purposes only and do not limit the scope of the present 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, rous Kaims et al, molecular biology reference Manual and J. Sambuque, D.W. Lassel, huang Pentang et al: a relevant section of the molecular cloning laboratory Manual (third edition, 8 months 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 following examples.
The pCDNA3.1/hygro (+) plasmid vectors used in the examples were purchased from Invitrogen, the pLKO.1-puro plasmid vectors were purchased from Sigma, the pET28a plasmid vectors were purchased from Novagen, and the pYES2.1TOPO TA plasmid vectors were purchased from Invitrogen. The primers for PCR used in the examples were all synthesized, purified and correctly identified by mass spectrometry by the Shanghai strapdown bioengineering technology, inc. The Taq DNA polymerase used in the examples was purchased from assist in Shanghai, sheng Biotech Co., ltd, primeSTAR DNA polymerase was purchased from TaKaRa, and the corresponding polymerase buffer and dNTPs 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 (T4 PNK), and T7 RNA polymerase were purchased from Fermentas, and supplied with buffers. Kanamycin (Kanamycin) was purchased from Ameresco; ampicillin (Amp) was purchased from Ameresco. The expression plasmids constructed in the examples were subjected to sequencing, which was performed by Jellier sequencing.
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, hieffClone TM One Step cloning kit was purchased from Shanghai assist saint Biotech, inc., and Eastep Super Total RNA extraction kit was purchased from Promega. BL21 (DE 3, 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. 384-well and 96-well fluorescence detection blackboards were purchased from Grenier. DFHBI-1T, DFHO were purchased from Lucerna, inc. All chemicals used in the examples were purchased from chemical reagents companies, such as titan technology, bidi medicine, loyan, etc., and were used without further purification. The dry solvents used in the experimental procedure, such as methanol, dichloromethane, etc., were purchased directly from Tatanchitechnology and used without further treatment.
The main instruments used in the examples: synergyNeo2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), microfuge22R bench-type high-speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, germany), living body imaging system (Kodak, USA), photometer (Japan and light), nucleic acid electrophoresis instrument (Poweron color Co.), bruker Avance 600 (400 MHz) type nuclear magnetic resonance instrument, micromass GCTTM type mass spectrometer, micromass LCTTM type mass spectrometer, leica SP8 laser confocal microscope and Zeiss Elyra PS.1 super-resolution imaging microscope.
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
(I) preparation of aptamer molecules:
the cDNA corresponding to the RNA to be detected was amplified using a primer containing a T7 promoter, and the RNA was obtained by transcription using the double-stranded cDNA recovered with T7 RNA polymerase (purchased from Fermentas, inc.) as a template. 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 and mixing, and after centrifugation at 10000rpm for 5min, the supernatant was collected. 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% pre-cooled anhydrous ethanol for 2 times. After the ethanol is volatilized, adding a proper 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.
Functional detection of aptamer (II)
The Paprika or Paprika mutant aptamer molecules were prepared according to general protocol (one), 2.5. Mu.M aptamer molecules were mixed with 0.5. Mu.M fluorophore molecules in assay buffer (40mM HEPES, pH 7.4, 125mM KCl,5mM MgCl. Sup.5 mM HEPES, pH 7.4, 125mM KCl, etc.) 2 5% DMSO), and detecting by using a Synergy Neo2 multifunctional plate reader to obtain the maximum excitation peak and the maximum emission peak of fluorescence of the aptamer-fluorophore molecular complex. And detecting the fluorescence intensity of the aptamer-fluorophore molecule complex under the maximum excitation and emission conditions by using a Synergy Neo2 multifunctional microplate reader, measuring a control sample (1 mu M fluorophore molecule without the aptamer) under the same conditions, and calculating the ratio of the fluorescence intensities. For example, a complex formed between 2.5. Mu.M of the Paprika-1 aptamer and 0.5. Mu.M of the I-5-57 fluorophore molecule has a fluorescence excitation peak maximum of 594nm and an emission peak maximum of 658nm. Detecting the fluorescence intensity of the compound at 195 under the excitation of 594 +/-10 nm and the emission condition of 658 +/-10 nm by using a Synergy Neo2 multifunctional enzyme-linked immunosorbent assay67, whereas the control (0.5. Mu.M of the I-5-57 fluorophore molecule) had a fluorescence intensity of 11 under the same assay conditions, the Paprika-1 aptamer had a fold activation of the I-5-57 fluorophore molecule of 1779.
(III) construction of recombinant plasmid based on homologous recombination method
1. Preparing a linearized vector: selecting proper cloning sites, linearizing the vector, and preparing the linearized vector by enzyme digestion or reverse PCR amplification.
PCR amplification for insert preparation: by introducing 15-25bp (excluding enzyme cutting sites) of the homologous sequences at the tail ends of the linearized vector into the 5' ends of the forward and reverse PCR primers of the insert, 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. Linearized vector and insert concentration determination: the linearized vector and insert amplification products were subjected to several equal-volume dilution gradients, and 1. Mu.L of each of the original and diluted products was subjected to agarose gel electrophoresis, and the band intensities were compared with DNA molecular weight standards (DNA Marker) to determine their approximate concentrations.
4. Recombination reactions
The optimal vector usage amount of the recombination reaction system is 0.03pmol; 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 0.09pmol.
X and Y are used to calculate linearized vector and insert, respectively, according to the formulas. After the system preparation is finished, the components are mixed uniformly and react for 20min at 50 ℃. When the insert is > 5kb, the incubation time can be extended to 25min. After the reaction was complete, it was recommended to cool the reaction tube on ice for 5min. The reaction product can be directly transformed, or can be stored at-20 ℃ and be thawed and transformed when needed.
(IV) cell culture and transfection:
the 293T/17 cells in this example were all in CO 2 IncubatorThe COS-7 cells were cultured in high glucose medium (RPMI) containing 10% Fetal Bovine Serum (FBS), streptomycin and penicillin 2 Culturing in culture box with 10% Fetal Bovine Serum (FBS), streptomycin and penicillin high glucose medium (DMEM), and subculturing when the growth reaches 80-90% confluency. At the time of transfection, use(purchased from Promega) was performed according to the instructions.
(V) fluorescence imaging:
the main imaging experiment in the examples was performed using a Leica SP8 confocal laser microscope equipped with white laser, using HCXPLAPO 63x1.47 oil lens and HyD detector.
Example 1 Secondary Structure of Paprika aptamer molecules
The secondary structure of the Paprika aptamer was analyzed using mFold online RNA structure analysis software. Paprika comprises 2 stem structures, 2 loop structures and 1 stem loop structure (fig. 1A). For the sequence of one of the stem and stem-loop, paprika-1 (SEQ ID NO: 1) predicted secondary structure (FIG. 1B).
Example 2 fluorescent activation Effect of different Paprika-1 mutants on the fluorophore molecules of I-5-57.
To examine the fluorescence activation effect of different Paprika-1 mutants on the I-5-57 fluorophore molecules, paprika-1 mutant RNAs containing different base mutations were prepared by point mutation of the sequences shown in Table 1 according to the usual experimental method (one), and 0.5. Mu.M of the I-5-57 mutant RNAs were incubated with 2.5. Mu.M of the different Paprika-1 mutant RNAs, respectively, and their fluorescence activation fold ratios on the I-5-57 fluorophore molecules were determined according to the usual experimental method (two). The detection result shows that most of the Paprika-1 mutants containing single base mutation have stronger fluorescence activation effect (more than or equal to 2 times) on I-5-57 (Table 2). Part of the Paprika-1 mutants containing 2-5 base mutations still retained the strong fluorescence activation (> 20 fold) effect on I-5-57 (Table 3). In conclusion, many single-and multi-base mutants of Paprika-1 can still retain the aptamer function of activating the I-5-57 fluorophore molecule.
TABLE 2 activating Effect of Paprika-1 mutant having Single base mutation on I-5-57
Note: paprika-1 in table 2 is a polypeptide having the sequence of SEQ ID NO: 1; other aptamers are point mutations in the Paprika-1 sequence at nucleotide positions corresponding to Paprika-1 of FIG. 1B.
TABLE 3 activating Effect of Paprika-1 mutant having multiple base mutation on I-5-57
Mutant | Multiple of activation | Mutant | Activation multiple |
Paprika-1 | 1779 | U4A/G12A/G13U | 362 |
A5U/U25A | 252 | U4A/G12A/G16A | 278 |
A5C/U25G | 168 | U4A/G12A/G16A | 298 |
A5G/U25C | 152 | U4A/G12A/A22C | 314 |
G6A/C24U | 175 | G12A/G13U/G16A | 318 |
G6U/C24A | 121 | G12A/G13U/U20C | 308 |
G6C/C24G | 152 | G12A/G13U/A22C | 298 |
U4A/G12A | 254 | G13U/G16A/U20C | 215 |
U4A/G13U | 396 | G13U/G16A/A22C | 198 |
U4A/G16A | 287 | G16A/U20C/A22C | 187 |
U4A/U20C | 102 | U4A/G12A/G13U/G16A/U20C | 102 |
U4A/A22C | 396 | U4A/G12A/G13U/G16A/A22C | 149 |
G12A/G13U | 176 | U4A/G13U/G16A/U20C/A22C | 156 |
G12A/G16A | 258 | U4A/G12A/G16A/U20C/A22C | 97 |
G12A/U20C | 326 | U4A/G12A/G13U/U20C/A22C | 145 |
G12A/A22C | 317 | A5U/U25A/U4A/G12A/G13U | 34 |
G13U/G16A | 289 | A5U/U25A/U4A/G12A/G16A | 67 |
G13U/U20C | 265 | A5U/U25A//U4A/G12A/G16A | 122 |
G13U/A22C | 382 | G6A/C24U/U4A/G12A/A22C | 57 |
G16A/U20C | 194 | G6A/C24U/G12A/G13U/G16A | 201 |
G16A/A22C | 125 | G6A/C24U/G12A/G13U/U20C | 121 |
U20C/A22C | 389 | G6A/C24U/G12A/G13U/A22C | 165 |
Example 3 Secondary Structure of Paprika-1 aptamer molecule
The secondary structure of the Paprika-1 (SEQ ID NO: 1) aptamer was analyzed using the mFold online RNA structure analysis software (FIG. 1B). Secondary structure of the F30-Paprika-1 (SEQ ID NO: 2) aptamer obtained by ligation using F30 scaffold RNA (FIG. 2A). Secondary structure of tRNA-Paprika-1 (SEQ ID NO: 3) aptamer obtained by tRNA scaffold RNA ligation (FIG. 2B).
Example 4 characterization of the spectral Properties of the Paprika-1-I-5-57 Complex
To determine whether the Paprika-1-I-5-57 complex can be activated by UV light, paprika-1 (SEQ ID NO: 2) RNA was prepared according to the general Experimental procedure (one). mu.M I-5-57 was incubated with 10. Mu.M Paprika-1. The detection results showed that the solution of the fluorophore I-5-57 alone, the solution of the fluorophore I-5-57 with the addition of the negative control RNA, or the solution of the mixture of the fluorophore Paprika-1 and the fluorophore I-5-57 containing RNase A showed no significant fluorescence under the irradiation of ultraviolet light, whereas the solution of the mixture of the fluorophore Paprika-1 and the fluorophore I-5-57 showed bright red fluorescence under the irradiation of ultraviolet light (FIG. 3A).
To examine the spectroscopic properties of the Paprika-1-I-5-57 complex, paprika-1 (SEQ ID NO: 1) RNA was prepared according to the general Experimental method (I). mu.M I-5-57 was incubated with 5. Mu.M Paprika-1. The detection result showed that the maximum excitation light of the Paprika-1-I-5-57 complex was 594nm and the maximum emission light was 658nm (FIG. 3B).
To detect the difference between the light absorption of the Paprika-1-I-5-57 complex and the light absorption of the I-5-57 fluorophore molecule itself, 2. Mu.M of I-5-57 was incubated with 10. Mu.M of Paprika-1, or 5. Mu.M of I-5-57 alone, and the light absorption of the I-5-57 and Paprika-1-I-5-57 complexes, respectively, was detected. The detection result shows that the maximum light absorption of the I-5-57 is 539nm, while the maximum light absorption of the Paprika-1-I-5-57 complex is greatly red-shifted relative to the I-5-57 per se, and the maximum light absorption is 588nm (figure 3C).
To determine the binding constant of Paprika-1 to I-5-57, their fluorescence values were determined by incubating Paprika-1 at 2nM with different concentrations of I-5-57. The assay result showed that Paprika-1 binds to I-5-57 with a binding constant of 2.2. + -. 0.2nM (FIG. 3D).
Example 5 Paprika-1-I-5-57 Complex stability characterization
To examine the stability of the Paprika-1-I-5-57 complex at different pH values, the Paprika-1-I-5-57 complex was placed in a different pH environment for 60min, and the fluorescence value was examined. The detection result shows that the Paprika-1-I-5-57 complex keeps high fluorescence signals in the pH range of 5-9 (FIG. 4A), which indicates that the Paprika-1-I-5-57 complex has good pH stability.
To detect Paprika-1-I-5-57 complex vs Mg 2+ Ion dependence, 1. Mu.M Paprika-1 and 5. Mu.M I-5-57, respectively, were incubated in buffer solutions containing different magnesium ion contents. The detection result shows that the concentration of magnesium ions has little influence on the fluorescence of the Paprika-1-I-5-57 complex (FIG. 4B), which indicates that the Paprika-1-I-5-57 complex has good magnesium ion stability.
To examine the temperature stability of Paprika-1, the temperature was measured in a medium containing 5mM Mg 2+ 10 μ M I-5-57 was incubated with 1 μ M Paprika-1, and then left at different temperatures for 5min to detect fluorescence. The results showed that the Tm value of Paprika-1-I-5-57 was 53.6 deg.C (FIG. 4C), indicating that the Paprika-1-I-5-57 complex had excellent temperature stability.
To detect Paprika-1-I-5-57 complex pair K + Ion dependence, 10. Mu.M I-5-57 and 1. Mu.M Paprika-1 were incubated in a buffer containing 100mM KCl or 100mM LiCl, respectively, treated at 70 ℃ for 5min and then left at room temperature for 15min or more, and fluorescence values were measured under different conditions. From previous literature reports, the stability of the G quadruplex structure is very dependent on K + The presence of ions. The experimental results show that the fluorescence of the Paprika-1-I-5-57 complex is independent of K + The presence of ions (FIG. 4D) suggests that the G quadruplex structure is absent in the Paprika-1 structure.
Example 6 characterization of I-5-57 analogs
F30-Paprika-1 RNA aptamer molecules are prepared according to the common experimental method (I), and are used for detecting the basic properties of the I-5-57 analogue combined with Paprika, including fluorescence spectrum and fluorescence activation multiple, and the detection results are shown in Table 4, and as can be seen from the data in the table, F30-Paprika-1 can activate the fluorescence of the I-5-57 analogue to different degrees.
Table 4: determination of physicochemical Properties of F30-Paprika-1 RNA aptamer molecules binding to different fluorescent molecules
Example 7 activating Effect of base-modified Paprika-1 on I-5-57
To examine the activating effect of base-modified Paprika-1 on I-5-57, paprika-2 (SEQ ID NO:4, sequence: GGUGUAGUAU) containing base modification was synthesizedGACGTTCGCGTCThe underlined bases of aggaagauugaucuccggc are deoxyribonucleotide bases) and Paprika-3 (SEQ ID NO:5, the sequence: GGUGUAGUAUGACGUUCGCThe underlined bases in GUCAGGAAGAAUUGAUCUCGGCC are 2'-F modified bases (synthesized by Shanghai Jima pharmaceutical technology, inc.), and they contain a stem-loop structure base replaced with a deoxyribonucleotide (bases shaded in FIG. 5A) and a part of the bases modified with 2' -F (bases shaded in FIG. 5B), respectively. The fluorescence activation effect of these base-modified Paprika-1 on the fluorophore molecule I-5-57 was examined according to the general experimental method (II). The detection result shows that the Paprika-2 and Paprika-3 modified by the base can still significantly activate the fluorescence of the I-5-57 fluorophore molecules (FIG. 5C).
Example 8 Paprika-1 concatemers
To examine the activation effect of the Paprika-1 concatemer on I-5-57 fluorescence, paprika-1 was concatenated in different forms, including the following three:
(1) In the "tandem 1" mode (FIG. 6A), the "head" and "tail" of the Paprika-1 structure are connected in a "head-to-tail" connection mode, so as to obtain nPaprika-1 (wherein n is Paprika-1 which can be any copy). In this example, cDNAs encoding F30-2Paprika-4 and F30-4Paprika-4 (sequences encoding RNA aptamers are SEQ ID NO:6 and SEQ ID NO:7, respectively) were synthesized in their entirety, and after PCR amplification, aptamer RNA was prepared according to the general experimental method (one), and after incubating 0.1. Mu.M RNA aptamers with 10. Mu.M I-5-57, the fluorescence intensity was measured according to the general experimental method (two). The detection result shows that the fluorescence of the nPaprika-4-I-5-57 is increased along with the increase of n, and although the fluorescence of the nPaprika-4-I-5-57 is not increased in an equal fold way, the fluorescence is still much higher than that of the nPaprika-1-I-5-57 (FIG. 6D), which indicates that the fluorescence intensity of the PAPprika-1-I-5-57 complex can be improved in a series 1 mode.
(2) In the "tandem 2" mode (FIG. 6B), paprika-1 was connected in tandem as a structural unit, thereby obtaining nxPaprika-1 (where n is Paprika-1 which may be an arbitrary copy). In this example, cDNA encoding 2xPaprika-5 and 4xPaprika-5 (the sequences encoding RNA aptamers are SEQ ID NO: 8, SEQ ID NO:9, respectively) were synthesized in their entirety, and nucleic acid aptamer RNA was prepared according to the general experimental method (one), and after incubating 0.1. Mu.M RNA aptamer with 10. Mu.M I-5-57, the fluorescence intensity was measured according to the general experimental method (two). The detection result shows that the fluorescence of the nxPaprika-1-I-5-57 is increased along with the increase of n (FIG. 6E), which indicates that the fluorescence intensity of the Paprika-1-I-5-57 complex can be improved in a mode of 'tandem connection 2'.
(3) The "tandem 3" system (FIG. 6C) is a system in which the aforementioned "tandem 1" and "tandem 2" are combined, and nprepika-1 obtained by the "tandem 1" is concatenated as a constitutional unit in the "tandem 2" system, thereby obtaining n1 x n2 paprepa-1 (where n1 and n2 are paprepa-1 which can be arbitrarily copied). In this example, cDNAs encoding 2X2Paprika-4 and 4X2Paprika-4 (the sequences encoding RNA aptamers are SEQ ID NOS: 10 and SEQ ID NO:11, respectively) were synthesized in their entirety, aptamer RNAs were prepared according to the general experimental method (one), and after incubating 0.1. Mu.M of the RNA aptamers with 20. Mu.M of I-5-57, the fluorescence intensity was measured according to the general experimental method (two). The detection result shows that the fluorescence intensity of the Paprika-1 concatemer-I-5-57 complex obtained by the mode of 'tandem 3' is obviously higher than that of the Paprika-1-I-5-57 (figure 6F), which indicates that the fluorescence intensity of the Paprika-1-I-5-57 complex can be improved by the mode of 'tandem 3'.
Example 9 use of F30-Paprika-1-I-5-57 Complex for labeling of RNA in bacteria
To examine the effect of F30-Paprika-1-I-5-57 in bacteria, a bacterial expression plasmid expressing F30-Paprika-1 (SEQ ID NO: 2) was first constructed. F30-Paprika-1 in example 2 was amplified using primers, the promoter and the multiple cloning site region were removed by amplifying pET28a using primers, and the F30-Paprika-1 DNA fragment obtained by the amplification was ligated to the pET28a linearized vector according to the experimental method (III), and the resulting recombinant plasmid was named pET28a-T7-F30-Paprika-1.
The primers used for amplifying the F30-Paprika-1 fragment were:
upstream primer (P1):
upstream primer (P2): 5' CCACATACTCTGATGATCCCGGTAGTAGGTCCTTCGGGACCGGAAGAATTGATCTG CG 3
Downstream primer (P3): 5' TTGCCATGAATGATCCGGCCGAGATCAATTTCCGTCCC
Primers used to amplify the pET28a vector for linearization were:
upstream primer (P4): 5' GGCCGGATCATTCATGGCAATAGCATAACCCCTTGGGCC 3
Downstream primer (P5): 5' GAACCCACTACACTACACTATGGCAACCTATAGTGAGTCGTATTTC-3
BL21 (DE 3, star) E.coli strain was transformed with pET28a-T7-F30-Paprika-1 recombinant plasmid, and single clone was selected and cultured at 37 ℃ and OD 600 About =0.2, 1mM IPTG was added to induce the expression of F30-Paprika-1, and the cells were harvested after 4 hours and resuspended in 2. Mu.M I-5-57-containing PBS solution. BL21 (DE 3, star) E.coli transformed with pET28a empty vector was used as a control. The results showed that only when F30-Paprika-1 was expressed and in the presence of I-5-57, the bacteria showed bright red fluorescence (FIG. 7), indicating that the Paprika-1-I-5-57 complex can be used for fluorescent labeling of RNA in bacteria.
Example 10 use of circular-Paprika-1 and I-5-57 and analogs thereof for labeling RNA in mammalian cells
In order to examine the expression of circular-Paprika-1 and I-5-57 in mammalian cells, a mammalian cell expression plasmid expressing circular-Paprika-1 (SEQ ID No: 12) was constructed. Synthesizing a DNA fragment of circular-Paprika-1 by using the whole gene, and amplifying by using primers P6 and P7 as templates, wherein the primers for amplifying the circular-Paprika-1 fragment are as follows:
upstream primer (P6): 5' GGGCCGCACTCGGTCCC
Downstream primer (P7): 5' GCGTGGACTGTACCTCCCAC
Primers used to amplify pLKO.1puro vector for linearization were:
upstream primer (P8): 5' GTGGAGGGTACAGTCCCACGCTTTTTTTGAATTTCGACCTCG
Downstream primer (P9):
these fragments were inserted into pLKO.1puro vector by the experimental method (III) to obtain an expression vector designated as pLKO.1-circular-Paprika-1, which expresses circular-Paprika-1.
pLKO.1-circular-Paprika-1 plasmid is transfected into 293T/17 cells, 1 mu M I-5-57 is added after 36h to mark circular-Paprika-1, cells which do not express corresponding aptamers are used as a control, and the marking effect is detected by an experimental method (V). The results showed that the circular-Paprika-1-I-5-57 complex exhibited a very bright red fluorescence (FIG. 8).
Example 11 use of F30-Paprika-1 and I-5-57 and analogs thereof for labeling RNA in mammalian cells
To examine the markers of F30-Paprika-1 and I-5-57 for RNA in mammalian cells, a mammalian cell expression plasmid expressing F30-Paprika-1 (SEQ ID NO: 2) was constructed. F30-Paprika-1 in example 9 was amplified with primers P1, P2 and P3, respectively.
Primers used to amplify the plko.1puro vector to linearize it were:
upstream primer (P10): 5' CTCGGCCGGATCATTCATGGCAATTTTTTTTTTGAATTTCGACCTCGAG-3
Downstream primer (P11): 5' GCGAACCCACTACACTACACTAATGGCAATCTAGAGTTCGTCCTTTCCAC-
These fragments were inserted into pLKO.1puro vector by the experimental method (III) to obtain an expression vector designated as pLKO.1-F30-Paprika-1, which expresses F30-Paprika-1.
The pLKO.1-F30-Paprika-1 plasmid is transfected into 293T/17 cells, 1 mu M I-5-57 is added after 36h to mark the F30-Paprika-1, and the marking effect is detected by an experimental method (V) by taking cells not expressing the corresponding aptamer as a control. The results showed that the F30-Paprika-1-I-5-57 complex exhibited a very bright red fluorescence (FIG. 9).
Example 12 labeling of U6 spliceosome RNA in Living cells with Paprika-1-I-5-57 as tag
To verify that Paprika-1 and I-5-57 serve as tags to label U6 spliceosome RNA in living cells, a mammalian cell expression plasmid expressing U6-Paprika-1 (SEQ ID NO: 13) was constructed. DNA sequence gene fragment of total gene synthesis U6-Paprika-1
The primers used for amplifying the U6-Paprika-1 fragment are as follows:
upstream primer (P12): 5' GTGCTCGCTTCGGCAGCACCAC-3
Downstream primer (P13): 5' CAAATATGGAACGCTTCAC-3
Primers used to amplify the plko.1puro vector to linearize it were:
upstream primer (P14): 5' CGTGAAGTCTTCCAATTTTTTTGTTTTTGAATTCTCGACCTCG-
Downstream primer (P15): 5' GTATATGTGCTGCCGAAGCGAGCACTCTAGAGTTTCCGCTTTCC-
These fragments were inserted into pLKO.1puro vector by the experimental method (III) to obtain an expression vector designated as pLKO.1-U6-Paprika-1, which expresses U6-Paprika-1. The pLKO.1-U6-Paprika-1 plasmid and pCDN3.1/hygro (+) -SART3-BFP (Chen et al. Nature biotechnology 2019.37, 1287-1293) plasmid are co-transfected into 293T/17 cells, 0.5. Mu.M I-5-57 is added after 36h for labeling, and the labeling effect is detected by the experimental method (V). The results show that Paprika-1 and SART3-BFP form a co-localization on kahal (Cajal body), the highlight in the figure being kahal, demonstrating that the function of kahal is indicated by Paprika-1-I-5-57 in U6 interaction with SART3-BFP, consistent with literature reports (figure 10) (Chen et al. Nature biotechnology 2019.37 1287-1293.
Example 13 labeling of mRNA in Living cells with Paprika-1-I-5-57 as a tag
To verify that Paprika-1 and I-5-57 serve as tags to tag the fibrillar actin ACTB mRNA in living cells, mammalian cell expression plasmids were constructed. pCDNA3.1/hygro (+) -ACTB and pCDNA3.1/hygro (+) -ACTB-Paprika-1 were constructed according to the reported literature (Chen et al. Nature biotechnology 2019.37, 1287-1293), and these plasmids express ACTB and ACTB-Paprika-1, respectively (SEQ ID NO: 14).
First, an expression plasmid of chimeric RNA in which Paprika-1 was fused with different RNAs was constructed. Because the Paprika-1 fragment is short, the Paprika-1 gene fragment obtained by primer bridging can be inserted into pCDNA3.1/hygro (+) vector by means of primer homologous recombination so as to obtain pCDNA3.1/hygro (+) -Paprika-1 recombinant plasmid.
The primers used for amplifying the pCDNA3.1/hygro (+) -Paprika-1 fragment are as follows:
upstream primer (P16):
downstream primer (P17):
and (2) synthesizing an ACTB gene fragment (ACTB RNA sequence: ACCESSION NM _ 001101) from the whole gene, amplifying the ACTB gene fragment by using a primer, and inserting the fragment into a pCDNA3.1/hygro (+) -Paprika-1 vector linearized by using an experimental method (III) to obtain a pCDNA3.1/hygro (+) -ACTB-Paprika-1 recombinant plasmid encoding ACTB-Paprika-1 chimeric RNA.
Primers used to amplify the ACTB fragment were:
upstream primer (P18): 5' ATGGATGATGATATCGCCGC-3
Downstream primer (P19): 5' CTAGAAGCATTTTGCGGTGGAC-3
The primers used for the amplification and linearization of the pCDNA3.1/hygro (+) -ACTB-Paprika-1 vector were:
upstream primer (P20): 5' CGTCCACCGCAAATTGCTTCTAGCTCGAGGCCGGTATGTAGTATG-3
Downstream primer (P21): 5' GCGGCGATATCATCCATGCATCGAGCTCGGTACCAAG-3
The plasmids pCDNA3.1/hygro (+) -ACTB and pCDNA3.1/hygro (+) -ACTB-Paprika-1 are respectively transfected into COS-7 cells, 0.5 mu M I-5-57 is respectively added after 24h for marking, and the marking effect is detected by an experimental method (V). The results showed that Paprika-1-I-5-57 was useful as ACTB mRNA marker (FIG. 11).
Example 14 Paprika-1 Label for extraction and purification of RNA
In order to examine the application of Paprika-1 in RNA extraction and purification, COS-7 cells were transfected with pCDNA3.1/hygro (+) -ACTB-Paprika-1 recombinant plasmid from example 13, and 24h later the cells were harvested and total RNA of the cells was extracted using Eastep Super Total RNA extraction kit (Promega). The extracted total RNA was dissolved in a solution containing 40mM HEPES, pH 7.4, 125mM KCl,5mM MgCl 2 The cells were incubated at 70 ℃ for 10min in the buffer solution of (1) and then left at room temperature for 30min or more.
After washing 500uL Activated Thiol Sepharose 4B (GE Healthcare) twice with 500. Mu.L PBS, 10mM TCEP (Sigma) in PBS was added and incubated at room temperature for 1h. After washing twice with 500. Mu.L of PBS, a maleimide-containing I-5-57 fluorophore molecule (Mal-I-5-57) was added thereto, and the mixture was reacted at room temperature for 30min and washed three times with 500. Mu.L of PBS. Incubating the treated total RNA with the treated beads at room temperature, centrifuging at 4000rpm for 2min after 30min, discarding the supernatant, and treating with 40mM HEPES, pH 7.4, 125mM KCl, and 5mM MgCl 2 The agarose beads were washed 6 times with the buffer solution and the supernatant was centrifuged off each time. The microbeads were resuspended in DEPC water, treated at 70 ℃ for 10min, centrifuged at 4000rpm for 2min, and the supernatant was collected. Adding 1/1 of the supernatant0 volume of NaAc,2.5 times volume of absolute ethyl alcohol, placing in a refrigerator at-80 ℃ for 20min, centrifuging at 14000rpm at 4 ℃ for 10min, leaving precipitate, discarding the supernatant, washing the precipitate with a precooled 70% ethanol solution, centrifuging at 14000rpm at 4 ℃ for 10min, leaving precipitate, discarding the supernatant, and repeating the steps once. And (3) placing the precipitate at room temperature for 5min, and adding a small volume of DEPC water to resuspend the precipitate after the ethanol is volatilized.
Respectively detecting the fluorescence of the supernatant after cell disruption and the final eluent after high-temperature elution after incubation with the fluorophore I-5-57, and taking the disrupted supernatant of the blank cell as a control. The detection result shows that the fluorescence of the eluent after incubation with I-5-57 is obviously higher than that of the supernatant after disruption before loading (figure 12), which indicates that ACTB-Paprika-1 RNA is well enriched, and the Paprika-1 can be used as a label for RNA separation and purification.
Example 15 Synthesis of I-5-57 and analogs thereof
The following describes in detail specific embodiments of the present invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
5-bromothiophene-2-carbaldehyde (0.5g, 2.6 mmol) and 2-methylaminoethanol (0.78g, 10.4 mmol) were dissolved in 10ml of water, heated in an oil bath at 100 ℃ under the protection of Ar, reacted overnight, after the reaction was completed, cooled to room temperature, extracted with dichloromethane 3 times, washed with saturated saline, dried with anhydrous sodium sulfate, the organic phase was spin-dried, and the residue was subjected to column chromatography to obtain 0.4g of compound 1 with a yield of 84.0%.1H NMR (400mhz, dmso-d 6) δ 9.40 (s, 1H), 7.65 (d, J =4.5hz, 1h), 6.12 (d, J =4.5hz, 1h), 4.86 (t, J =5.4hz, 1h), 3.62 (q, J =5.5hz, 2h), 3.47 (t, J =5.6hz, 2h), 3.09 (s, 3H).
(I-5-1)
Dissolving the compound 1 (0.2g, 1.08mmol) and 2, 4-oxazolidinedione (0.13g, 1.30mmol) in 50ml of absolute ethyl alcohol, adding a catalytic amount of piperidine, heating in an oil bath under the protection of Ar at 90 ℃, reacting for 3h, cooling to room temperature, separating out a large amount of solid in the system, filtering, washing a filter cake twice with cold ethanol, and drying in vacuum to obtain 0.2g of a yellow compound (I-5-1) with the yield of 71.4%.1H NMR (400mhz, dmso-d 6) δ 12.09 (s, 1H), 7.81 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 3.62 (q, J =5.5hz, 2h), 3.46 (t, J =5.6hz, 2h), 3.09 (s, 3H).
(I-5-2)
The synthesis method of reference (I-5-1) gave a yield of 75.2%.1H NMR (400mhz, dmso-d 6) δ 13.09 (s, 1H), 7.71 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 3.62 (q, J =5.5hz, 2h), 3.46 (t, J =5.6hz, 2h), 3.34 (s, 3H).
The yield was 71.6% according to the method for synthesizing (I-5-1). 1H NMR (400mhz, dmso-d 6) δ 14.09 (s, 1H), 7.01 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 3.62 (q, J =5.5hz, 2h), 3.46 (t, J =5.6hz, 2h), 3.09 (s, 3H).
Dissolving a compound 2 (0.5g, 1.66mmol) in acetonitrile, adding sodium azide (1.08g, 16.6 mmol), heating in an oil bath at 60 ℃, reacting for 24h, extracting with dichloromethane and water after the reaction of the raw material compound 2 is finished, combining organic phases, carrying out spin drying to obtain a yellow solid, dissolving the yellow solid with tetrahydrofuran and water (10: 1), adding triphenylphosphine (0.52g, 1.98mmol) for reduction, reacting for 4h, carrying out spin drying on tetrahydrofuran after the reaction is finished, extracting with dichloromethane and water, combining organic phases, carrying out spin drying on a crude product, and carrying out silica gel column chromatography on the crude product to obtain 0.25g of a compound 3 with the yield of 50.3%. 1H NMR (400mhz, dmso-d 6) δ 14.09 (s, 1H), 7.01 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 3.62 (q, J =5.5hz, 2h), 3.46 (t, J =5.6hz, 2h), 3.09 (s, 3H).
(I-5-3)
Dissolving the compound 3 (0.25g, 0.84mmol) in dichloromethane, adding dichloromethane-dissolved methanesulfonyl chloride (0.114g, 1.0 mmol) under the protection of nitrogen, reacting at room temperature for 30min, extracting with dichloromethane and water when the compound 3 is completely reacted, combining the organic phases, and spin-drying to obtain a crude product, wherein the crude product is subjected to silica gel column chromatography to obtain (I-5-3) as a yellow solid 0.19g, and the yield is 59.4%.1H NMR (400mhz, dmso-d 6) δ 14.09 (s, 1H), 7.01 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 3.62 (q, J =5.5hz, 2h), 3.46 (t, J =5.6hz, 2h), 3.09 (s, 3H), 2.93 (s, 3H).
(I-5-4)
The synthesis method of reference (I-5-1) was carried out in a yield of 73.2%.1H NMR (400mhz, dmso-d 6) δ 16.09 (s, 1H), 7.91 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 3.62 (q, J =5.5hz, 2h), 3.46 (t, J =5.6hz, 2h), 3.09 (s, 3H).
(I-5-5)
The synthesis method of reference (I-5-1) gave a yield of 74.3%.1H NMR (400mhz, dmso-d 6) δ 12.10 (s, 1H), 7.71 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 4.24 (q, 2H), 3.62 (q, J =5.5hz, 2h), 3.46 (t, J =5.6hz, 2h), 3.09 (s, 3H), 1.20 (t, 3H).
Dissolving 5-bromothiophene-2-carbaldehyde (0.5g, 2.6 mmol) in 10ml of diethanolammonium, heating in an oil bath under the protection of Ar at 100 ℃, reacting overnight, cooling to room temperature after the reaction is finished, extracting with dichloromethane for 3 times, washing with saturated salt water, drying with anhydrous sodium sulfate, spin-drying an organic phase, and carrying out column chromatography on a residue to obtain 0.32g of a compound 4 with the yield of 57.1%. 1H NMR (400mhz, dmso-d 6) δ 9.40 (s, 1H), 7.65 (d, J =4.5hz, 1h), 6.12 (d, J =4.5Hz, 1H), 4.86 (t, J =5.4Hz, 1h), 3.62 (q, J =5.5hz, 4h), 3.47 (t, J =5.6Hz, 4h), 3.09 (s, 3H).
(I-5-6)
The synthesis method of reference (I-5-1) was carried out at a yield of 70.4%.1H NMR (400mhz, dmso-d 6) δ 12.10 (s, 1H), 11.20 (s, 1H), 7.51 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 4.87 (t, J =5.3hz, 1h), 3.62 (q, J =5.5hz, 4h), 3.46 (t, J =5.6hz, 4h), 3.09 (s, 3H).
Reference is made to the synthesis of compound 1 in 92.5% yield. 1H NMR (400mhz, dmso-d 6) δ 10.40 (s, 1H), 7.65 (d, J =4.5hz, 1h), 6.12 (d, J =4.5hz, 1h), 3.09 (s, 6H).
(I-5-7)
The synthesis method of reference (I-5-1) gave a yield of 89.4%.1H NMR (400mhz, dmso-d 6) δ 12.09 (s, 1H), 7.81 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 3.34 (s, 3H), 3.09 (s, 6H).
(I-5-8)
The yield was 90.2% according to the synthesis method of (I-5-1). 1H NMR (400mhz, dmso-d 6) δ 14.08 (s, 1H), 7.51 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 3.09 (s, 6H).
(I-5-9)
The yield was 79.5% according to the method for synthesizing (I-5-1). 1H NMR (400mhz, dmso-d 6) δ 13.01 (s, 1H), 7.71 (s, 1H), 7.43 (d, J =4.4hz, 1h), 6.15 (d, J =4.4hz, 1h), 3.09 (s, 6H).
(I-5-10)
The synthesis method of reference (I-5-1) gave a yield of 80.5%.1H NMR (400mhz, dmso-d 6) δ 15.01 (s, 1H), 7.91 (s, 1H), 7.44 (d, J =4.4hz, 1h), 6.17 (d, J =4.4hz, 1h), 3.09 (s, 6H).
(I-5-11)
The yield was 82.7% according to the method for synthesizing (I-5-1). 1H NMR (400mhz, dmso-d 6) δ 12.05 (s, 1H), 7.56 (s, 1H), 7.44 (d, J =4.4hz, 1h), 6.17 (d, J =4.4hz, 1h), 3.09 (s, 6H).
(I-5-12)
The synthesis method of reference (I-5-1) gave a yield of 85.2%.1H NMR (400mhz, dmso-d 6) δ 13.65 (s, 1H), 7.66 (s, 1H), 7.44 (d, J =4.4hz, 1h), 6.17 (d, J =4.4hz, 1h), 3.09 (s, 6H).
(I-5-13)
The synthesis method of reference (I-5-1) gave a yield of 89.1%.1H NMR (400mhz, dmso-d 6) δ 12.14 (s, 1H), 7.59 (d, J =8.6hz, 2h), 6.77 (d, J =8.7hz, 2h), 6.61 (s, 1H), 4.74 (d, J =13.1hz, 1h), 4.24 (q, 2H), 3.56 (d, J =5.9hz, 2h), 3.47 (t, J =6.0hz, 2h), 3.01 (s, 3H), 1.20 (t, 3H).
(I-5-14)
The synthesis method of reference (I-5-1) was carried out in a yield of 73.6%.1H NMR (400mhz, dmso-d 6) 13.14 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 6.90 (s, 1H), 6.80 (d, J =8.7hz, 2h), 4.75 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.04 (s, 3H).
(I-5-15)
The synthesis method of reference (I-5-1) gave a yield of 85.6%.1H NMR (400mhz, dmso-d 6) 13.84 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 6.82 (s, 1H), 6.80 (d, J =8.7hz, 2h), 4.75 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.5l (d, J =5.7hz, 2h), 3.04 (s, 3H).
(I-5-16)
The synthesis method of reference (I-5-1) gave a yield of 86.7%.1H NMR (400mhz, dmso-d 6) 19.94 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 6.96 (s, 1H), 6.80 (d, J =8.7hz, 2h), 4.75 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.04 (s, 3H).
(I-5-17)
The synthesis method of reference (I-5-1) gave a yield of 83.8%.1H NMR (400mhz, dmso-d 6) 12.11 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 7.23 (s, 1H), 6.80 (d, J =8.7hz, 2h), 4.75 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.04 (s, 3H).
(I-5-18)
The yield was 84.8% according to the method for synthesizing (I-5-1). 1H NMR (400mhz, dmso-d 6) 12.12 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 6.93 (s, 1H), 6.80 (d, J =8.7hz, 2h), 4.75 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.04 (s, 3H).
(I-5-19)
The synthesis method of reference (I-5-1) was carried out at a yield of 81.2%.1H NMR (400MHz, DMSO-d 6) delta 12.57 (s, 1H), 7.63 (s, 1H), 7.48-7.35 (m, 2H), 6.91-6.79 (m, 2H), 3.04 (s, 6H).
(I-5-20)
The synthesis method of reference (I-5-1) gave a yield of 83.4%.1H NMR (400MHz, DMSO-d 6) Δ 13.87 (s, 1H), 7.49 (s, 1H), 7.45-7.35 (m, 2H), 6.9l-6.59 (m, 2H), 4.24 (t, J =5.28, 2H), 3.04 (s, 6H), 2.63 (t, J =5.28, 2H).
(I-5-21)
The synthesis method of reference (I-5-1) gave a yield of 74.2%.1H NMR (400MHz, DMSO-d 6) delta 14.07 (s, 1H), 7.89 (s, 1H), 7.55-7.25 (m, 2H), 6.71-6.39 (m, 2H), 3.06 (s, 6H).
(I-5-22)
The synthesis method of reference (I-5-1) gave a yield of 90.8%.1H NMR (400MHz, DMSO-d 6) delta 16.27 (s, 1H), 7.90 (s, 1H), 7.65-7.45 (m, 2H), 6.81-6.41 (m, 2H), 3.05 (s, 6H).
(I-5-23)
The yield was 88.4% according to the method for synthesizing (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 11.57 (s, 1H), 7.74 (s, 1H), 7.65-7.45 (m, 2H), 6.81-6.41 (m, 2H), 3.05 (s, 6H).
(I-5-24)
The synthesis method of reference (I-5-1) gave a yield of 83.1%.1H NMR (400MHz, DMSO-d 6) delta 12.87 (s, 1H), 7.94 (s, 1H), 7.65-7.32 (m, 2H), 6.78-6.41 (m, 2H), 3.04 (s, 6H).
Compound 6
4-bromo-2-fluorobenzaldehyde (0.5 g,2.5 mmol) is dissolved in 15mL of N-methyl-N-hydroxyethylamine, copper powder (6.4 mg, 0.01mmol), cuprous iodide (19mg, 0.01mmol), potassium phosphate (0.63g, 2.96mmol) are added, the mixture is heated in an oil bath at 80 ℃ under the protection of Ar overnight, after the reaction is finished, the mixture is cooled to room temperature, the system is poured into 50mL of water, dichloromethane is extracted for 3 times, organic phases are combined, the solvent is evaporated by rotary evaporation, and the yellow product compound 6 of 0.35g is obtained by column chromatography, wherein the yield is 77.8%.1H NMR (400mhz, dmso-d 6) 10.21 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 6.93 (s, 1H), 4.55 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.02 (s, 3H).
(I-5-25)
The yield was 85.2% according to the method for synthesizing (I-5-1). 1H NMR (400MHz, DMSO-d 6) Δ 11.07 (s, 1H), 7.72 (s, 1H), Δ 7.67 (d, J =8.6Hz, 2H), 6.93 (s, 1H), 4.55 (s, 1H), 3.57 (d, J =5.9Hz, 2H), 3.51 (d, J =5.7Hz, 2H), 3.02 (s, 3H).
Compound 7
Reference compound 6 was synthesized in 79.2% yield. 1H NMR (400mhz, dmso-d 6) δ 10.21 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 6.93 (s, 1H), 4.55 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.02 (s, 3H).
The synthesis method of reference (I-5-1) was carried out in a yield of 88.8%.1H NMR (400mhz, dmso-d 6) δ 12.27 (s, 1H), 7.62 (s, 1H), δ 7.57 (d, J =8.6hz, 2h), 6.93 (s, 1H), 4.55 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.02 (s, 3H).
(I-5-26)
Compound 8 (0.5g, 1.34mmol) was dissolved in 20ml DMF, 3- (2-hydroxyethoxy) propionic acid (0.22g, 1.64mmol), pybap (1.39g, 2.67mmol) were added, the reaction was stirred at room temperature overnight, after the starting compound 8 had reacted completely, extraction was carried out three times with dichloromethane, the organic phases were combined and dried by spin-drying to give the crude product, which was chromatographed on a silica gel column to give (I-5-26) as a yellow solid 0.52g in 78.8% yield. 1H NMR (400mhz, dmso-d 6) δ 12.27 (s, 1H), 7.62 (s, 1H), δ 7.57 (d, J =8.6Hz, 2h), 6.93 (s, 1H), 4.55 (s, 1H), 3.73 (m, 6H), 3.57 (d, J =5.9Hz, 2H), 3.51 (d, J =5.7Hz, 2h), 3.02 (s, 3H), 2.42 (m, 2H).
The synthesis method of reference (I-5-1) gave a yield of 80.1%.1H NMR (400MHz, DMSO-d 6) Δ 13.16 (s, 1H), 7.73 (s, 1H), Δ 7.67 (d, J =8.6Hz, 2H), 6.93 (s, 1H), 4.55 (s, 1H), 3.57 (d, J =5.9Hz, 2H), 3.51 (d, J =5.7Hz, 2H), 3.02 (s, 3H), 2.43 (s, 3H).
(I-5-27)
Reference compound (I-5-26) was synthesized in a yield of 38.2%.1H NMR (400mhz, dmso-d 6) δ 13.16 (s, 1H), 7.73 (s, 1H), δ 7.67 (d, J =8.6hz, 2h), 6.93 (s, 1H), 4.55 (s, 1H), 3.70 (m, 2H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.48 (m, 6H), 3.02 (s, 3H), 2.43 (s, 3H), 2.35 (m, 2H).
(I-5-28)
The yield was 98.1% according to the synthesis method of (I-5-1). 1H NMR (400mhz, dmso-d 6) δ 15.86 (s, 1H), 7.83 (s, 1H), δ 7.79 (d, J =8.6hz, 2h), 7.03 (s, 1H), 4.55 (s, 1H), 3.57 (d, J =5.9hz, 2h), 3.51 (d, J =5.7hz, 2h), 3.02 (s, 3H), 2.43 (s, 3H).
(I-5-29)
The synthesis method of reference (I-5-1) gave a yield of 89.3%.1H NMR (400mhz, dmso-d 6) δ 11.26 (s, 1H), 7.63 (s, 1H), δ 7.59 (d, J =8.6hz, 1h), 7.03 (d, J =8.6hz, 1h), 6.68 (s, 1H), 3.61 (t, J =5.3hz, 2h), 2.98 (t, J =5.3hz, 2h), 2.75 (s, 3H).
(I-5-30)
The synthesis method of reference (I-5-1) was carried out in a yield of 78.5%.1H NMR (400mhz, dmso-d 6) δ 11.26 (s, 1H), 7.63 (s, 1H), δ 7.59 (s, 1H), 6.68 (s, 1H), 3.61 (t, J =5.3hz, 2h), 2.98 (t, J =5.3hz, 2h), 2.75 (s, 3H).
Dissolving 3, 4-dihydro-2H-benzo [ b ] [1,4] thiazine (0.30g, 2mmol) in 20mL of DMF, adding cesium carbonate (0.78 g,2.4 mmol), methyl iodide (0.31g, 2.2mmol), heating in an oil bath at 65 ℃ under the protection of Ar, reacting for 4H, cooling to room temperature after the reaction is finished, pouring the system into 50mL of water, extracting with dichloromethane for 3 times, combining organic phases, evaporating the solvent by rotation, and separating by column chromatography to obtain 0.29g of a product with the yield of 90.6%.1H NMR (400MHz, DMSO-d 6) delta 6.98-6.94 (m, 1H), 6.94-6.88 (m, 1H), 6.67 (dd, J =8.1,1.2Hz, 1H), 6.57 (td, J =7.5,1.2Hz, 1H), 3.57-3.42 (m, 2H), 3.13-3.00 (m, 2H), 2.87 (s, 3H).
Compound 13
Adding 10ml of DMF into a three-neck flask, placing the mixture into an ice bath for cooling for 5min, dropwise adding 0.2ml of phosphorus oxychloride, stirring for 1h under the ice bath condition, dissolving a compound 4 into the DMF, dropwise adding the compound into the system, stirring for 0.5h under the Ar protection and ice bath conditions, slowly raising the system to room temperature, continuing stirring for 5h, after the reaction is finished, adding a saturated sodium carbonate solution to adjust the pH to be =10.0, stirring overnight under the room temperature condition, separating out an organic phase the next day, extracting an aqueous phase with 50ml of dichloromethane for three times, combining the organic phases, washing twice with saturated saline, drying the organic phase with anhydrous sodium sulfate, rotationally evaporating the solvent to dryness, and performing column chromatography on the residue to obtain 0.25g of a yellow solid with the yield of 75.3%.1H NMR (400MHz, DMSO-d 6) delta 10.11 (s, 1H), 6.98-6.94 (m, 1H), 6.94-6.88 (m, 1H), 6.57 (td, J =7.5,1.2Hz, 1H), 3.57-3.42 (m, 2H), 3.13-3.00 (m, 2H), 2.87 (s, 3H).
(I-5-31)
The yield was 89.8% according to the synthesis method of (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 13.61 (s, 1H), 7.46 (s, 1H), 7.28-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.76-3.57 (m, 5H), 3.13-3.05 (m, 2H), 3.03 (s, 3H).
(I-5-32)
The yield was 69.5% according to the method for synthesizing (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 14.21 (s, 1H), 7.58 (s, 1H), 7.38-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.76-3.57 (m, 2H), 3.13-3.05 (m, 2H), 3.53 (s, 3H), 3.03 (s, 3H).
(I-5-33)
The synthesis method of reference (I-5-1) gave a yield of 85.8%.1H NMR (400MHz, DMSO-d 6) delta 11.75 (s, 1H), 7.42 (s, 1H), 7.38-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.76-3.57 (m, 2H), 3.13-3.05 (m, 2H), 3.03 (s, 3H).
(I-5-34)
The yield was 99.2% according to the synthesis method of (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 16.05 (s, 1H), 7.51 (s, 1H), 7.38-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.76-3.57 (m, 2H), 3.13-3.05 (m, 2H), 3.03 (s, 3H).
(I-5-35)
The yield was 75.3% according to the method for synthesizing (I-5-1). 1H NMR (400mhz, dmso-d 6) δ 12.17 (s, 1H), 7.46 (s, 1H), 7.38-7.15 (m, 2H), 6.82 (d, J =8.7hz, 1h), 4.07 (q, J =5.5hz, 3h), 3.76-3.57 (m, 2H), 3.13-3.05 (m, 2H), 3.03 (s, 3H), 1.20 (t, J =5.5hz, 3h).
Compound 14
Compound 14 was synthesized according to the method for synthesizing compound 13, in 58.5% yield. 1H NMR (400MHz, DMSO-d 6) delta 10.26 (s, 1H), 6.98-6.94 (m, 1H), 6.94-6.88 (m, 1H), 6.57 (td, J =7.5,1.2Hz, 1H), 3.38-3.32 (m, 2H), 3.05-2.80 (m, 2H), 2.69 (s, 3H).
(I-5-36)
The synthesis method of reference (I-5-1) was carried out in a yield of 69.3%.1H NMR (400MHz, DMSO-d 6) delta 14.52 (s, 1H), 7.44 (s, 1H), 7.38-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.56-3.47 (m, 2H), 3.23-3.05 (m, 2H), 3.03 (s, 3H).
(I-5-37)
The synthesis method of reference (I-5-1) gave a yield of 89.9%.1H NMR (400MHz, DMSO-d 6) Δ 13.42 (s, 1H), 7.46 (s, 1H), 7.38-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.56-3.47 (m, 2H), 3.23-3.05 (m, 2H), 3.03 (s, 3H).
(I-5-38)
The synthesis method of reference (I-5-1) gave a yield of 80.1%.1H NMR (400mhz, dmso-d 6) δ 16.72 (s, 1H), 7.56 (s, 1H), 7.48-7.15 (m, 2H), 6.82 (d, J =8.7hz, 1h), 4.40 (t, J =5.1hz, 2h), 3.56-3.47 (m, 2H), 3.23-3.05 (m, 2H), 3.03 (s, 3H), 2.61 (t, J =5.1hz, 2h).
(I-5-39)
The synthesis method of reference (I-5-1) gave a yield of 85.8%.1H NMR (400MHz, DMSO-d 6) Δ 11.54 (s, 1H), 7.26 (s, 1H), 7.24-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.56-3.47 (m, 2H), 3.23-3.05 (m, 2H), 3.03 (s, 3H), 2.79 (m, 2H).
(I-5-40)
The synthesis method of reference (I-5-1) gave a yield of 76.1%.1H NMR (400MHz, DMSO-d 6) delta 12.78 (s, 1H), 7.26 (s, 1H), 7.24-7.15 (m, 2H), 6.82 (d, J =8.7Hz, 1H), 3.59 (s, 3H), 3.56-3.47 (m, 2H), 3.23-3.05 (m, 2H), 3.03 (s, 3H), 2.79 (m, 2H).
Compound 15
Compound 15 was synthesized according to the method for synthesizing compound 13 in 69.7% yield. 1H NMR (400mhz, dmso-d 6) δ 10.21 (s, 1H), 7.33 (dd, J =8.8,2.2hz, 1h), 7.15 (d, J =2.2hz, 1h), 6.63 (d, J =8.8hz, 1h), 5.47 (d, J =1.5hz, 1h), 2.87 (s, 3H), 1.96 (d, J =1.4hz, 3h), 1.34 (s, 6H).
(I-5-41)
The synthesis method of reference (I-5-1) was carried out at a yield of 95.1%.1H NMR (400mhz, dmso-d 6) δ 13.57 (s, 1H), 7.51 (s, 1H), 7.33 (dd, J =8.8,2.2hz, 1h), 7.15 (d, J =2.2hz, 1h), 6.63 (d, J =8.8hz, 1h), 5.47 (d, J =1.5hz, 1h), 2.87 (s, 3H), 1.96 (d, J =1.4hz, 3h), 1.34 (s, 6H).
(I-5-42)
The synthesis method of reference (I-5-1) gave a yield of 74.6%.1H NMR (400mhz, dmso-d 6) δ 13.86 (s, 1H), 7.41 (s, 1H), 7.33 (dd, J =8.8,2.2hz, 1h), 7.15 (d, J =2.2hz, 1h), 6.63 (d, J =8.8hz, 1h), 5.47 (d, J =1.5hz, 1h), 2.87 (s, 3H), 1.96 (d, J =1.4hz, 3h), 1.34 (s, 6H).
(I-5-43)
The synthesis method of reference (I-5-1) was carried out in a yield of 93.2%.1H NMR (400mhz, dmso-d 6) δ 15.72 (s, 1H), 7.65 (s, 1H), 7.33 (dd, J =8.8,2.2hz, 1h), 7.15 (d, J =2.2hz, 1h), 6.63 (d, J =8.8hz, 1h), 5.47 (d, J =1.5hz, 1h), 2.87 (s, 3H), 1.96 (d, J =1.4hz, 3h), 1.34 (s, 6H).
(I-5-44)
The yield was 75.9% according to the method for synthesizing (I-5-1). 1H NMR (400mhz, dmso-d 6) δ 11.02 (s, 1H), 7.35 (s, 1H), 7.33 (dd, J =8.8,2.2hz, 1h), 7.15 (d, J =2.2hz, 1h), 6.63 (d, J =8.8hz, 1h), 5.47 (d, J =1.5hz, 1h), 4.07 (q, J =5.4hz, 2h), 2.87 (s, 3H), 1.96 (d, J =1.4hz, 3h), 1.34 (s, 6H) 1.20 (t, J =5.4hz, 2h).
(I-5-45)
The synthesis method of reference (I-5-1) gave a yield of 78.9%.1H NMR (400mhz, dmso-d 6) δ 15.72 (s, 1H), 7.48 (s, 1H), 7.33 (dd, J =8.8,2.2hz, 1h), 7.15 (d, J =2.2hz, 1h), 6.63 (d, J =8.8hz, 1h), 5.47 (d, J =1.5hz, 1h), 2.87 (s, 3H), 1.96 (d, J =1.4hz, 3h), 1.34 (s, 6H).
Compound 16
Reference compound 13 was synthesized in 89.4% yield. 1H NMR (400MHz, DMSO-d 6) delta 10.25 (s, 1H), 6.95 (s, 2H), 3.25 (dd, J =6.6,4.9Hz, 4H), 2.68 (t, J =6.3Hz, 4H), 2.05-1.57 (m, 4H). (I-5-46)
The synthesis method of reference (I-5-1) gave a yield of 72.7%.1H NMR (400MHz, DMSO-d 6) delta 12.74 (s, 1H), 7.61 (s, 1H), 6.95 (s, 2H), 3.59 (s, 3H), 3.25 (dd, J =6.6,4.9Hz, 4H), 2.68 (t, J =6.3Hz, 4H), 2.05-1.57 (m, 4H).
(I-5-47)
The synthesis method of reference (I-5-1) gave a yield of 86.7%.1H NMR (400MHz, DMSO-d 6) delta 12.58 (s, 1H), 7.46 (s, 1H), 6.95 (s, 2H), 3.25 (dd, J =6.6,4.9Hz, 4H), 2.68 (t, J =6.3Hz, 4H), 2.05-1.57 (m, 4H).
(I-5-48)
The yield was 92.3% according to the method for synthesizing (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 15.79 (s, 1H), 7.38 (s, 1H), 6.95 (s, 2H), 3.25 (dd, J =6.6,4.9Hz, 4H), 2.68 (t, J =6.3Hz, 4H), 2.05-1.57 (m, 4H).
(I-5-49)
The yield was 78.3% according to the method for synthesizing (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 11.23 (s, 1H), 7.35 (s, 1H), 6.95 (s, 2H), 3.25 (dd, J =6.6,4.9Hz, 4H), 2.68 (t, J =6.3Hz, 4H), 2.05-1.57 (m, 4H).
(I-5-50)
The synthesis method of reference (I-5-1) was carried out at a yield of 71.2%.1H NMR (400MHz, DMSO-d 6) delta 12.21 (s, 1H), 7.31 (s, 1H), 6.95 (s, 2H), 3.25 (dd, J =6.6,4.9Hz, 4H), 2.68 (t, J =6.3Hz, 4H), 2.05-1.57 (m, 4H).
Thieno [3,2-b ] thiophene (10g, 71.31mmol) was added to a 250ml three-necked flask, 120ml of anhydrous tetrahydrofuran was added to dissolve the thiophene, the resulting solution was cooled to-78 ℃, 49ml of 1.6M n-butyllithium was added dropwise to the system, the temperature was maintained at-78 ℃ for 2 hours, cooling was stopped, the mixture was slowly returned to room temperature, the mixture was stirred overnight, after the reaction was completed, 40ml of water was added under ice-bath to quench the reaction, the system was poured into 200ml of dichloromethane, extraction was carried out, the aqueous phase was extracted three times with dichloromethane, the organic phase was dried with anhydrous sodium sulfate, the solvent was spin-dried, and the residue was subjected to column chromatography to give 10.7g of the compound. The yield was 90%.1H NMR (400MHz, chloroform-d) Δ 9.97 (s, 1H), 7.94 (s, 1H), 7.70 (d, J =5.3Hz, 1H), 7.33 (d, J =5.3Hz, 1H).
Compound 18
Compound 9 (10g.59.5mmol) is dissolved in 120ml of a dry mixed solvent of DMF and acetic acid (1: 1), NBS (11.66g, 65.5mmol) is added, oil bath heating is carried out under the protection of Ar at 120 ℃ for reflux overnight, after the reaction is finished, ethyl acetate and water are extracted for three times, organic phases are combined and dried, the solvent is dried in a spinning mode, and the residual column chromatography is carried out to obtain 11.2g of a product, wherein the yield is 78%.1H NMR (400MHz, chloroform-d) Δ 10.01 (s, 1H), 7.70 (d, J =5.3Hz, 1H), 7.33 (d, J =5.3Hz, 1H).
Compound 19
Reference is made to the synthesis of compound 1 in 85% yield. 1H NMR (400MHz, DMSO-d 6) delta 10.15 (s, 1H), 7.87 (s, 1H), 6.39 (s, 1H), 3.09 (s, 6H).
(I-5-51)
The yield was 90.2% according to the synthesis method of (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 12.25 (s, 1H), 8.017 (s, 1H), 87 (s, 1H), 6.39 (s, 1H), 3.09 (s, 6H).
(I-5-52)
The synthesis method of reference (I-5-1) gave a yield of 96.1%.1H NMR (400MHz, DMSO-d 6) delta 15.35 (s, 1H), 7.91 (s, 1H), 7.87 (s, 1H), 6.59 (s, 1H), 3.09 (s, 6H).
(I-5-53)
The yield was 71.3% according to the method for synthesizing (I-5-1). 1H NMR (400MHz, DMSO-d 6) delta 11.35 (s, 1H), 7.96 (s, 1H), 7.57 (s, 1H), 6.59 (s, 1H), 3.09 (s, 6H).
(I-5-54)
The synthesis method of reference (I-5-1) was carried out in a yield of 78.5%.1H NMR (400MHz, DMSO-d 6) delta 11.35 (s, 1H), 7.64 (s, 1H), 7.57 (s, 1H), 6.59 (s, 1H), 3.09 (s, 6H).
Dissolving 2-bromo dithiophene (0.438g, 2mmol) in 15 mLN-methyl-N-hydroxyethyl amine, adding copper powder (6.4 mg,0.01mmo 1), cuprous iodide (19mg, 0.01mmo 1), tripotassium phosphate (0.850g, 4 mmol), heating in an oil bath at 80 ℃ under the protection of Ar overnight, after the reaction is finished, cooling to room temperature, pouring the system into 50mL of water, extracting with dichloromethane for 3 times multiplied by 50mL, combining organic phases, evaporating the solvent by rotation, and separating by column chromatography to obtain 0.362g of yellow product with the yield of 85%. 1H-NMR (400MHz, CDCl3): δ =7.92 (s, 1H), 7.63 (d, 1h, j = 5.2hz), 7.31 (d, 1h, j = 5.2hz), 3.85 (t, 2h, j =5.6 hz), 3.60 (t, 2h, j =5.6 hz), 3.10 (s, 3H).
Dissolving the compound 20 (0.426g and 2mmol) in 50ml of anhydrous dichloromethane, adding 1 ml of triethylamine, slowly dropwise adding acetic anhydride (0.3ml and 3mmol) under ice bath conditions, after dropwise adding, slowly raising the temperature of the system to room temperature, stirring for 3h, after reaction, adding 100ml of water, separating an organic phase, extracting an aqueous phase twice by using 50ml of dichloromethane, combining the organic phases, drying the anhydrous sodium sulfate, rotationally evaporating the solvent to dryness, and directly using the residue in the next step without further purification.
Dissolving the residue in 50ml of dichloromethane, adding 5ml of dimethylformamide, adding 2ml of phosphorus oxychloride under ice bath conditions, stirring for 0.5h under Ar protection conditions, slowly raising the system to room temperature, continuing stirring for 5h, after the reaction is finished, adding a saturated sodium carbonate solution to adjust the pH to be =10.0, stirring overnight under room temperature conditions, separating an organic phase on the next day, extracting an aqueous phase with 50ml of dichloromethane for three times, combining the organic phases, washing the organic phase with saturated saline water twice, drying the organic phase with anhydrous sodium sulfate, evaporating the solvent by rotation, and carrying out column chromatography on the residue to obtain 0.285g of yellow solid with the yield of 59%.1H-NMR (400MHz, CDCl3): δ =10.01 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H) 3.85 (t, 2h, j =5.6 hz), 3.60 (t, 2h, j =5.6 hz), 3.10 (s, 3H).
(I-5-55)
The yield was 58.1% according to the synthesis method of (I-5-1). 1H-NMR (400MHz, CDCl3): δ =12.31 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H), 6.96 (s, 1H), 3.85 (t, 2h, J =5.6 Hz), 3.60 (t, 2H, J =5.6 Hz), 3.10 (s, 3H).
(I-5-56)
The synthesis method of reference (I-5-1) gave a yield of 70.1%.1H-NMR (400MHz, CDCl3): δ =13.56 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H), 6.96 (s, 1H), 3.85 (t, 2h, J =5.6 Hz), 3.60 (t, 2H, J =5.6 Hz), 3.10 (s, 3H).
(I-5-57)
The synthesis method of reference (I-5-1) gave a yield of 86.1%.1H-NMR (400MHz, CDCl3): δ =13.45 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H), 6.91 (s, 1H), 3.85 (t, 2h, J =5.6 Hz), 3.60 (t, 2H, J =5.6 Hz), 3.10 (s, 3H).
(I-5-58)
The synthesis method of reference (I-5-1) gave a yield of 96.8%.1H-NMR (400MHz, CDCl3): δ =17.23 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H), 6.91 (s, 1H), 3.85 (t, 2h, J =5.6 Hz), 3.60 (t, 2H, J =5.6 Hz), 3.10 (s, 3H).
Synthetic method of compound 22 was synthesized with reference to the synthetic method of compound 21, with a yield of 52.1%.1H-NMR (400 MHz, CDCl3): δ =10.01 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H) 3.85 (t, J =5.6Hz, 4H), 3.60 (t, J =5.6Hz, 4H).
(I-5-59)
The synthesis method of reference (I-5-1) gave a yield of 86.1%.1H-NMR (400MHz, CDCl3): δ =7.92 (s, 1H), 7.63 (s, 1H), 6.91 (s, 1H), 3.85 (t, J =5.6hz, 4H), 3.60 (t, J =5.6hz, 4H), 3.59 (s, 3H).
(I-5-60)
The synthesis method of reference (I-5-1) was carried out in a yield of 88.6%.1H-NMR (400MHz, CDCl3): δ =7.92 (s, 1H), 7.63 (s, 1H), 6.65 (s, 1H), 3.85 (t, J =5.6hz, 4H), 3.60 (t, J =5.6hz, 4H), 3.45 (s, 3H).
Compound 23
Synthetic method for compound 23 reference was made to the method for compound 19 in 82.3% yield. .1H-NMR (400 MHz, CDCl 3): δ =9.81 (s, 1H), 7.68 (d, 1h, j = 7.88hz), 7.55 (d, 1h, j = 7.88hz), 7.25 (d, 2h, j = 8.00hz), 6.78 (d, 2h, j = 8.00hz), 3.86 (t, 2h, j = 4.80hz), 3.56 (t, 2h, j =4.80 Hz), 3.06 (s, 3H).
(I-5-61)
The synthesis method of reference (I-5-1) gave a yield of 84.1%.1H-NMR (400MHz, CDCl3): δ =13.21 (s, 1H), 7.68 (d, 1h, j = 7.88hz), 7.55 (d, 1h, j = 7.88hz), 7.25 (d, 2h, j = 8.00hz), 6.80 (s, 1H), 6.78 (d, 2h, j = 8.00hz), 3.86 (t, 2h, j = 4.80hz), 3.56 (t, 2h, j = 4.80hz), 3.06 (s, 3H).
Synthetic method for compound 24 was synthesized with reference to the synthetic method for compound 19, in 59.3% yield. 1H-NMR (400 MHz, CDCl3): δ =10.01 (s, 1H), 7.68 (d, 1h, J = 7.88hz), 7.55 (d, 1h, J = 7.88hz), 7.25 (d, 1h, J = 7.65hz), 6.78 (d, 1h, J = 7.65hz), 3.86 (t, 2h, J = 4.80hz), 3.56 (t, 2H, J =4.80 Hz), 3.06 (s, 3H).
(I-5-62)
The yield was 78.3% according to the method for synthesizing (I-5-1). 1H-NMR (400MHz, CDCl3): δ =11.21 (s, 1H), 7.68 (d, 1h, j = 7.88hz), 7.55 (d, 1h, j = 7.88hz), 7.25 (d, 1h, j = 7.65hz), 6.82 (s, 1H), 6.78 (d, 1h, j = 7.65hz), 3.86 (t, 2h, j = 4.80hz), 3.56 (t, 2h, j = 4.80hz), 3.06 (s, 3H).
Synthetic method for compound 25 was synthesized in 58.9% yield with reference to the synthetic method for compound 19. .1H-NMR (400 MHz, CDCl3): δ =10.35 (s, 1H), 7.68 (d, 1h, j = 7.88hz), 7.55 (d, 1h, j = 7.88hz), 7.25 (d, 2h, j = 8.23hz), 6.78 (d, 2h, j = 8.23hz), 3.86 (t, 2h, j = 5.02hz), 3.56 (t, 2h, j = 5.02hz), 3.06 (s, 3H).
(I-5-63)
The synthesis method of reference (I-5-1) gave a yield of 74.7%.1H-NMR (400MHz, CDCl3): δ =7.68 (d, 1h, j = 7.88hz), 7.55 (d, 1h, j = 7.88hz), 7.25 (d, 2h, j = 8.23hz), 6.82 (s, 1H), 6.78 (d, 2h, j = 8.23hz), 3.86 (t, 2h, j = 5.02hz), 3.56 (t, 2h, j = 5.02hz), 3.52 (s, 3H), 3.06 (s, 3H).
Compound 26
Synthesis of compounds reference was made to the synthesis of compound 21 with a yield of 49.6%.1H-NMR (400 MHz, CDCl 3): δ =9.92 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1h), 3.61 (t, J =8.0hz, 2h), 3.34 (t, J =8.0hz, 2h), 3.10 (s, 3H).
(I-5-64)
The synthesis method of reference (I-5-1) was carried out in a yield of 90.1%.1H-NMR (400MHz, CDCl3): δ =12.56 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.85 (s, 1H), 6.82 (d, J =9.1hz, 1h), 3.61 (t, J =8.0hz, 2h), 3.34 (t, J =8.0hz, 2h), 3.10 (s, 3H).
(I-5-65)
The synthesis method of reference (I-5-1) gave a yield of 72.3%.1H-NMR (400MHz, CDCl3): δ =11.01 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.85 (s, 1H), 6.82 (d, J =9.1hz, 1h), 3.61 (t, J =8.0hz, 2h), 3.34 (t, J =8.0hz, 2h), 3.01 (s, 3H).
(I-5-66)
The yield was 95.3% according to the method for synthesizing (I-5-1). 1H-NMR (400MHz, CDCl3): δ =16.01 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.82 (d, J =9.1hz, 1h), 6.65 (s, 1H), 3.61 (t, J =8.0hz, 2h), 3.34 (t, J =8.0hz, 2h), 3.01 (s, 3H).
Compound 27
Synthesis of compounds reference was made to the synthesis of compound 21, with a yield of 60.3%.1H-NMR (400 MHz, CDCl 3): δ =9.92 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (d, J =2.0hz, 1h), 6.82 (d, J =9.1,2.3hz, 1h), 3.05 (s, 6H).
(I-5-67)
The synthesis method of reference (I-5-1) gave a yield of 75.5%.1H-NMR (400MHz, CDCl3): δ =12.06 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (d, J =2.0hz, 1h), 6.85 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1h), 3.05 (s, 6H).
Synthetic method of compound 28 reference was made to the synthetic method of compound 21 with a yield of 50.6%.1H-NMR (400 MHz, CDCl3): δ =10.02 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =9.0Hz, 1h), 6.92 (d, J =2.0Hz, 1h), 6.82 (d, J =9.1,2.3Hz, 1h), 3.61 (t, J =8.0Hz, 4H), 3.34 (t, J =8.0Hz, 4H).
(I-5-68)
The synthesis method of reference (I-5-1) was carried out in a yield of 88.3%.1H-NMR (400MHz, CDCl3): δ =7.81 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (d, J =2.0hz, 1h), 6.85 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1h), 3.61 (t, J =8.0hz, 4h), 3.52 (s, 3H), 3.34 (t, J =8.0hz, 4h).
Synthetic method of compound 29 reference was made to the synthetic method of compound 21 in 49.9% yield. 1H-NMR (400 MHz, CDCl3): δ =9.92 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1h), 3.61 (t, J =8.0hz, 2h), 3.34 (t, J =8.0hz, 2h), 3.10 (s, 3H).
(I-5-69)
The yield was 75.3% according to the method for synthesizing (I-5-1). 1H-NMR (400MHz, CDCl3): δ =12.31 (s, 1H), 7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.85 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1H), 3.61 (t, J =8.0hz, 2h), 3.34 (t, J =8.0hz, 2h), 3.10 (s, 3H).
(I-5-70)
The synthesis method of reference (I-5-1) gave a yield of 48.8%.1H-NMR (400MHz, CDCl3): δ =7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.85 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1h), 4.24 (t, J =5.2hz, 2h), 3.61 (t, J =8.0hz, 2h), 3.34 (t, J =8.0hz, 2h), 3.10 (s, 3H), 2.63 (t, J =5.2hz, 2h).
(I-5-71)
The yield was 78.9% according to the method for synthesizing (I-5-1). 1H-NMR (400MHz, CDCl3): δ =7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.85 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1h), 3.61 (t, J =8.0hz, 2h), 3.52 (s, 3H), 3.34 (t, J =8.0hz, 2h), 3.10 (s, 3H).
(I-5-72)
The synthesis method of reference (I-5-1) gave a yield of 98.4%.1H-NMR (400MHz, CDCl3): δ =7.68 (d, J =9.0hz, 1h), 6.92 (s, 1H), 6.85 (s, 1H), 6.82 (d, J =9.1,2.3hz, 1h).
Compound 30
Synthesis of compound 30 reference was made to the synthesis of compound 19 with a yield of 42.5%.1H-NMR (400 MHz, CDCl3): δ =9.92 (s, 1H), 7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 6.78 (m, 2H), 3.82 (t, 4h, j =5.6 hz), 3.54 (t, 4h, j =5.6 hz), 1.42 (s, 6H).
(I-5-73)
The synthesis method of reference (I-5-1) was carried out in a yield of 88.9%.1H-NMR (400MHz, CDCl3): δ =12.31 (s, 1H), 7.82 (s, 1H), 7.71 (m, lH), 7.60 (m, 2H), 6.85 (s, 1H), 6.78 (m, 2H), 3.82 (t, 4H, j =5.6 hz), 3.54 (t, 4H, j =5.6 hz), 1.42 (s, 6H).
Compound 31
Synthetic method of compound 31 reference was made to the synthetic method of compound 19 in 82.7% yield. 1H-NMR (400 MHz, CDCl3): δ =9.92 (s, lH), 7.82 (s, 1H), 7.7l (m, 1H), 7.60 (m, 2H), 6.78 (m, 2H), 3.62 (s, 6H), 1.42 (s, 6H).
(I-5-74)
The synthesis method of reference (I-5-1) was carried out in a yield of 78.3%.1H-NMR (400MHz, CDCl3): δ =11.23 (s, 1H), 7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 6.85 (s, 1H), 6.78 (m, 2H), 3.62 (s, 6H), 1.42 (s, 6H).
Compound 32
Synthetic method of compound 32 was synthesized with reference to the synthetic method of compound 19, in a yield of 63.0%.1H-NMR (400 MHz, CDCl3): δ =9.92 (s, lH), 7.82 (s, 1H), 7.7l (m, 1H), 7.60 (m, 2H), 6.78 (m, 2H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-75)
The yield was 98.3% according to the method for synthesizing (I-5-1). 1H-NMR (400MHz, CDCl3): δ =14.89 (s, 1H), 7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 6.85 (s, 1H), 6.78 (m, 2H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H). (I-5-76)
The yield was 72.8% according to the method for synthesizing (I-5-1). 1H-NMR (400MHz, CDCl3): δ =12.03 (s, 1H), 7.82 (s, 1H), 7.71 (m, lH), 7.60 (m, 2H), 6.82 (s, 1H), 6.78 (m, 2H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H). Compound 33
Synthetic method of compound 33 reference was made to the synthetic method of compound 19 in 68.1% yield. 1H-NMR (400 MHz, CDCl3): δ =9.92 (s, 1H), 7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-77)
The synthesis method of reference (I-5-1) gave a yield of 70.1%.1H-NMR (400MHz, CDCl3): δ =12.05 (s, 1H), 7.82 (s, 1H), 7.71 (m, lH), 7.60 (m, 2H), 6.82 (s, 1H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-78)
The synthesis method of reference (I-5-1) gave a yield of 70.2%.1H-NMR (400MHz, CDCl3): δ =11.25 (s, 1H), 7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 6.82 (s, 1H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-79)
The synthesis method of reference (I-5-1) gave a yield of 80.6%.1H-NMR (400MHz, CDCl3): δ =13.01 (s, 1H), 7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 6.82 (s, 1H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
Compound 34
Synthetic method for compound 34 reference was made to the synthetic method for compound 19 in 58.9% yield. 1H-NMR (400 MHz, CDCl3): δ =9.92 (s, 1H), 7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 3.82 (t, J =5.6hz, 4H), 3.54 (t, J =5.6hz, 4H), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-80)
The synthesis method of reference (I-5-1) gave a yield of 90.6%.1H-NMR (400MHz, CDCl3): δ =7.82 (s, 1H), 7.71 (m, 1H), 7.60 (m, 2H), 3.82 (t, J =5.6hz, 4H), 3.54 (t, J =5.6hz, 4H), 3.55 (s, 3H), 1.42 (s, 6H).
Compound 35
Synthetic method of compound 35 was synthesized with reference to the synthetic method of compound 19, in 59.7% yield. 1H-NMR (400 MHz, CDCl3): δ =10.02 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 3.82 (t, J =5.6Hz, 2H), 3.54 (t, J =5.6Hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-81)
The synthesis method of reference (I-5-1) was carried out at a yield of 70.2%.1H-NMR (400MHz, CDCl3): δ =7.82 (s, 1H), 7.71 (s, 1H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-82)
The synthesis method of reference (I-5-1) was carried out at a yield of 95.4%.1H-NMR (400MHz, CDCl3): δ =14.21 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
(I-5-83)
The yield was 95.1% according to the synthesis method of (I-5-1). 1H-NMR (400MHz, CDCl3): δ =12.21 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 3.82 (t, J =5.6hz, 2h), 3.54 (t, J =5.6hz, 2h), 3.05 (s, 3H), 1.42 (s, 6H).
Compound 36
Synthetic method for compound 36 reference was made to the synthetic method for compound 19 in 63.5% yield. 1H-NMR (400 MHz, CDCl3): δ =10.02 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 3.82 (t, J =5.6Hz, 4H), 3.54 (t, J =5.6Hz, 4H), 1.42 (s, 6H).
(I-5-84)
The synthesis method of reference (I-5-1) was carried out in a yield of 88.3%.1H-NMR (400MHz, CDCl3): δ =7.82 (s, 1H), 7.71 (s, 1H), 3.82 (t, J =5.6hz, 4H), 3.54 (t, J =5.6hz, 4H), 3.55 (s, 3H) 1.42 (s, 6H).
It should be understood that the amounts used, reaction conditions, etc. in the various examples herein are approximate unless otherwise indicated, and may be varied somewhat as is practical to achieve similar results. 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 in terms of exemplary preferred embodiments, those of ordinary skill in the art will recognize that the invention can be practiced with methods and materials similar to those described herein with the same or similar results, and that various changes or modifications can be made to the invention without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> Nanying (Shanghai) Biotechnology Ltd
<120> RNA detection and quantification method
<130> 072-2108062IP
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 1
gguguaguau gacguucgcg ucaggaagaa uugaucucgg cc 42
<210> 2
<211> 100
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 2
uugccaugug uauguggguu cgcccacaua cucugaugau ccgguguagu agguccuucg 60
ggaccggaag aauugaucuc ggccggauca uucauggcaa 100
<210> 3
<211> 141
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 3
gcccggauag cucagucggu agagcagcgc uggguguagu agguguagua gguccuucgg 60
gaccggaaga auugaucucg gccggaagaa uugaucucgg cccagcgcgg guccaggguu 120
caagucccug uucgggcgcc a 141
<210> 4
<211> 42
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 4
gguguaguau gacguucgcg ucaggaagaa uugaucucgg cc 42
<210> 5
<211> 42
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 5
gguguaguau gacguucgcg ucaggaagaa uugaucucgg cc 42
<210> 6
<211> 138
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 6
uugccaugug uauguggguu cgcccacaua cucugaugau ccgguguagu agguccggug 60
uaguaggucc uucgggaccg gaagaauuga ucucggccgg accggaagaa uugaucucgg 120
ccggaucauu cauggcaa 138
<210> 7
<211> 214
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 7
uugccaugug uauguggguu cgcccacaua cucugaugau ccgguguagu agguccggug 60
uaguaggucc gguguaguag guccggugua guagguccuu cgggaccgga agaauugauc 120
ucggccggac cggaagaauu gaucucggcc ggaccggaag aauugaucuc ggccggaccg 180
gaagaauuga ucucggccgg aucauucaug gcaa 214
<210> 8
<211> 99
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 8
gguguaguag guccuucggg accggaagaa uugaucucgg cccaaaacaa aacaaaaggu 60
guaguagguc cuucgggacc ggaagaauug aucucggcc 99
<210> 9
<211> 213
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 9
gguguaguag guccuucggg accggaagaa uugaucucgg cccaaaacaa aacaaaaggu 60
guaguagguc cuucgggacc ggaagaauug aucucggccc aaaacaaaac aaaaggugua 120
guagguccuu cgggaccgga agaauugauc ucggcccaaa acaaaacaaa agguguagua 180
gguccuucgg gaccggaaga auugaucucg gcc 213
<210> 10
<211> 185
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 10
gguguaguag guccggugua guagguccuu cgggaccgga agaauugauc ucggccggac 60
cggaagaauu gaucucggcc caaaacaaaa caaaacaaaa caaaaggugu aguagguccg 120
guguaguagg uccuucggga ccggaagaau ugaucucggc cggaccggaa gaauugaucu 180
cggcc 185
<210> 11
<211> 395
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 11
gguguaguag guccggugua guagguccuu cgggaccgga agaauugauc ucggccggac 60
cggaagaauu gaucucggcc caaaacaaaa caaaacaaaa caaaaggugu aguagguccg 120
guguaguagg uccuucggga ccggaagaau ugaucucggc cggaccggaa gaauugaucu 180
cggcccaaaa caaaacaaaa caaaacaaaa gguguaguag guccggugua guagguccuu 240
cgggaccgga agaauugauc ucggccggac cggaagaauu gaucucggcc caaaacaaaa 300
caaaacaaaa caaaaggugu aguagguccg guguaguagg uccuucggga ccggaagaau 360
ugaucucggc cggaccggaa gaauugaucu cggcc 395
<210> 12
<211> 201
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 12
gggccgcacu cgccgguccc aagcccggau aaaaugggag ggggcgggaa accgccuaac 60
caugccgagu gcggccgcgg uguaguaggu ccuucgggac cggaagaauu gaucucggcc 120
guggccgcgg ucggcgugga cuguagaaca cugccaaugc cggucccaag cccggauaaa 180
aguggagggu acaguccacg c 201
<210> 13
<211> 159
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 13
gugcucgcuu cggcagcaca uauacuaaaa uuggaacgau acagagaaga uuagcauggc 60
cccucgaaga ggguguagua gguccuucgg gaccggaaga auugaucucg gcccucuucg 120
aggaugacac gcaaauucgu gaagcguucc auauuuuug 159
<210> 14
<211> 1186
<212> RNA
<213> Artificial Sequence (Synthetic Sequence)
<400> 14
auggaugaug auaucgccgc gcucgucguc gacaacggcu ccggcaugug caaggccggc 60
uucgcgggcg acgaugcccc ccgggccguc uuccccucca ucguggggcg ccccaggcac 120
cagggcguga uggugggcau gggucagaag gauuccuaug ugggcgacga ggcccagagc 180
aagagaggca uccucacccu gaaguacccc aucgagcacg gcaucgucac caacugggac 240
gacauggaga aaaucuggca ccacaccuuc uacaaugagc ugcguguggc ucccgaggag 300
caccccgugc ugcugaccga ggccccccug aaccccaagg ccaaccgcga gaagaugacc 360
cagaucaugu uugagaccuu caacacccca gccauguacg uugcuaucca ggcugugcua 420
ucccuguacg ccucuggccg uaccacuggc aucgugaugg acuccgguga cggggucacc 480
cacacugugc ccaucuacga gggguaugcc cucccccaug ccauccugcg ucuggaccug 540
gcuggccggg accugacuga cuaccucaug aagauccuca ccgagcgcgg cuacagcuuc 600
accaccacgg ccgagcggga aaucgugcgu gacauuaagg agaagcugug cuacgucgcc 660
cuggacuucg agcaagagau ggccacggcu gcuuccagcu ccucccugga gaagagcuac 720
gagcugccug acggccaggu caucaccauu ggcaaugagc gguuccgcug cccugaggca 780
cucuuccagc cuuccuuccu gggcauggag uccuguggca uccacgaaac uaccuucaac 840
uccaucauga agugugacgu ggacauccgc aaagaccugu acgccaacac agugcugucu 900
ggcggcacca ccauguaccc uggcauugcc gacaggaugc agaaggagau cacugcccug 960
gcacccagca caaugaagau caagaucauu gcuccuccug agcgcaagua cuccgugugg 1020
aucggcggcu ccauccuggc cucgcugucc accuuccagc agauguggau cagcaagcag 1080
gaguaugacg aguccggccc cuccaucguc caccgcaaau gcuucuagcu cgaggccgcg 1140
guguaguaug acguucgcgu caggaagaau ugaucucggc cgcggc 1186
Claims (25)
1. A nucleic acid aptamer molecule comprising the following nucleotide sequence (a), (b) or (c):
(a) The nucleotide sequence is: n is a radical of 1 UGUAGUAN 9 -N 10 -N 11 GGAAGAAUUGAUCUCGGN 29 In which N is 1 、N 9 、N 10 、N 11 And N 29 Represents a nucleotide fragment of length ≧ 1, and N 1 And N 29 At least one pair of bases in the nucleotide sequence form a complementary pair, N 9 And N 11 At least one pair of bases in the nucleotide sequence forms complementary pairing;
(b) A nucleotide sequence having at least 58% identity to the nucleotide sequence defined in (a);
(c) Not including N in the nucleotide sequence defined in (a) 1 、N 9 、N 10 、N 11 And N 29 The 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 58%,63%,67%,71%,75%,79%,83%,94%,96%,98% or 100% identity to the Paprika structural nucleotide sequence of (a).
3. The aptamer molecule of claim 1 wherein nucleotide sequence (c) does not include N in the Paprika structural nucleotide sequence defined in (a) 1 、N 9 、N 10 、N 11 And N 29 The aptamer molecule of (4) through substitution, deletion and/or addition of 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 nucleotides.
4. The aptamer molecule of claim 3, wherein nucleotide sequence (c) does not include N in the nucleotide sequence defined in (a) 1 、N 9 、N 10 、N 11 And N 29 The 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) 1 And N 29 Complementary pairing, N 1 The orientation of the nucleotide sequence is 5'-3', N 29 The orientation of the nucleotide sequence is 3'-5'; n is a radical of 9 And N 11 Complementary pairing, N 9 The orientation of the nucleotide sequence is 5'-3', N 11 The orientation of the nucleotide sequence is 3'-5'.
6. The aptamer molecule of claim 5 wherein N is 1 And N 29 When the length of at least one fragment in (a) is more than or equal to 5 nucleotide bases, then N 1 And N 29 At least two pairs of nucleotide bases in the nucleotide sequence form complementary pairing; when N is present 9 And N 11 When the length of at least one fragment in (1) is more than or equal to 5 nucleotide bases, then N 9 And N 11 At least two pairs of bases in the nucleotide sequenceForming a complementary pair.
7. The nucleic acid aptamer molecule of any one of claims 1to 6, wherein the substitution of a nucleotide for the structure of formula Paprika is selected from one of the following: U2A, U2C, U2G, G3A, G3U, G3C, U4A, U4C, U4G, A5U, A5C, A5G, G6A, G6U, G6C, U7A, U7C, U7G, A8U, A8C, A8G, G12A, G12U, G12C, G13A, G13U, G13C, A14U, A14C, A14G, A15U, A15C, A15G, G16A, G16U, G16C, A17U, A17C, A17G, A18U, A18C, A18G, U19A, U19C U19G, U20A, U20C, U20G, G21A, G21U, G21C, A22U, A22C, A22G, U23A, U23C, U23G, C24A, C24U, C24G, U25A, U25C, U25G, C26A, C26U, C26G, G27A, G27U, G27C, G28A, G28U, G28C, A5U/U25A, A5C/U25G, A5G/U25C, G6A/C24U, G6U/C24A, G6C/C24G, U4A/G12A, U4A/G13U, U U4A/G16A, U4A/U20C, U4A/A22C, G12A/G13U, G12A/G16A, G12A/U20C, G12A/A22C, G13U/G16A, G13U/U20C, G13U/A22C, G16A/U20C, G16A/A22C, U20C/A22C, U4A/G12A/G13U, U4A/G12A/G16A, U4A/G12A/A22C, G12A/G13U/G16A, G12A/G13U/G16C, G12A/G13U/U20C, G12A/G13U/A22C G13U/G16A/U20C, G13U/G16A/A22C, G16A/U20C/A22C, U4A/G12A/G13U/G16A/U20C, U4A/G12A/G13U/G16A/A22C, U4A/G13U/G16A/U20C/A22C, U4A/G12A/G13U/U20C/A22C, A5U/U25A/U4A/G12A/G13U, A5U/U25A/U4A/G12A/G16A, G6A/C24U/U4A/G12A/A22C, G6A/C24U/G12A/G13U/G16A, G6A/C24U/G12A/G13U/U20C, G6A/C24U/G12A/G13U/A22C.
8. The aptamer molecule of claim 7 wherein the substitution for a nucleotide of the general formula Paprika is selected from one of the following groups: U2A, U2C, U2G, U4A, U4C, U4G, A5C, A5G, G6U, U7A, U7C, U7G, A8U, A8C, A8G, G12A, G12U, G12C, G13A, G13U, G13C, A14U, A14C, A14G, A15U, A15C, A15G, G16A, G16U, G16C, A17U, A17C, A17G, A18U, A18C, U19A, U19C, U19G, U20A, U20C, U20G, G21A G21U, A22C, A22G, U23A, U23C, U23G, C24A, C24U, C24G, U25A, U25C, C26A, C26U, G27A, G27U, G27C, G28A, G28U, G28C, A5U/U25A, A5C/U25G, A5G/U25C, G6A/C24U, G6U/C24A, G6C/C24G, U4A/G12A, U4A/G13U, U4A/G16A, U4A/U20C, U4A/A22C, G12A/G13U G12A/G16A, G12A/U20C, G12A/A22C, G13U/G16A, G13U/U20C, G13U/A22C, G16A/U20C, G16A/A22C, U20C/A22C, U4A/G12A/G13U, U4A/G12A/G16A, U4A/G12A/A22C, G12A/G13U/G16A, G12A/G13U/U20C, G12A/G13U/A22C, G13U/G16A/U20C, G13U/G16A/A22C G16A/U20C/A22C, U4A/G12A/G13U/G16A/U20C, U4A/G12A/G13U/G16A/A22C, U4A/G13U/G16A/U20C/A22C, U4A/G12A/G13U/U20C/A22C, A5U/U25A/U4A/G12A/G13U, A5U/U25A/U4A/G12A/G16A, G6A/C24U/U4A/G12A/A22C, G6A/C24U/G12A/G13U/G16A, G6A/C24U/G12A/G13U/U20C, G6A/C24U/G12A/G13U/A22C.
9. The aptamer molecule of claim 8, wherein the substitution of a nucleotide of the general formula Paprika is selected from one of the following groups: U2A, U2C, U2G, U4A, U4C, U4G, A5C, A5G, G6U, U7C, U7G, A8U, A8C, A8G, G12A, G12U, G12C, G13A, G13U, G13C, A14U, A14C, A14G, A15U, A15C, A15G, G16A, G16U, G16C, A17U, A17C, A17G, U19A, U19C, U20A, U20C, U20G, G21U, A22C, A22G U23A, U23C, U23G, C24A, C24G, U25C, C26U, G27A, G27U, G27C, G28A, G28U, G28C, A5U/U25A, A5C/U25G, A5G/U25C, G6A/C24U, G6U/C24A, G6C/C24G, U4A/G12A, U4A/G13U, U4A/G16A, U4A/U20C, U4A/A22C, G12A/G13U, G12A/G16A, G12A/U20C, G12A/A22C G13U/G16A, G13U/U20C, G13U/A22C, G16A/U20C, G16A/A22C, U20C/A22C, U4A/G12A/G13U, U4A/G12A/G16A, U4A/G12A/A22C, G12A/G13U/G16A, G12A/G13U/U20C, G12A/G13U/A22C, G13U/G16A/U20C, G13U/G16A/A22C, G16A/U20C/A22C, U4A/G12A/G13U/G16A/U20C, U4A/G12A/U20A 20C U4A/G12A/G13U/G16A/A22C, U4A/G13U/G16A/U20C/A22C, U4A/G12A/G13U/U20C/A22C, A5U/U25A/U4A/G12A/G13U, A5U/U25A/U4A/G12A/G16A, G6A/C24U/U4A/G12A/A22C, G6A/C24U/G12A/G13U/G16A, G6A/C24U/G12A/G13U/U20C, G6A/C24U/G12A/G13U/A22C.
10. The aptamer molecule of claims 1-9 wherein N in nucleotide sequence (a) 1 And N 29 The 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 said aptamer function is such that the aptamer increases the fluorescence intensity of the fluorophore molecule under excitation light of a suitable wavelength by at least a factor of 2, at least a factor of 5 to 10, at least a factor of 20 to 50, at least a factor of 100 to 200, at least a factor of 200 to 1000, or at least a factor of 1000 to 5000.
14. The aptamer molecule according to claim 1, further comprising concatemers that can bind multiple fluorophore molecules, wherein said 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: 1.2, 3,4,5,6,7,8,9, 10, 11, 12, 13 and 14.
15. A complex of an aptamer molecule and a fluorophore molecule, wherein the aptamer molecule is according to any one of claims 1to 14 and the fluorophore molecule has the structure of formula (I):
electron donor moiety-D is-NX 1-X2, X1 is selected from hydrogen, alkyl, or modified alkyl, X2 is selected from hydrogen, alkyl, or modified alkyl, X1, X2 are optionally linked to each other, forming together with the N atom a lipo-heterocycle;
the conjugated system E is formed by at least one conjugated connection selected from double bonds, triple bonds, aromatic rings and aromatic heterocycles, wherein each contained hydrogen atom is optionally and independently substituted by a substituent selected from halogen atoms, hydroxyl groups, amino groups, primary amino groups, secondary amino groups, hydrophilic groups, alkyl groups and modified alkyl groups, and the substituents are optionally connected with each other to form an alicyclic ring or an alicyclic heterocyclic ring;
x1, X2, independently, are joined to the conjugated structure E to form a lipoheterocycle;
the electron acceptor A moiety has a cyclic structure represented by the following formula (I-1-a):
ra is independently selected from hydrogen, halogen atoms, nitro groups, alkyl groups, aryl groups, heteroaryl groups, hydrophilic groups or modified alkyl groups; r b Independently selected from hydrogen, halogen atom, hydroxyl, carboxyl, amino, nitro, alkyl, aryl, heteroaryl, hydrophilic group or modified alkyl or a group formed by conjugated connection of double bond and at least one of aromatic ring and aromatic heterocycle;
each Y 1 Independently selected from-O-, -S-, - (S = O) -, and- (NR) i ) -, wherein R i Selected from hydrogen, amino, alkyl or modified alkyl;
each Y is 2 Independently selected from = O, = S = O and = NR i Wherein R is i Selected from hydrogen, alkyl or modified alkyl;
each Y 3 Independently selected from = O, = S = O and = NR i Wherein R is i Selected from hydrogen, alkyl or modified alkyl;
or, each Y 3 Independently = C (R) e ) (CN); wherein R is e Selected from hydrogen, ester group, amide group, sulfonic group, sulfonamide group, sulfonic ester group;
wherein,
said "alkyl" is C 1 -C 30 Linear or branched alkyl of (a); preferably, is C 1 -C 10 A linear or branched alkyl group; preferably, is C 1 -C 7 A linear or branched alkyl group; preferably, is C 1 -C 5 A linear or branched alkyl group; <xnotran> , , , , , , , , , ,1- ,2- ,3- , ,1- , , ,1- ,2- ,3- , ,1,1- ,2,2- ,3,3- ,1,2- ,1,3- ,2,3- ,2- , ,2- ,3- ,2,2- ,3,3- ,2,3- ,2,4- ,3- 2,2,3- ; </xnotran>
The "modified alkyl" is an alkyl in which any carbon atom is substituted with a halogen atom, -O-, -OH, -CO-, -CS-, -NO 2 、-CN、-S-、-SO 2 -、-(S=O)-、A group obtained by replacing at least one group of phenyl, phenylene, primary amino, secondary amino, tertiary amino, quaternary ammonium base, saturated or unsaturated monocyclic or bicyclic cyclic hydrocarbon, biaryl heterocyclic ring and bridged alicyclic ring, wherein the modified alkyl has 1to 30 carbon atoms, and a carbon-carbon single bond of the modified alkyl is optionally and independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond;
the carbon atom is replaced, and the carbon atom or the carbon atom and the hydrogen atom on the carbon atom are replaced by the corresponding group;
the alicyclic ring is a saturated or unsaturated 4-to 10-membered monocyclic or polycyclic alicyclic ring;
the "aliphatic heterocycle" is a saturated or unsaturated 4-to 10-membered monocyclic or polycyclic aliphatic heterocycle containing at least one heteroatom selected from N, O, S and Si on the ring, and the aliphatic heterocycle containsWhen S atom is present, it is-S-, -SO-or-SO 2 -; the aliphatic heterocyclic ring is optionally substituted with a halogen atom, a nitro group, an alkyl group, an aryl group, a hydrophilic group and a modified alkyl group;
the "aryl or aromatic ring" is a 5-to 10-membered monocyclic or fused bicyclic aromatic group;
the heteroaryl or the aromatic heterocycle is a 5-to 10-membered monocyclic or fused bicyclic heteroaromatic group containing at least one heteroatom selected from N, O, S or Si on the ring;
the halogen atoms are respectively and independently selected from F, cl, br and I;
the "hydrophilic group" is a hydroxyl group, a sulfonic group, a carboxyl group, a phosphite group, a primary amino group, a secondary amino group or a tertiary amino group;
the bridged lipoheterocycle is a 5-20-membered bridged lipoheterocycle containing at least one heteroatom selected from N, O or S on the ring;
the "ester group" is an R (C = O) OR' group;
said "phosphite" RP (= O) (OH) 2 group;
the "sulfonic acid group" is RSO 3 A H group;
the "sulfonate group" is RSO 3 An R' group;
the "sulfonic acid amino group" is RSO 2 A NR' R "group;
the "primary amino group" is RNH 2 A group;
said "secondary amino" is an RNHR' group;
the "tertiary amino" group is an RNR' R "group;
said "quaternary ammonium salt group" R 'R "R'" N + group;
each R, R' is independently a single bond, an alkyl group, an alkylene group, a modified alkyl group, or a modified alkylene group, the modified alkyl or modified alkylene is C1-C10 (preferably C1-C6) alkyl or alkylene, any carbon atom of which is selected from-O-, (II) -one of-OH, -CO-, -CS-, - (S = O) -, in place of the resulting group;
optionally, the modified alkyl or modified alkylene is each independently selected from the group consisting of-OH, -O-, and ethylene glycol monoElement (- (CH) 2 CH 2 O) n-), C1-C8 alkyl, C1-C8 alkoxy, C1-C8 acyloxy, C1-C8 haloalkyl monosaccharide groups, disaccharide groups, polysaccharide groups, -O-CO-, -NH-CO-, - (-NH-CHR "" CO-) n-, -SO 2 -O-、-SO-、-SO 2 -NH-, -S-S-, -CH = CH-, a halogen atom, a cyano group, a nitro group, an o-nitrophenyl group, a phenacyl group, a phosphate group, wherein n is 1to 100, preferably 1to 50, more preferably 1to 30, more preferably 1to 10; r "" is H or the residue of an alpha amino acid; said "phosphate group" has the definition as described above;
the "monosaccharide units" are saccharide units that can no longer be simply hydrolyzed to smaller sugar molecules;
the disaccharide unit is a saccharide unit formed by dehydration of two monosaccharides;
the polysaccharide unit is a saccharide unit formed by dehydration of more than ten monosaccharides;
optionally, the C1-C8 alkyl is methyl, ethyl, propyl, isopropyl, the C1-C8 alkoxy is methoxy, ethoxy, propoxy, isopropoxy, the C1-C8 acyloxy is acetoxy, ethyl, propyl, isopropyl, the C1-C8 haloalkyl is trifluoromethyl, chloromethyl, bromomethyl;
optionally, the lipoheterocycle is selected from azetidine, pyrrolidine, piperidine, tetrahydrofuran, tetrahydropyran, morpholine, thiomorpholine.
Alternatively, the conjugated system E is selected from the structures of the following formulas (I-2-1) to (I-2-39):
alternatively, the conjugated system E is conjugated with-NX 1 -X 2 To form a lipid represented by the following (I-3-1) (I-3-5)Heterocyclic ring:
alternatively, the conjugated system E is with-NX 1 -X 2 The structure shown in the following (I-3-6) is formed:
optionally, the electron acceptor moiety is one selected from the following formulae (I-4-1) to (I-4-42):
17. the composite of claim 16, wherein the first polymer is a polymer, wherein the fluorophore molecule is selected from the group consisting of I-5-1, I-5-2, I-5-3, I-5-4, I-5-5, I-5-6, I-5-7, I-5-8, I-5-9, I-5-10, I-5-11, I-5-12, I-5-13, I-5-14, I-5-15, I-5-16, I-5-17, I-5-18, I-5-19, I-5-20, I-5-21, I-5-22, I-5-23, I-5-24I-5-25, I-5-26, I-5-27, I-5-28, I-5-29, I-5-30, I-5-31, I-5-32, I-5-33, I-5-34, I-5-35, I-5-36, I-5-37, I-5-38, I-5-39, I-5-40, I-5-41, I-5-42, I-5-43, I-5-44, I-5-45, I-5-46, I-5-47, I-5-48, I-5-49, I-5-50, I-5-51, I-5-52, I-5-53, I-5-54, I-5-55, I-5-56, I-5-57, I-5-58, I-5-59, I-5-60, I-5-61, I-5-62, I-5-63, I-5-64, I-5-65, I-5-66, I-5-67, I-5-68, I-5-69, I-5-70, I-5-71, I-5-72, I-5-73, I-5-74, I-5-75, I-5-76, I-5-77, I-5-78, I-5-79, I-5-80, I-5-81, I-5-82, I-5-83, I-5-84.
18. The complex of any one of claims 15-17, 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 and 14.
19. A complex according to any one of claims 15 to 18 for use in the detection or labelling of a target nucleic acid molecule in vitro or in vivo.
20. A DNA molecule that transcribes the nucleic acid aptamer molecule of any one of claims 1to 14.
21. An expression vector comprising the DNA molecule of claim 20.
22. A host cell comprising the expression system of claim 21.
23. A kit comprising the aptamer molecule of any one of claims 1to 14 and/or the expression vector of claim 21 and/or the host cell of claim 22 and/or the complex of any one of claims 15 to 18.
24. A method of detecting a target molecule comprising the steps of:
a) Adding the complex of any one of claims 15-18 to a solution comprising the target molecule;
b) Exciting the complex with light of a suitable wavelength;
c) Detecting the fluorescence of the complex.
25. A method for extracting and purifying RNA comprising extracting and purifying RNA using the complex of any one of claims 15-18.
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